Mohamed Rahmani

The major form of cell death in normal as well as malignant cells is apoptosis, which is a programmed process highly regulated by the BCL-2 family of proteins. This includes the antiapoptotic proteins (BCL-2, BCL-XL, MCL-1, BCLW, and BFL-1) and the proapoptotic proteins, which can be divided into two groups: the effectors (BAX, BAK, and BOK) and the BH3-only proteins (BIM, BAD, NOXA, PUMA, BID, BIK, HRK). Notably, the BCL-2 antiapoptotic proteins are often overexpressed in malignant cells. While this offers survival advantages to malignant cells and strengthens their drug resistance capacity, it also offers opportunities for novel targeted therapies that selectively kill such cells. This review provides a comprehensive overview of the extensive preclinical and clinical studies targeting BCL-2 proteins with various BCL-2 proteins inhibitors with emphasis on venetoclax as a single agent, as well as in combination with other therapeutic agents. This review also discusses recent advances, challenges focusing on drug resistance, and future perspectives for effective targeting the Bcl-2 family of proteins in cancer.

BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome <i>c</i> and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2 inactivation and caspase-independent down-regulation of Mcl-1. Inducible expression of a constitutively active MEK1 construct did not prevent Mcl-1 down-regulation, suggesting that this event is not related to MEK/ERK pathway inactivation. Furthermore, BAY 43-9006 did not induce major changes in Mcl-1 mRNA levels monitored by real-time PCR or <i>Mcl-1</i> promoter activity demonstrated by luciferase reporter assays, but it did enhance Mcl-1 down-regulation in actinomycin D-treated cells. Inhibition of protein synthesis by cycloheximide or proteasome function with MG132 and pulse-chase studies with [³⁵S]methionine demonstrated that BAY 43-9006 did not diminish Mcl-1 protein stability, nor did it enhance Mcl-1 ubiquitination, but instead markedly attenuated Mcl-1 translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor. Finally, ectopic expression of Mcl-1 in leukemic cells markedly inhibited BAY 43-9006-mediated cytochrome <i>c</i> cytosolic release, caspase-9, -7, and -3 activation, as well as cell death, indicating that Mcl-1 operates upstream of cytochrome <i>c</i> release and caspase activation. Together, these findings demonstrate that BAY 43-9006 mediates cell death in human leukemia cells, at least in part, through down-regulation of Mcl-1 via inhibition of translation.

Abstract Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor κB (NF-κB) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies. (Blood. 2003;102:3765-3774)

The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-xL, and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting. The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-xL, and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting. Chronic myelogenous leukemia (CML) 1The abbreviations used are: CML, chronic myelogenous leukemia; RT, reverse transcriptase; FITC, fluorescein isothiocyanate; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HA, hemagglutinin; PARP, poly [ADP-ribose] polymerase; bcr, breakpoint cluster region; siRNA, small interfering RNA; dsRNAi, double-stranded RNA interference; DiOC6, 3,3-dihexyloxacarbocyanine; PP2, 4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine; AIF, apoptosis-inducing factor. is a hematopoietic stem cell disorder in which 90% of patients display a reciprocal translocation involving chromosomes 9 and 22, resulting in what has been designated the Philadelphia (Ph) chromosome. This translocation stems from a head-to-tail fusion of the breakpoint cluster region (bcr) at band q11 of chromosome 22 with the proto-oncogene (c-ABL) at band q34 of chromosome 9. This leads in turn to the expression in virtually all Ph+ CML patients of the chimeric fusion protein p210 Bcr/Abl, a constitutively active tyrosine kinase (1Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1125) Google Scholar). The etiologic role of the BCR/ABL oncogenic tyrosine kinase in the pathogenesis of CML has been well documented (2McLaughlin J. Chianese E. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6558-6562Crossref PubMed Scopus (306) Google Scholar, 3Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1929) Google Scholar). Expression of Bcr/Abl not only contributes to leukemic transformation but also represents a barrier to the successful treatment of this disorder. 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Reiffers J.J. Shea T.C. Chapuis B. Coutre S. Tura S. Morra E. Larson R.A. Saven A. Peschel C. Gratwohl A. Mandelli F. Ben-Am M. Gathmann I. Capdeville R. Paquette R.L. Druker B.J. Blood. 2002; 99: 3530-3539Crossref PubMed Scopus (1067) Google Scholar, 15Kantarjian H. Sawyers C. Hochhaus A. Guilhot F. Schiffer C. Gambacorti-Passerini C. Niederwieser D. Resta D. Capdeville R. Zoellner U. Talpaz M. Druker B. Goldman J. O'Brien S.G. Russell N. Fischer T. Ottmann O. Cony-Makhoul P. Facon T. Stone R. Miller C. Tallman M. Brown R. Schuster M. Loughran T. Gratwohl A. Mandelli F. Saglio G. Lazzarino M. Russo D. Baccarani M. Morra E. International STI571 CML Study Group N. Engl. J. Med. 2002; 346: 645-652Crossref PubMed Scopus (1835) Google Scholar). Resistance to imatinib mesylate stems from BCR/ABL gene amplification, leading to overexpression of Bcr/Abl protein (16le Coutre P. Tassi E. Varella-Garcia M. Barni R. Mologni L. Cabrita G. Marchesi E. Supino R. Gambacorti-Passerini C. Blood. 2000; 95: 1758-1766Crossref PubMed Google Scholar, 17Gorre M.E. Mohammed M. Ellwood K. Hsu N. Paquette R. Rao P.N. Sawyers C.L. Science. 2001; 293: 876-880Crossref PubMed Scopus (2756) Google Scholar), or point mutations in the BCR/ABL gene, resulting in a single amino acid substitution (e.g. Thr-315 → Ile or Glu-255 → Lys or Glu-255 → Val) within the ATP pocket of the Abl kinase domain known to be essential for imatinib mesylate binding (17Gorre M.E. Mohammed M. Ellwood K. Hsu N. Paquette R. Rao P.N. Sawyers C.L. Science. 2001; 293: 876-880Crossref PubMed Scopus (2756) Google Scholar, 18Shah N.P. Nicoll J.M. Nagar B. Gorre M.E. Paquette R.L. Kuriyan J. Sawyers C.L. Cancer Cell. 2002; 2: 117-125Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar). In addition, mutations outside of the kinase domain that allosterically inhibit imatinib mesylate binding to the Bcr/Abl protein have now been described (19Azam M. Latek R.R. Daley G.Q. Cell. 2003; 112: 831-843Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Recently, a putatively Bcr/Abl-independent form of imatinib mesylate resistance has been reported by several groups (20Nimmanapalli R. O'Bryan E. Huang M. Bali P. Burnette P.K. Loughran T. Tepperberg J. Jove R. Bhalla K. Cancer Res. 2002; 62: 5761-5769PubMed Google Scholar, 21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar). Specifically, Bcr/Abl-positive CML cells cultured in the continuous presence of imatinib mesylate (20Nimmanapalli R. O'Bryan E. Huang M. Bali P. Burnette P.K. Loughran T. Tepperberg J. Jove R. Bhalla K. Cancer Res. 2002; 62: 5761-5769PubMed Google Scholar, 21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar), or obtained from certain CML patients who have progressed while receiving imatinib mesylate (22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar), display a decline in Bcr/Abl protein and/or mRNA levels and a corresponding increase in expression/activity of the Lyn and Hck kinase (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar). Lyn and Hck represent members of the Src tyrosine kinase family and have been implicated in the regulation of cell survival and proliferation, among numerous other functions (23Lionberger J.M. Wilson M.B. Smithgall T.E. J. Biol. Chem. 2000; 275: 18581-18585Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Bates R.C. Edwards N.S. Burns G.F. Fisher D.E. Cancer Res. 2001; 61: 5275-5283PubMed Google Scholar). Lyn exists in two isoforms, p56 and p53, derived from alternatively spliced mRNAs (25Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar). The catalytic activity of Lyn is tightly regulated through tyrosine phosphorylation at the conserved Tyr-508 (26Shangary S. Lerner E.C. Zhan Q. Corey S.J. Smithgall T.E. Baskaran R. Exp. Cell Res. 2003; 289: 67-76Crossref PubMed Scopus (11) Google Scholar). Evidence derived from both murine and human model systems suggests that Lyn is a potentially important downstream target of Bcr/Abl (27Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar); moreover, Lyn has been shown to be activated in Bcr/Abl-positive cells obtained from CML patients in blast crisis as well as in HL-60 human leukemia cells transfected with Bcr/Abl (28Ptasznik A. Urbanowska E. Chinta S. Costa M.A. Katz B.A. Stanislaus M.A. Demir G. Linnekin D. Pan Z.K. Gewirtz A.M. J. Exp. Med. 2002; 196: 667-678Crossref PubMed Scopus (90) Google Scholar). Together, such findings raise the possibility that activation of Lyn can subserve the anti-apoptotic functions of Bcr/Abl, including conditions in which expression of Bcr/Abl is diminished, for whatever reason. Although the role that Lyn plays in protecting cells from lethal stimuli has been examined in some detail (29Grishin A.V. Azhipa O. Semenov I. Corey S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10172-10177Crossref PubMed Scopus (65) Google Scholar), the functional relationship that exists between this Src kinase and Bcr/Abl remains to be fully elucidated. Moreover, no information exists concerning interactions hyperactivated Lyn might have with Bcl-2 family members. To address these issues, we have examined the molecular profile of human CML cells that have become resistant to imatinib mesylate in association with the loss of Bcr/Abl and activation of the Lyn kinase. Here we report that in such cells activation of Lyn is associated with a pronounced increase in the levels of the anti-apoptotic protein Bcl-2 and that disruption of Lyn activation through either pharmacologic or genetic strategies has a significant functional impact on Bcl-2 expression and resistance to mitochondria-dependent apoptosis. In addition, pharmacological or genetic disruption of Bcl-2 function increases the susceptibility of these resistant cells to lethality of imatinib mesylate or the topoisomerase II inhibitor VP-16. These findings may have implications for the development of new therapeutic strategies directed against leukemia cells exhibiting novel forms of imatinib mesylate resistance. Cells and Reagents—LAMA84 were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Imatinib-resistant LAMA84 cells (LAMA-R) were generated by culturing cells in gradually increasing concentrations of imatinib mesylate (beginning at 0.1 μm and increasing in stepwise increments of 0.1 μm) until a level of 1 μm (30Yu C. Krystal G. Dent P. Grant S. Clin. Cancer Res. 2002; 8: 2976-2984PubMed Google Scholar). Parental K562 and imatinib-resistant K562-R cells (generated as described above for LAMA-R cells) were kindly provided by Dr. L. Varticovski (Tufts University School of Medicine, Boston). Cells were cultured in 10% fetal bovine serum/RPMI 1640 medium as described previously (30Yu C. Krystal G. Dent P. Grant S. Clin. Cancer Res. 2002; 8: 2976-2984PubMed Google Scholar). K562-R and LAMA-R were maintained in the medium containing 1 μm imatinib mesylate and were washed free of drug before all experimental procedures. All experiments were performed utilizing logarithmically growing cells (4–6 × 105 cells/ml). Imatinib mesylate (Gleevec, STI-571), provided by Dr. Elizabeth Buchdunger (Novartis Pharmaceuticals, Basel, Switzerland), was dissolved in Me2SO at a stock concentration of 50 mm, stored at –20 °C, and subsequently diluted with serum-free RPMI medium prior to use. 4-Amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine (PP2), a selective inhibitor of the Src family tyrosine kinases (31Wilson M.B. Schreiner S.J. Choi H.J. Kamens J. Smithgall T.E. Oncogene. 2002; 21: 8075-8088Crossref PubMed Scopus (120) Google Scholar), 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3), a negative control for PP2, and ethyl-2-amino-6-bromo-4-[1-cyano-2-ethoxy-2-oxoethyl]-4H-chromene-3-carboxylate (HA14-1), a cell-permeable and low molecular weight Bcl-2 inhibitory ligand (32Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Crossref PubMed Scopus (1184) Google Scholar, 33An J. Chen Y. Huang Z. J. Biol. Chem. 2004; 279: 19133-19140Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), were purchased from Calbiochem and Biomol (Plymouth Meeting, PA). cpm-1285 (a cell-permeable Bcl-2-binding peptide derived from the BH3 domain encompassing residues 140–165) and its negative control peptide cpm-1285mt (a mutant in which Leu-151 is replaced by Ala) (34Wang J.L. Zhang Z.J. Choksi S. Shan S. Lu Z. Croce C.M. Alnemri E.S. Korngold R. Huang Z. Cancer Res. 2000; 60: 1498-1502PubMed Google Scholar) were provided by Calbiochem. Etoposide (VP-16), a topoisomerase II inhibitor, was obtained from Sigma. These agents were dissolved in Me2SO as a stock solution and stored at –80 °C. In all experiments, the final concentration of Me2SO did not exceed 0.1%. Transient and Stable Transfection with cDNAs—cDNAs encoding full-length, kinase-defective (K275D, in which Asp is substituted for Lys-275 in the ATP-binding pocket), and constitutively active (Y508F, in which Tyr-508 is substituted in the conserved tail of the C terminus with Phe) Lyn were subcloned into pcDNA3 containing an HA tag (26Shangary S. Lerner E.C. Zhan Q. Corey S.J. Smithgall T.E. Baskaran R. Exp. Cell Res. 2003; 289: 67-76Crossref PubMed Scopus (11) Google Scholar). Bcl-2 cDNA (wild type) in pUSEamp was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). LAMA84 and K562 cells (1 × 106 per condition) were transiently transfected with three forms of Lyn cDNA (i.e. wild type, K275D, and Y508F) and Bcl-2 cDNA, respectively, using the Amaxa Nucleofector Device (program T-16) with Cell Line-specific Nucleofector Kit V (Amaxa GmbH, Cologne, Germany). Alternatively, K562 and LAMA84 cells were transfected with K275D and Y508F constructs as well as their empty vector counterparts (pcDNA3), respectively, and stably transfected clones were selected by limited dilution using G418. Bcl-2 RNA Interference and Antisense Oligonucleotides—1 × 106 LAMA84 and LAMA-R cells were transfected with 2 μg of Bcl-2-annealed dsRNAi oligonucleotide (5′-GUACAUCCAUUAUAAGCUGTT-3′/3′-TTCAUGUAGGUAAUAUUCGAC-5′, Orbigen, San Diego, CA) and SignalSilence Control siRNA (Cell Signaling, Beverly, MA), respectively, using the Amaxa Nucleofector Device (program T-16, Kit V). Alternatively, LAMA84 and LAMA-R cells were transfected with 5 μgof a Bcl-2 antisense oligonucleotide (5′-TCTCCCAGCGTGCGCCAT-3′, G3139, Calbiochem) or a scrambled control oligonucleotide (5′-TACCGCGTGCGACCCTCT-3′, G3622, Calbiochem) as described for transfection with dsRNAi. Transfection efficiency (>80% at 24 h post-transfection) was monitored by using fluorescein-labeled dsRNAi or antisense oligonucleotide and flow cytometric analysis. Bcl-2 protein levels were determined by Western blot analysis. Apoptosis and Viability—The extent of apoptosis was evaluated by annexin V-FITC staining and flow cytometry. Briefly, 1 × 106 cells were stained with annexin V-FITC (Pharmingen) and 5 μg/ml propidium iodide (PI, Sigma) in 1× binding buffer (10 mm Hepes/NaOH, pH 7.4, 140 mm NaOH, 2.5 mm CaCl2) for 15 min at room temperature in the dark. The samples were analyzed by flow cytometry within 1 h to determine the percentage of cells displaying annexin V+ (early apoptosis) or annexin V+/PI+ staining (late apoptosis). The ViaCount assay was performed to evaluate cell viability in the experiments involving transient transfection. 1 × 105 cells were stained with Guava ViaCount Reagent, and the percentage of viable cells was determined using a Guava Personal Cytometer (Gauva Technologies, Hayward, CA) as per the manufacturer's instructions. The ViaCount assay distinguishes between viable and nonviable cells, based on the differential permeability of DNA-binding dyes in the ViaCount Reagent, and was found to correlate closely with the results of annexin/PI staining. Mitochondrial Membrane Potential (Δψm)—2 × 105 cells were stained with 40 nm 3,3-dihexyloxacarbocyanine (DiOC6; Molecular Probes Inc., Eugene, OR) in phosphate-buffered saline at 37 °C for 20 min and then analyzed by flow cytometry. The percentage of cells exhibiting decreased level of DiOC6 uptake, which reflects loss of Δψm, was determined using FACScan from BD Biosciences. Cell Growth and Survival (MTT Assay)—5 × 104 (in 100 μl volume)/well cells were seeded into 96-well plates and incubated with 20 μl of CellTiter 96® AQueous One Solution (Promega, Madison, WI) as per the manufacturer's instructions, and the absorbance at 490 nm was recorded using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). Western Blot—Whole-cell pellets were lysed in SDS sample buffer, and 30 μg of protein for each condition was subjected to Western blot analysis following the procedures described in detail previously (35Dai Y. Yu C. Singh V. Tang L. Wang Z. McInistry R. Dent P. Grant S. Cancer Res. 2001; 61: 5106-5115PubMed Google Scholar). Where indicated, the blots were reprobed with antibodies against β-actin (rabbit polyclonal, Transduction Laboratories, Lexington, KY) or α-tubulin (mouse monoclonal, Calbiochem) to ensure equal loading and transfer of proteins. The following antibodies were used as primary antibodies: phospho-Bcr (Tyr-177) antibody (rabbit polyclonal, Cell Signaling), c-Abl antibody (mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA), STAT5 antibody (rabbit polyclonal, Santa Cruz Biotechnology), Bcl-xL antibody (rabbit polyclonal, Cell Signaling), anti-PARP (poly [ADP-ribose] polymerase, mouse monoclonal, Biomol), phospho-Lyn (Tyr-507) antibody (rabbit polyclonal, Cell Signaling), Lyn antibody (rabbit polyclonal, Cell Signaling), anti-human Bcl-2 oncoprotein (mouse monoclonal, Dako, Carpinteria, CA), Bax antibody (rabbit polyclonal, Santa Cruz Biotechnology), XIAP antibody (mouse monoclonal, Transduction Laboratories), phospho-CrkL (Tyr-207) antibody (rabbit polyclonal, Cell Signaling), CrkL (32H4) antibody (mouse monoclonal, Cell Signaling), phospho-Hck (Tyr-411) antibody (rabbit polyclonal, Santa Cruz Biotechnology), anti-Hck antibody (rabbit polyclonal, Upstate Biotechnology, Inc.), and HA probe (rabbit polyclonal, Santa Cruz Biotechnology). In some cases, the density of blots was quantified using FluoChem 8800 Imaging System (Alpha Innotech, San Leandro, CA) and VideoTesT-Master software (VideoTesT, Ltd., St. Petersburg, Russia). Translocation of Bax, Cytochrome c, and AIF—4 × 106 cells were washed in phosphate-buffered saline and lysed by incubating for 30 s in lysis buffer (75 mm NaCl, 8 mm Na2HPO4, 1 mm NaH2PO4, 1 mm EDTA, and 350 μg/ml digitonin). After centrifuged at 12,000 × g for 1 min, the supernatant (cytosolic fraction) was collected in an equal volume of 2× sample buffer, and the pellet (mitochondria-rich fraction) was lysed by sonication in 1× SDS sample buffer. For both cytosolic and pellet fractions, the proteins were quantified, and 30 μg of protein per condition was separated by 15% SDS-PAGE and subjected to Western blot, as described above, using Bax antibody, cytochrome c antibody (mouse monoclonal, Santa Cruz Biotechnology), and AIF antibody (mouse monoclonal, Santa Cruz Biotechnology) as primary antibody. RT-PCR—Total RNA was isolated from 1 × 106 cells using RNeasy mini kit (Qiagen, Valencia, CA) with QIAshredder spin column (Qiagen) as per the manufacturer's instructions. 1 μg per condition of total RNA was subjected to RT-PCR using One-step RT-PCR kit (Qiagen) and PTC-200 Peltier Thermal cycler (MJ Research, Reno, NV). The primers (forward, 5′-CGACTTCGCCGAGATGTCCAGGCAG-3′; reverse, 5′-GACCCACGGATAGACCCGGTGTTCA-3′) were used for Bcl-2. RT-PCR was performed under the conditions as follows: reverse transcription at 50 °C for 30 min, initial PCR activation step at 95 °C for 15 min, three-step cycling (denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min) for 30 cycles, and final extension at 72 °C for 10 min. The reactions were run, in parallel, for actin as endogenous control. PCR products of Bcl-2 (388 bp) were analyzed in 2% agarose gel with ethidium bromide. Real Time Quantitative RT-PCR—Total RNA was prepared as described above. The real time RT-PCR was performed on the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) using the TaqMan One-step PCR Master Mix Reagents kit (P/N, 4309169). All the samples were tested in triplicate under the conditions recommended by the fabricant. The cycling conditions are as follows: 48 °C/30 min; 95 °C/10 min; and 40 cycles of 95 °C/15 s and 60 °C/1 min. The cycle threshold was determined to provide the optimal standard curve values (0.98 to 1.0). The probes (5′-CCTGGTGGACAACATCGCCCTGT-3′) and primers for Bcl-2 (forward, 5′-CCTGGTGGACAACATCGCCCTGT-3′; reverse, 5′-GCCGGTTCAGGTACTCAGTCAT-3′) were designed using the Primer Express 2.0 version. The probes were labeled at the 5′ end with 6-carboxyfluorescein and at the 3′ end with 6-carboxytetramethylrhodamine. Ribosomal RNA (18 S rRNA) from the Pre-developed TaqMan Assay Reagents (P/N, 4310893E) was used as endogenous control. Statistical Analysis—For analysis of apoptosis, Δψm, MTT, viability, and real time quantitative RT-PCR, values represent the means ± S.D. for at least three separate experiments performed in triplicate. The significance of differences between experimental variables was determined by using the Student's t test. Imatinib-resistant CML Cells Exhibit Loss of Bcr/Abl and Down-regulation of Bcr/Abl Downstream Targets—The response of K562 and LAMA84 cells and their resistant counterparts (K562-R and LAMA-R) to imatinib mesylate was compared. Following an exposure to 0.5–10.0 μm imatinib, K562 (48 h) and LAMA84 cells (24 h) displayed progressive increases in annexin V/PI positivity, whereas their resistant counterparts were minimally affected (Fig. 1, A and B). Thus, both the K562-R and LAMA-R cell lines displayed marked resistance to imatinib mesylate-mediated lethality. Attempts were then made to establish the basis for imatinib mesylate resistance in the two resistant cell lines. Parental K562 and LAMA84 cells, as well as their resistant counterparts, were exposed to 1 μm imatinib mesylate for 48 (K562 and K562-R) and 24 h (LAMA84 and LAMA-R), after which Western blot analysis was performed. As shown in Fig. 1C, both K562-R and LAMA-R cells displayed a dramatic reduction in levels of total and phosphorylated Bcr/Abl, as well as markedly diminished expression of Stat5 and Bcl-xL, well described Bcr/Abl downstream targets (36Horita M. Andreu E.J. Benito A. Arbona C. Sanz C. Benet I. Prosper F. Fernandez-Luna J.L. J. Exp. Med. 2000; 191: 977-984Crossref PubMed Scopus (325) Google Scholar), compared with parental cells. Treatment of both sensitive and resistant cells with imatinib mesylate resulted in reductions in Bcl-xL levels, whereas expression of the inhibitor of apoptosis protein XIAP was only diminished following imatinib mesylate exposure in sensitive cells. Consistent with the results shown in Fig. 1, A and B, imatinib mesylate-induced PARP cleavage was largely abrogated in K562-R and LAMA-R cells. Thus, both K562-R and LAMA-R cells appeared to exhibit a Bcr/Abl-independent form of imatinib mesylate resistance, analogous to that previously described by Donato et al. (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar) in K562 cells and patient samples. Bcr/Abl-independent Imatinib Resistance Is Associated with Marked Activation of Lyn and Increased Expression of Bcl-2— Expression of phosphorylated Bcr/Abl and related proteins in resistant cells was examined next. As shown in Fig. 2A, levels of phospho-Bcr/Abl (activated) were dramatically reduced in K562-R and LAMA-R compared with their parental counterparts. Furthermore, consistent with earlier results involving K562 cells (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar), phosphorylation of the Src kinase Lyn was markedly increased in both resistant cell lines

NF-κB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-κB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IκBα, were strikingly diminished by either coadministration of the IκBα phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IκBα superrepressor. Inhibition of NF-κB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl l-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-κB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-κB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-κB-dependent antiapoptotic proteins, and stress-related JNK1 activation.

The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I-induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and Akt. Expression of constitutively active Akt protected against SK1-I-induced apoptosis. Notably, SK1-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK1-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK1 warrant attention as potential additions to the therapeutic armamentarium in leukemia.

AbstractSorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1α markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1α or XBP1, disruption of PERK activity, or inhibition of eIF2α phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib. We thank J. A. Diehl (University of Pennsylvania) for providing us with the PERKΔC construct, C. Hetz and L. H. Glimcher (Harvard Medical School) for providing us with the HA-IRE1α construct, and Mark Lynch (Bayer) and John Wright (Cancer Treatment and Evaluation Program, NCI) for critically reading the manuscript and providing us with sorafenib.This work was supported by awards CA 63753, CA 93738, and CA 100866 from the National Cancer Institute; award 6045-03 from the Leukemia and Lymphoma Society of America; and awards from the Department of Defense and the V Foundation.SUPPLEMENTAL MATERIAL

Abstract Interactions between histone deacetylase inhibitors (HDACIs) and the alkyl-lysophospholipid perifosine were examined in human leukemia cells. Coadministration of sodium butyrate, suberoylanilide hydroxamic acid (SAHA), or trichostatin with perifosine synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and a marked decrease in cell growth in U937 as well as HL-60 and Jurkat leukemia cells. These events were associated with inactivation of extracellular signal-regulated kinase (ERK) 1/2 and Akt, p46 c-jun-NH2-kinase (JNK) activation, and a pronounced increase in generation of ceramide and reactive oxygen species (ROS). They were also associated with up-regulation of Bak and a marked conformational change in Bax accompanied by membrane translocation. Ectopic expression of Bcl-2 delayed but was ultimately ineffective in preventing perifosine/HDACI-mediated apoptosis. Enforced expression of constitutively active mitogen-activated protein kinase kinase (MEK) 1 or myristoylated Akt blocked HDACI/perifosine-mediated ceramide production and cell death, suggesting that MEK/ERK and Akt inactivation play a primary role in these phenomena. However, inhibition of JNK activation (e.g., by the JNK inhibitor SP600125) did not attenuate sodium butyrate/perifosine-induced apoptosis. In addition, the free radical scavenger N-acetyl-l-cysteine attenuated ROS generation and apoptosis mediated by combined treatment. Finally, the acidic sphingomyelinase inhibitor desipramine attenuated HDACI/perifosine-mediated ceramide and ROS production as well as cell death. Together, these findings indicate that coadministration of HDACIs with perifosine in human leukemia cells leads to Akt and MEK/ERK disruption, a marked increase in ceramide and ROS production, and a striking increase in mitochondrial injury and apoptosis. They also raise the possibility that combining these agents may represent a novel antileukemic strategy.

Introduction: Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers. Areas covered: Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments. Expert opinion: We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.

We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2alpha phosphorylation; suppression of eIF2alpha function abolished drug combination lethality. Cell killing was paralleled by PERK-and eIF2alpha-dependent lowering of c-FLIP-s protein levels and overexpression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRbeta and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Overexpression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2alpha- downward arrowc-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).

Abstract Effects of concomitant inhibition of the PI3K/AKT/mTOR pathway and Bcl-2/Bcl-xL (BCL2L1) were examined in human myeloid leukemia cells. Tetracycline-inducible Bcl-2 and Bcl-xL dual knockdown sharply increased PI3K/AKT/mTOR inhibitor lethality. Conversely, inducible knockdown or dominant-negative AKT increased, whereas constitutively active AKT reduced lethality of the Bcl-2/Bcl-xL inhibitor ABT-737. Furthermore, PI3K/mTOR inhibitors (e.g., BEZ235 and PI-103) synergistically increased ABT-737–mediated cell death in multiple leukemia cell lines and reduced colony formation in leukemic, but not normal, CD34+ cells. Notably, increased lethality was observed in four of six primary acute myelogenous leukemia (AML) specimens. Responding, but not nonresponding, samples exhibited basal AKT phosphorylation. PI3K/mTOR inhibitors markedly downregulated Mcl-1 but increased Bim binding to Bcl-2/Bcl-xL; the latter effect was abrogated by ABT-737. Combined treatment also markedly diminished Bax/Bak binding to Mcl-1, Bcl-2, or Bcl-xL. Bax, Bak, or Bim (BCL2L11) knockdown or Mcl-1 overexpression significantly diminished regimen-induced apoptosis. Interestingly, pharmacologic inhibition or short hairpin RNA knockdown of GSK3α/β significantly attenuated Mcl-1 downregulation and decreased apoptosis. In a systemic AML xenograft model, dual tetracycline-inducible knockdown of Bcl-2/Bcl-xL sharply increased BEZ235 antileukemic effects. In a subcutaneous xenograft model, BEZ235 and ABT-737 coadministration significantly diminished tumor growth, downregulated Mcl-1, activated caspases, and prolonged survival. Together, these findings suggest that antileukemic synergism between PI3K/AKT/mTOR inhibitors and BH3 mimetics involves multiple mechanisms, including Mcl-1 downregulation, release of Bim from Bcl-2/Bcl-xL as well as Bak and Bax from Mcl-1/Bcl-2/Bcl-xL, and GSK3α/β, culminating in Bax/Bak activation and apoptosis. They also argue that combining PI3K/AKT/mTOR inhibitors with BH3 mimetics warrants attention in AML, particularly in the setting of basal AKT activation and/or addiction. Cancer Res; 73(4); 1340–51. ©2012 AACR.

Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda-7/IL-24-mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda-7/IL-24-induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda-7/IL-24-mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda-7/IL-24 (Ad.5/3-mda-7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda-7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3-mda-7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda-7/IL-24-mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.

Interactions between the multikinase inhibitor sorafenib and the BH3-mimetic obatoclax (GX15-070) were examined in human acute myeloid leukemia (AML) cells. Treatment with sorafenib/obatoclax induced pronounced apoptosis in and reduced the clonogenic growth of multiple AML lines and primary AML cells but not normal CD34(+) cells. Sorafenib triggered rapid and pronounced Mcl-1 down-regulation accompanied by enhanced binding of Bim to Bcl-2 and Bcl-xL, effects that were abolished by obatoclax coadministration. Notably, shRNA knockdown of Bim, Bak, or Bax, but not Noxa, significantly attenuated obatoclax/sorafenib lethality, whereas ectopic expression of Mcl-1 exerted a protective effect. Furthermore, exposure of leukemia cells to sorafenib and obatoclax markedly induced autophagy, reflected by rapid and pronounced LC3 processing and LC3-green fluorescent protein (GFP) punctate formation. Multiple autophagy inhibitors or VPS34 knockdown, significantly potentiated sorafenib/obatoclax lethality, indicating a cytoprotective role for autophagy in this setting. Finally, studies in a xenograft mouse model revealed that combined sorafenib/obatoclax treatment markedly reduced tumor growth and significantly prolonged survival in association with Mcl-1 down-regulation and apoptosis induction, whereas agents administered individually had only modest effects. These findings suggest that combining sorafenib with agents that inhibit Mcl-1 and Bcl-2/Bcl-xL such as obatoclax may represent a novel and potentially effective strategy in AML.

Inhibitors targeting BCL-2 apoptotic proteins have significant potential for the treatment of acute myeloid leukemia (AML); however, complete responses are observed in only 20% of patients, suggesting that targeting BCL-2 alone is insufficient to yield durable responses. Here, we assessed the efficacy of coadministration of the PI3K/mTOR inhibitor GDC-0980 or the p110β-sparing PI3K inhibitor taselisib with the selective BCL-2 antagonist venetoclax in AML cells. Tetracycline-inducible downregulation of BCL-2 significantly sensitized MV4-11 and MOLM-13 AML cells to PI3K inhibition. Venetoclax/GDC-0980 coadministration induced rapid and pronounced BAX mitochondrial translocation, cytochrome c release, and apoptosis in various AML cell lines in association with AKT/mTOR inactivation and MCL-1 downregulation; ectopic expression of MCL-1 significantly protected cells from this regimen. Combined treatment was also effective against primary AML blasts from 17 patients, including those bearing various genetic abnormalities. Venetoclax/GDC-0980 markedly induced apoptosis in primitive CD34+/38-/123+ AML cell populations but not in normal hematopoietic progenitor CD34+ cells. The regimen was also active against AML cells displaying intrinsic or acquired venetoclax resistance or tumor microenvironment-associated resistance. Either combinatorial treatment markedly reduced AML growth and prolonged survival in a systemic AML xenograft mouse model and diminished AML growth in two patient-derived xenograft models. Venetoclax/GDC-0980 activity was partially diminished in BAK-/- cells and failed to induce apoptosis in BAX-/- and BAX-/-BAK-/- cells, whereas BIM-/- cells were fully sensitive. Similar results were observed with venetoclax alone in in vitro and in vivo systemic xenograft models. Collectively, these studies demonstrate that venetoclax/GDC-0980 exhibits potent anti-AML activity primarily through BAX and, to a lesser extent, BAK. These findings argue that dual BCL-2 and PI3K inhibition warrants further evaluation in AML.Significance: Combined treatment with clinically relevant PI3K and BCL-2 inhibitors may prove effective in the treatment of acute myeloid leukemia. Cancer Res; 78(11); 3075-86. ©2018 AACR.

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.

Abstract Purpose: The aim of this study is to explore the efficacy and define mechanisms of action of coadministration of the PI3K/mTOR inhibitor BEZ235 and pan-HDAC inhibitor panobinostat in diffuse large B-cell lymphoma (DLBCL) cells. Experimental Design: Various DLBCL cells were exposed to panobinostat and BEZ235 alone or together after which apoptosis and signaling/survival pathway perturbations were monitored by flow cytometry and Western blot analysis. Genetic strategies defined the functional significance of such changes, and xenograft mouse models were used to assess tumor growth and animal survival. Results: Panobinostat and BEZ235 interacted synergistically in ABC-, GC-, and double-hit DLBCL cells and MCL cells but not in normal CD34+ cells. Synergism was associated with pronounced AKT dephosphorylation, GSK3 dephosphorylation/activation, Mcl-1 downregulation, Bim upregulation, increased Bcl-2/Bcl-xL binding, diminished Bax/Bak binding to Bcl-2/Bcl-xL/Mcl-1, increased γH2A.X phosphorylation and histone H3/H4 acetylation, and abrogation of p21CIP1 induction. BEZ235/panobinostat lethality was not susceptible to stromal/microenvironmental forms of resistance. Genetic strategies confirmed significant functional roles for AKT inactivation, Mcl-1 downregulation, Bim upregulation, and Bax/Bak in synergism. Finally, coadministration of BEZ235 with panobinostat in immunocompromised mice bearing SU-DHL4–derived tumors significantly reduced tumor growth in association with similar signaling changes observed in vitro, and combined treatment increased animal survival compared with single agents. Conclusions: BEZ235/panobinostat exhibits potent anti-DLBCL activity, including in poor-prognosis ABC- and double-hit subtypes, but not in normal CD34+ cells. Synergism is most likely multifactorial, involving AKT inactivation/GSK3 activation, Bim upregulation, Mcl-1 downregulation, enhanced DNA damage, and is operative in vivo. Combined PI3K/mTOR and HDAC inhibition warrants further attention in DLBCL. Clin Cancer Res; 20(18); 4849–60. ©2014 AACR.

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.

Interactions between proteasome and cyclin-dependent kinase inhibitors have been examined in human leukemia cells in relation to induction of apoptosis. Simultaneous exposure (24 h) of U937 myelomonocytic leukemia cells to 100 nM flavopiridol and 300 nM MG-132 resulted in a marked increase in mitochondrial injury (cytochrome c, Smac/DIABLO release, loss of deltaPsi(m)), caspase activation, and synergistic induction of cell death, accompanied by a marked decrease in clonogenic potential. Similar effects were observed with other proteasome inhibitors (e.g., Bortezomib (VELCADE trade mark bortezomib or injection), lactacystin, LLnL) and cyclin-dependent kinase inhibitors (e.g., roscovitine), as well as other leukemia cell types (e.g., HL-60, Jurkat, Raji). In U937 cells, synergistic interactions between MG-132 and flavopiridol were associated with multiple perturbations in expression/activation of signaling- and survival-related proteins, including downregulation of XIAP and Mcl-1, activation of JNK and p34(cdc2), and diminished expression of p21(CIP1). The lethal effects of MG-132/flavopiridol were not reduced in leukemic cells ectopically expressing Bcl-2, but were partially attenuated in cells ectopically expressing dominant-negative caspase-8 or CrmA. Flavopiridol/proteasome inhibitor-mediated lethality was also significantly diminished by agents and siRNA blocking JNK activation. Lastly, coadministration of MG-132 with flavopiridol resulted in diminished DNA binding of NF-kappaB. Notably, pharmacologic interruption of the NF-kappaB pathway (e.g., by BAY 11-7082, PDTC, or SN-50) or molecular dysregulation of NF-kappaB (i.e., in cells ectopically expressing an IkappaBalpha super-repressor) mimicked the actions of proteasome inhibitors in promoting flavopiridol-induced mitochondrial injury, JNK activation, and apoptosis. Together, these findings indicate that proteasome inhibitors strikingly lower the apoptotic threshold of leukemic cells exposed to pharmacologic CDK inhibitors, and suggest that interruption of the NF-kappaB cytoprotective pathway and JNK activation both play key roles in this phenomenon. They also raise the possibility that combining proteasome and CDK inhibitors could represent a novel antileukemic strategy.

Abstract Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nm) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nm), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-β-d-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27KIP1, cyclins A, E, and D1, and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH2-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger l-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Abstract Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl+ human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor κB (NF-κB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-β-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl- human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies. (Blood. 2004;104:509-518)

Mechanism(s) by which the multikinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat interact to kill hepatic, renal, and pancreatic adenocarcinoma cells has been defined.Low doses of sorafenib and vorinostat interacted in vitro in a synergistic fashion to kill hepatic, renal, and pancreatic adenocarcinoma cells in multiple short-term viability (24-96 h) and in long-term colony formation assays. Cell killing was suppressed by inhibition of cathepsin proteases and caspase-8 and, to a lesser extent, by inhibition of caspase-9. Twenty-four hours after exposure, the activities of extracellular signal-regulated kinase 1/2, AKT, and nuclear factor-kappaB were only modestly modulated by sorafenib and vorinostat treatment. However, 24 h after exposure, sorafenib- and vorinostat-treated cells exhibited markedly diminished expression of c-FLIP-s, full-length BID, BCL-2, BCL-XL, MCL-1, XIAP, increased expression of BIM, and increased activation of BAX, BAK, and BAD. Expression of eIF2alpha S51A blocked sorafenib- and vorinostat-induced suppression of c-FLIP-s levels and overexpression of c-FLIP-s abolished lethality. Sorafenib and vorinostat treatment increased surface levels of CD95 and CD95 association with caspase-8. Knockdown of CD95 or FADD expression significantly reduced sorafenib/vorinostat-mediated lethality.These data show that combined exposure of epithelial tumor cell types to sorafenib and vorinostat diminishes expression of multiple antiapoptotic proteins and promotes activation of the CD95 extrinsic apoptotic and the lysosomal protease pathways, and that suppression of c-FLIP-s expression represents a critical event in transduction of the proapoptotic signals from CD95 to promote mitochondrial dysfunction and death.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect antitumor activity through inhibition of angiogenesis, stimulation of an antitumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.

We examined the interaction between the multikinase inhibitor sorafenib and histone deacetylase inhibitors. Sorafenib and vorinostat synergized (sorafenib + vorinostat) to kill HCT116 and SW480 cells. In SW480 cells, sorafenib + vorinostat increased CD95 plasma membrane levels and promoted death-inducing signal complex (DISC) formation, and drug toxicity was blocked by knockdown of CD95 or overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s). In SW620 cells that are patient-matched to SW480 cells, sorafenib + vorinostat toxicity was significantly lower, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing, whereas knockdown of LASS6 in SW480 cells suppressed CD95 activation. Knocking down LASS6 expression also suppressed CD95 activation in hepatoma, pancreatic, and ovarian cancer cells. In HCT116 cells, sorafenib + vorinostat treatment caused DISC formation without reducing c-FLIP-s expression and did not increase CD95 plasma membrane levels; sorafenib + vorinostat exposure killed HCT116 cells via an intrinsic pathway/caspase 9-dependent mechanism. In HCT116 cells, knockdown of CD95 enhanced sorafenib + vorinostat lethality, which correlated with less drug-induced CD95-dependent autophagy. Sorafenib + vorinostat treatment activated the c-Jun NH(2)-terminal kinase pathway, which was causal in promoting dissociation of Beclin1 from BCL-2, and in promoting autophagy. Knockdown of Beclin1 expression blocked autophagy and enhanced drug toxicity. Our data demonstrate that treatment of colon cancer cells with sorafenib + vorinostat activates CD95 via de novo ceramide synthesis that promotes viability via autophagy or degrades survival via either the extrinsic or intrinsic pathways.

Effects of the PI-3 kinase inhibitor LY294002 (LY) have been examined in relation to responses of human leukemia cells to histone deacetylase inhibitors (HDIs). Coexposure of U937 cells for 24 h to marginally toxic concentrations of LY294002 (e.g., 30 microM) and sodium butyrate (SB; 1 mM) resulted in a marked increase in mitochondrial damage (e.g., cytochrome c and Smac/DIABLO release, loss of DeltaPsi(m)), caspase activation, and apoptosis. Similar results were observed in Jurkat, HL-60, and K562 leukemic cells and with other HDIs (e.g., SAHA, MS-275). Exposure of cells to SB/LY was associated with Bcl-2 and Bid cleavage, XIAP and Mcl-1 downregulation, and diminished CD11b expression. While LY blocked SB-mediated Akt activation, enforced expression of a constitutively active (myristolated) Akt failed to attenuate SB/LY-mediated lethality. Unexpectedly, treatment of cells with SB+/-LY resulted in a marked reduction in phosphorylation (activation) of p44/42 mitogen-activated protein (MAP) kinase. Moreover, enforced expression of a constitutively active MEK1 construct partially but significantly attenuated SB/LY-induced apoptosis. Lastly, cotreatment with LY blocked SB-mediated induction of p21(CIP1/WAF1); moreover, enforced expression of p21(CIP1/WAF1) significantly reduced SB/LY-mediated apoptosis. Together, these findings indicate that LY promotes SB-mediated apoptosis through an AKT-independent process that involves MEK/MAP kinase inactivation and interference with p21(CIP1/WAF1) induction.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.

The effects of 2-Methoxyestradiol (2ME)-induced apoptosis was examined in human leukemia cells (U937 and Jurkat) in relation to mitochondrial injury, oxidative damage, and perturbations in signaling pathways. 2ME induced apoptosis in these cells in a dose-dependent manner associated with release of mitochondrial proteins (cytochrome c, AIF), generation of reactive oxygen species (ROS), downregulation of Mcl-1 and XIAP, and inactivation (dephosphorylation) of Akt accompanied by activation of JNK. In these cells, enforced activation of Akt by a constitutively active myristolated Akt construct prevented 2ME-mediated mitochondrial injury, XIAP and Mcl-1 downregulation, JNK activation, and apoptosis, but not ROS generation. Conversely, 2ME lethality was potentiated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Furthermore, in U937 cells, the hydrogen peroxide scavenger catalase and a superoxide dismutase (SOD) mimetic, TBAP, blocked these events, as well as Akt inactivation. Interruption of the JNK pathway by pharmacologic or genetic (e.g. siRNA) means attenuated 2ME-induced mitochondrial injury, XIAP and Mcl-1 downregulation, and apoptosis. Collectively, these findings suggest a hierarchical model of 2ME-related apoptosis induction in human leukemia cells in which 2ME-induced oxidative injury represents a primary event resulting in Akt inactivation, leading, in turn, to JNK activation, and culminating in XIAP and Mcl-1 downregulation, mitochondrial injury, and apoptosis. They also suggest that in human leukemia cells, the Akt pathway plays a critical role in mediating the response to oxidative stress induced by 2ME.

Abstract Interactions between pharmacologic NF-κB inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-κB/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138+ cells and between an inhibitory peptide of NF-κB (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-xL. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-xL but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IκB kinase (IKK)/IκB/NF-κB pathway warrants attention in MM. (Blood. 2004;103:2761-2770)

Abstract Interactions between the tyrphostin adaphostin and proteasome inhibitors (eg, MG-132 and bortezomib) were examined in multiple human leukemia cell lines and primary acute myeloid leukemia (AML) specimens. Cotreatment of Jurkat cells with marginally toxic concentrations of adaphostin and proteasome inhibitors synergistically potentiated mitochondrial damage (eg, cytochrome c release), caspase activation, and apoptosis. Similar interactions occurred in other human leukemia cell types (eg, U937, HL-60, Raji). These interactions were associated with a marked increase in oxidative damage (eg, ROS generation), down-regulation of the Raf/MEK/ERK pathway, and JNK activation. Adaphostin/MG-132 lethality as well as mitochondrial damage, down-regulation of Raf/MEK/ERK, and activation of JNK were attenuated by the free-radical scavenger NAC, suggesting that oxidative damage plays a functional role in antileukemic effects. Ectopic expression of Raf-1 or constitutively active MEK/ERK or genetic interruption of the JNK pathway significantly diminished adaphostin/MG-132-mediated lethality. Interestingly, enforced Raf or MEK/ERK activation partially diminished adaphostin/MG-132-mediated ROS generation, suggesting the existence of an amplification loop. Finally, the adaphostin/MG-132 regimen displayed similar toxicity toward 5 primary AML samples but not normal hematopoietic progenitors (eg, bone marrow CD34+ cells). Collectively, these findings suggest that potentiating oxidative damage by combining adaphostin with proteasome inhibitors warrants attention as an antileukemic strategy.

Interactions between the histone deacetylase (HDAC) inhibitors suberanoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein (Hsp) 90 antagonist 17-allylamino 17-demethoxygeldanamycin (17-AAG) have been examined in Bcr-Abl⁺ human leukemia cells (K562 and LAMA84), including those sensitive and resistant to STI571 (imatinib mesylate). Cotreatment with 17-AAG and SAHA or SB synergistically induced mitochondrial dysfunction (cytochrome <i>c</i> and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and growth inhibition. Similar effects were observed in LAMA84 cells and K562 cells resistant to STI571, as well as in CD34⁺ cells isolated from the bone marrows of three patients with chronic myelogenous leukemia. These events were associated with increased binding of Bcr-Abl, Raf-1, and Akt to Hsp70, and inactivation of extracellular signal-regulated kinase 1/2 and Akt. In addition, 17-AAG/SAHA abrogated the DNA binding and the transcriptional activities of signal transducer and activator of transcription (STAT) 5 in K562 cells, including those ectopically expressing a constitutively active STAT5A construct. Cotreatment with 17-AAG and SAHA also induced down-regulation of Mcl-1, Bcl-xL, and B-Raf; up-regulation of Bak; cleavage of 14-3-3 proteins; and a profound conformational change in Bax accompanied by translocation to the membrane fraction. Moreover, ectopic expression of Bcl-2 attenuated cell death induced by this regimen, implicating mitochondrial injury in the lethality observed. Together, these findings raise the possibility that combining HDAC inhibitors with the Hsp90 antagonist 17-AAG may represent a novel strategy against Bcr-Abl⁺ leukemias, including those resistant to STI571.

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway.

Mechanisms underlying apoptosis induced by concomitant interruption of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways were investigated in human leukemia cells. Inhibition of these pathways using the MEK inhibitor PD184352 or U0126 and the PI3K/Akt inhibitor perifosine strikingly induced apoptosis in multiple malignant human hematopoietic cells, and substantially reduced the colony-forming capacity of primary acute myeloblastic leukemia, but not normal CD34+ cells. These events were associated with pronounced Bim up-regulation, Mcl-1 down-regulation, marked Bak/Bax conformational change accompanied by Bax membrane translocation, and a pronounced increase in Bax/Bak association. Molecular studies using tet-inducible Akt, constitutively active MEK1, dominant-negative Akt, and MEK1 small interfering RNA revealed that inhibition of both MEK/ERK1/2 and Akt pathways plays a critical functional role in perifosine/PD184352-mediated lethality. Ectopic Mcl-1 expression potently inhibited perifosine/PD184352-induced apoptosis, as did Bak or Bax knockdown. Notably, knockdown of Bim, but not Bad, blocked Bak and Bax conformational change, inhibited Bax membrane translocation, diminished Bax/Bak binding, and sharply attenuated perifosine/PD184352-induced apoptosis. Finally, enforced expression of Bim significantly enhanced apoptosis induced by PI3K/Akt inhibitors, analogous to the effects of MEK1/2 inhibitors. Collectively, these findings suggest that Bim, and Mcl-1, but not Bad, integrate death signaling triggered by concomitant disruption of the PI3K/Akt and MEK1/2/ERK1/2 pathways in human leukemia cells.

We have further defined mechanism(s) by which 2-amino-<i>N</i>-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1<i>H</i>-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK^-/-), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knock-down or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R-like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK-/- cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B-dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.

We have defined some of the mechanisms by which the kinase inhibitor lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing followed by outgrowth of lapatinib-adapted cells. Adapted cells were resistant to serum starvation-induced cell killing and were cross-resistant to multiple therapeutic drugs. Lapatinib was competent to inhibit basal and epidermal growth factor (EGF)-stimulated ERBB1 phosphorylation in adapted cells. Coexpression of dominant-negative ERBB1 and dominant-negative ERBB2 inhibited basal and EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental and adapted cells. However, in neither parental nor adapted cells did expression of dominant-negative ERBB1 and dominant-negative ERBB2 recapitulate the cell death-promoting effects of lapatinib. Adapted cells had increased expression of MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK. Overexpression of BCL-XL protected parental cells from lapatinib toxicity. Knockdown of MCL-1 expression enhanced lapatinib toxicity in adapted cells that was reverted by knockdown of BAK expression. Inhibition of caspase function modestly reduced lapatinib toxicity in parental cells, whereas knockdown of apoptosis-inducing factor expression suppressed lapatinib toxicity. Thus, in HCT116 cells, lapatinib adaptation can be mediated by altered expression of pro- and antiapoptotic proteins that maintain mitochondrial function.

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double-stranded break (DSB) repair. Here, we show that depletion of CHD4 in acute myeloid leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ataxia-telangiectasia mutated pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic agent-induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34(+) hematopoietic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor-forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

Key Points The NAE inhibitor pevonedistat induces Chk1/Wee1 activation and the intra-S checkpoint, limiting its anti-AML efficacy. The HDAC inhibitor belinostat potentiates the in vitro and in vivo activity of pevonedistat in AML by disrupting the DDR.

Chronic pressure-overload (PO)- induced cardiomyopathy is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of the α isoform of glycogen synthase kinase-3 (GSK-3α) in PO-induced cardiac remodeling is unclear and its downstream molecular targets are largely unknown. To investigate the potential roles of GSK-3α, cardiomyocyte-specific GSK-3α conditional knockout (cKO) and control mice underwent trans-aortic constriction (TAC) or sham surgeries. Cardiac function in the cKOs and littermate controls declined equally up to 2 weeks of TAC. At 4 week, cKO animals retained concentric LV remodeling and showed significantly less decline in contractile function both at systole and diastole, vs. controls which remained same until the end of the study (6 wk). Histological analysis confirmed preservation of LV chamber and protection against TAC-induced cellular hypertrophy in the cKO. Consistent with attenuated hypertrophy, significantly lower level of cardiomyocyte apoptosis was observed in the cKO. Mechanistically, GSK-3α was found to regulate mitochondrial permeability transition pore (mPTP) opening and GSK-3α-deficient mitochondria showed delayed mPTP opening in response to Ca2+ overload. Consistently, overexpression of GSK-3α in cardiomyocytes resulted in elevated Bax expression, increased apoptosis, as well as a reduction of maximum respiration capacity and cell viability. Taken together, we show for the first time that GSK-3α regulates mPTP opening under pathological conditions, likely through Bax overexpression. Genetic ablation of cardiomyocyte GSK-3α protects against chronic PO-induced cardiomyopathy and adverse LV remodeling, and preserves contractile function. Selective inhibition of GSK-3α using isoform-specific inhibitors could be a viable therapeutic strategy to limit PO-induced heart failure.

Abstract Interactions between the protein kinase C (PKC) and Chk1 inhibitor UCN-01 and the heat shock protein 90 (Hsp90) antagonist 17-AAG have been examined in human leukemia cells in relation to effects on signal transduction pathways and apoptosis. Simultaneous exposure (30 hours) of U937 monocytic leukemia cells to minimally toxic concentrations of 17-AAG (eg, 400 nM) and UCN-01 (eg, 75 nM) triggered a pronounced increase in mitochondrial injury (ie, loss of mitochondrial membrane potential [Δψm]; cytosolic release of cytochrome c), caspase activation, and apoptosis. Synergistic induction of apoptosis was also observed in other human leukemia cell types (eg, Jurkat, NB4). Coexposure of human leukemia cells to 17-AAG and the PKC inhibitor bisindolylmaleimide (GFX) did not result in enhanced lethality, arguing against the possibility that the PKC inhibitory actions of UCN-01 are responsible for synergistic interactions. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and marked down-regulation of Raf-1, MEK1/2, and mitogen-activated protein kinase (MAPK). Coadministration of 17-AAG and UCN-01 did not modify expression of Hsp90, Hsp27, phospho-JNK, or phospho-p38 MAPK, but was associated with further p34cdc2 dephosphorylation and diminished expression of Bcl-2, Mcl-1, and XIAP. In addition, inducible expression of both a constitutively active MEK1/2 or myristolated Akt construct, which overcame inhibition of ERK and Akt activation, respectively, significantly attenuated 17-AAG/UCN-01–mediated lethality. Together, these findings indicate that the Hsp90 antagonist 17-AAG potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that interference with both the Akt and Raf-1/MEK/MAP kinase cytoprotective signaling pathways contribute to this phenomenon.

The effects of the multikinase inhibitor sorafenib (BAY 43-9006), an agent shown previously to induce apoptosis in human leukemia cells through inhibition of myeloid cell leukemia-1 (Mcl-1) translation, have been examined in Bcr/Abl⁺ leukemia cells resistant to imatinib mesylate (IM). When administered at pharmacologically relevant concentrations (10–15 μM), sorafenib potently induced apoptosis in imatinib mesylate-resistant cells expressing high levels of Bcr/Abl, cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, and CD34⁺ cells obtained from imatinib-resistant patients. In addition, Ba/F3 cells expressing mutations rendering them resistant to IM (e.g., E255K, M351T) or to IM, dasatinib, and nilotinib (T315I) remained fully sensitive to sorafenib. Induction of apoptosis by sorafenib was associated with rapid and pronounced down-regulation of Mcl-1 and diminished signal transducer and activator of transcription (STAT) 5 phosphorylation and reporter activity but only very modest and delayed inactivation of the Bcr/Abl downstream target Crkl. Moreover, transfection with a constitutively active STAT5 construct partially but significantly protected cells from sorafenib lethality. Ba/F3 cells expressing Bcr/Abl mutations were as sensitive to sorafenib-induced Mcl-1 down-regulation and dephosphorylation of STAT5 and eukaryotic initiation factor 4E as wild-type cells. Finally, stable knockdown of Bcl-2-interacting mediator of cell death (Bim) with short hairpin RNA in K562 cells significantly diminished sorafenib lethality, arguing strongly for a functional role of this proapoptotic Bcl-2 family member in the lethality of this agent. Together, these findings suggest that sorafenib effectively induces apoptosis in highly imatinib-resistant chronic myelogenous leukemia cells, most likely by inhibiting or down-regulating targets (i.e., STAT5 and Mcl-1) downstream or independent of Bcr/Abl.

Abstract Interactions between MEK1/2 inhibitors and the dual Abl/Src kinase inhibitor dasatinib (BMS-354825) were examined in chronic myeloid leukemia (CML) cell lines and primary specimens. Cotreatment of K562 or LAMA cells with subtoxic or marginally toxic concentrations of PD184352 (or U0126) and dasatinib synergistically potentiated mitochondrial damage, caspase activation, and apoptosis. Similar interactions were observed in CD34+ cells from one CML patient–derived but not in a normal human CD34+ bone marrow cell specimen. These interactions were associated with multiple perturbations in survival signaling pathways, including inactivation of Bcr/Abl, STAT5, and ERK1/2; down-regulation of Bcl-xL and Mcl-1; and dephosphorylation/activation of Bim. They were also associated with BAX/BAK conformational change, mitochondrial dysfunction, and caspase activation. Bim knockdown by shRNA suppressed BAX and BAK conformational change and protected cells from dasatinib/PD184352 lethality. Conversely, K562 cells ectopically expressing Mcl-1 or Bcl-xL were significantly less susceptible to dasatinib/PD184352 toxicity. Notably, the dasatinib/PD184352 regimen was active against leukemic cells exhibiting various forms of imatinib mesylate resistance, including Bcr/Abl overexpression, Lyn activation, and several Bcr/Abl kinase domain mutations (eg, E255K, M351T), but not T315I. Together, these findings suggest that strategies combining dasatanib with MEK1/2 inhibitors warrant further investigation in Bcr/Abl+ malignancies, particularly in the setting of imatinib mesylate–resistant disease.

The cytokine melanoma differentiation associated gene 7 (mda-7) was identified by subtractive hybridization as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Based on conserved structure, chromosomal location and cytokine-like properties, MDA-7, was classified as a member of the interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and rapid cell death. In addition, MDA-7/IL-24 has been noted to radiosensitize tumor cells which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress and suppress protein translation. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, strongly arguing that MDA-7/IL-24 could have significant therapeutic value. This review describes what is presently known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.

The novel cytokine melanoma differentiation associated gene-7 (mda-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared with non-transformed cells. On the basis of conserved structure, chromosomal location and cytokine-like properties, MDA-7, has now been classified as a member of the expanding interleukin (IL)-10 gene family and designated as MDA-7/IL-24. Multiple studies have shown that the expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in the corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which is partly because of the generation of reactive oxygen species and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 [Ad.mda-7 (INGN-241)] was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. This review describes what is known about the impact of MDA-7/IL-24 on tumor cell biology and its potential therapeutic applications.

Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1−/− mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis.

Signal transduction events regulating induction of apoptosis by the histone deacetylase inhibitors (HDIs) sodium butyrate (SB) and SAHA have been examined in Bcr/Abl+ human leukemia cells (K562, LAMA 84). Exposure of K562 cells to greater or less than 3.0 mM SB or 3.0 mM SAHA for 24-48 hr resulted in a marked induction of mitchondrial damage (e.g., cytochrome c release) and apoptosis, events associated with downregulation of Bcr/Abl and Raf-1, induction of p21CIP1, inactivation of MEK1/2, ERK1/2, and p70S6K, and a dramatic increase in JNK activation. HDI-mediated apoptosis was attenuated by pharmacologic JNK inhibitors and enhanced by the MEK1/2 inhibitor U0126 as well as by the JNK activator anisomycin. Interestingly, HDI-induced JNK activation was potentiated by pharmacologic MEK inhibition. Furthermore, HDI lethality was significantly diminished in cells ectopically expressing constitutively active MEK1, confirming a functional role for MEK/ERK inactivation in HDI-mediated apoptosis. Similar events were observed in Bcr/Abl+ LAMA 84 cells. Lastly, the free radical scavenger L-N-acetylcysteine (LNAC) attenuated HDI-mediated ROS generation, JNK activation, and apoptosis. Together, these findings support a model in which induction of apoptosis in Bcr/Abl+ cells by HDIs involves coordinate inactivation of the cytoprotective Raf/MEK/ERK pathway in conjunction with the ROS-dependent activation of JNK.

Abstract Interactions between the Chk1 inhibitor UCN-01 and the farnesyltransferase inhibitor L744832 were examined in human leukemia cells. Combined exposure of U937 cells to subtoxic concentrations of UCN-01 and L744832 resulted in a dramatic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary acute myeloid leukemia (AML) blasts. Coadministration of L744832 blocked UCN-01-mediated phosphorylation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), leading to down-regulation of phospho-cyclic adenosine monophosphate responsive element-binding protein (phospho-CREB) and -p90RSK and activation of p34cdc2 and stress-activated protein kinase/ERK kinase/c-Jun N-terminal kinase (SEK/JNK). Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -glycogen synthase kinase-3 (-GSK-3), -p70S6K, -mammalian target of rapamycin (-mTOR), -forkhead transcription factor (-FKHR), -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis but did not prevent activation of p34cdc2 and JNK or inactivation of MEK/ERK and Akt. Enforced expression of myristoylated Akt but not constitutively active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01 and, in so doing, dramatically increase mitochondria-dependent apoptosis. (Blood. 2005;105:1706-1716)

Abstract Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-xL and diminished the expression of cyclin D1 and p34cdc2. Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin–mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.

Interactions between the histone deacetylase inhibitors (HDACIs) suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (SB) and the heat shock protein (Hsp) 90 antagonist 17-allylamino-17-demethoxygeldanamycin (17-AAG) have been examined in human leukemia cells (U937). Coadministration of marginally toxic concentrations of 17-AAG with sublethal concentrations of SB or SAHA resulted in highly synergistic induction of mitochondrial damage (i.e., cytochrome c release), caspase-3 and -8 activation, and apoptosis. Similar interactions were noted in human promyelocytic (HL-60) and lymphoblastic (Jurkat) leukemia cells. These events were accompanied by multiple perturbations in signal transduction, cell cycle, and survival-related pathways, including early down-regulation of Raf-1, inactivation of extracellular signal-regulated kinase (ERK) 1/2 and mitogen-activated protein/ERK kinase (MEK) 1/2, diminished expression of phospho-Akt, and late activation of c-Jun-NH(2)-terminal kinase, but no changes in expression of phospho-p38 mitogen-activated protein kinase. Coadministration of 17-AAG blocked SAHA-mediated induction of the cyclin-dependent kinase inhibitor p21(CIP1) and resulted in reduced expression of p27(KIP1) and p34(cdc2). 17-AAG/SAHA-treated cells also displayed down-regulation of the antiapoptotic protein Mcl-1 and evidence of Bcl-2 cleavage. Enforced expression of doxycycline-inducible p21(CIP1) or constitutively active MEK1 significantly diminished 17-AAG/SAHA-mediated lethality, indicating that interference with ERK activation and p21(CIP1) induction play important functional roles in the lethal effects of this regimen. In contrast, enforced expression of constitutively active Akt failed to exert cytoprotective actions. Together, these findings indicate that coadministration of SAHA or SB with the Hsp90 antagonist 17-AAG in human leukemia cells leads to multiple perturbations in signaling, cell cycle, and survival pathways that culminate in mitochondrial injury and apoptosis. They also raise the possibility that combining such agents with Hsp90 antagonists may represent a novel antileukemic strategy.

AbstractMelanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by Yacoub et al. (Mol Cancer Ther 2008; In press) further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK-/- cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.Addendum to: Yacoub A, Park MA, Gupta P, Rahmani P, Zhang G, Hamed H, Hanna D, Sarkar D, Lebedeva, IV Emdad L, Sauane M, Vozhilla N, Spiegel S, Koumenis C, Graf M, Curiel DT, Grant S, Fisher PB, Dent P. Mol Cancer Ther 2008; In press.

Abstract Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7–induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal–regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH2-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7–induced phosphorylation of protein kinase R–like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R–like endoplasmic reticulum kinase that correlated with reduced c-jun NH2-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal–regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.[Mol Cancer Ther 2009;8(5):OF1–12]

As one of the current global health conundrums, COVID-19 pandemic caused a dramatic increase of cases exceeding 79 million and 1.7 million deaths worldwide. Severe presentation of COVID-19 is characterized by cytokine storm and chronic inflammation resulting in multi-organ dysfunction. Currently, it is unclear whether extrapulmonary tissues contribute to the cytokine storm mediated-disease exacerbation. In this study, we applied systems immunology analysis to investigate the immunomodulatory effects of SARS-CoV-2 infection in lung, liver, kidney, and heart tissues and the potential contribution of these tissues to cytokines production. Notably, genes associated with neutrophil-mediated immune response (e.g. CXCL1) were particularly upregulated in lung, whereas genes associated with eosinophil-mediated immune response (e.g. CCL11) were particularly upregulated in heart tissue. In contrast, immune responses mediated by monocytes, dendritic cells, T-cells and B-cells were almost similarly dysregulated in all tissue types. Focused analysis of 14 cytokines classically upregulated in COVID-19 patients revealed that only some of these cytokines are dysregulated in lung tissue, whereas the other cytokines are upregulated in extrapulmonary tissues (e.g. IL6 and IL2RA). Investigations of potential mechanisms by which SARS-CoV-2 modulates the immune response and cytokine production revealed a marked dysregulation of NF-κB signaling particularly CBM complex and the NF-κB inhibitor BCL3. Moreover, overexpression of mucin family genes (e.g. MUC3A, MUC4, MUC5B, MUC16, and MUC17) and HSP90AB1 suggest that the exacerbated inflammation activated pulmonary and extrapulmonary tissues remodeling. In addition, we identified multiple sets of immune response associated genes upregulated in a tissue-specific manner (DCLRE1C, CHI3L1, and PARP14 in lung; APOA4, NFASC, WIPF3, and CD34 in liver; LILRA5, ISG20, S100A12, and HLX in kidney; and ASS1 and PTPN1 in heart). Altogether, these findings suggest that the cytokines storm triggered by SARS-CoV-2 infection is potentially the result of dysregulated cytokine production by inflamed pulmonary and extrapulmonary (e.g. liver, kidney, and heart) tissues.

Interactions between the Bcr/Abl kinase inhibitor STI571 (Gleevec, imatinib mesylate) and histone deacetylase inhibitors (HDIs) have been examined in STI571-sensitive and -resistant Bcr/Abl(+) human leukemia cells (K562 and LAMA 84). Cotreatment of K562 cells with 250 nM imatinib mesylate and 2.0 micro M suberoylanilide hydroxamic acid (SAHA) for 24 h, exposures that were minimally toxic alone, resulted in a marked increase in mitochondrial damage (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor release), caspase activation, and apoptosis. Similar events were observed in other Bcr/Abl(+) cells (i.e., LAMA 84), and in cells exposed to STI571 in combination with the HDI sodium butyrate. Coexposure of cells to HDIs in conjunction with STI571 resulted in multiple perturbations in signaling and cell cycle-regulatory proteins, including down-regulation of Raf, phospho-mitogen-activated protein kinase kinase (MEK), phospho-extracellular signal-regulated kinase (ERK), phospho-Akt, phospho-signal transducers and activators of transcription 5, cyclin D1, and Mcl-1, accompanied by dephosphorylation and cleavage of retinoblastoma protein and a striking increase in phosphorylation of c-Jun NH(2)-terminal kinase. Coexposure of Bcr/Abl(+) cells to STI571 also blocked SAHA-mediated induction of p21(CIP1) and resulted in down-regulation of Bcr/Abl protein expression. STI571 and SAHA also interacted synergistically to induce apoptosis in STI571-resistant K562 and LAMA 84 cells that display increased Bcr/Abl protein expression. Lastly, inducible expression of a constitutively active MEK1/2 construct significantly attenuated SAHA/STI571-mediated apoptosis in K562 cells, implicating disruption of the Raf/MEK/ERK axis in synergistic antileukemic effects of this drug combination. Together, these findings indicate that combined exposure of Bcr/Abl(+) cells to the kinase inhibitor STI571 and HDIs leads to diverse perturbations in signaling and cell cycle-regulatory proteins, associated with a marked increase in mitochondrial damage and cell death. They also raise the possibility that this strategy may be effective in some Bcr/Abl(+) cells that are resistant to STI571 through increased Bcr/Abl expression.

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles.

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) have been examined in human leukemia cells in relation to effects on nuclear factor kappaB (NF-kappaB) activation. Exposure (24 h) of U937 human leukemia cells to NaB (1 mM) or SAHA (1.5 microM) resulted in a marked increase in NF-kappaB DNA binding, effects that were essentially abrogated by coadministration of flavopiridol (100 nM). These events were accompanied by a marked increase in mitochondrial injury, caspase activation, and apoptosis. Mutant cells expressing an IkappaBalpha super-repressor exhibited impairment of NF-kappaB DNA binding in response to HDACIs and a significant although modest increase in apoptosis. However, disruption of the NF-kappaB pathway also increased mitochondrial injury and caspase activation in response to flavopiridol and to an even greater extent to the combination of flavopiridol and HDACIs. Coadministration of flavopiridol with HDACIs down-regulated the X-linked inhibitor of apoptosis (XIAP), Mcl-1, and p21CIP1/WAF1 and activated c-Jun NH2-terminal kinase; moreover, these effects were considerably more pronounced in IkappaBalpha mutants. Similar responses were observed in U937 mutant cells stably expressing RelA/p65 small interfering RNA. In all cases, flavopiridol was significantly more potent than genetic interruption of the NF-kappaB cascade in promoting HDACI-mediated lethality. Together, these findings are consistent with the notion that although inhibition of NF-kappaB activation by flavopiridol contributes to antileukemic interactions with HDACIs, other NF-kappaB-independent flavopiridol actions (e.g., down-regulation of Mcl-1, XIAP, and p21CIP1/WAF1) play particularly critical roles in this phenomenon.

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.

Mechanisms of lethality of the three-substituted indolinone and putatively selective cyclin-dependent kinase (CDK)2 inhibitor 3-[1-(3<i>H</i>-imidazol-4-yl)-meth-(<i>Z</i>)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) were examined in human leukemia cells. Exposure of U937 and other leukemia cells to SU9516 concentrations ≥5 μM rapidly (i.e., within 4 h) induced cytochrome <i>c</i> release, Bax mitochondrial translocation, and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1. These effects were associated with inhibition of phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase (Pol) II on serine 2 but not serine 5. Reverse transcription-polymerase chain reaction analysis revealed pronounced down-regulation of Mcl-1 mRNA levels in SU9516-treated cells. Similar results were obtained in Jurkat and HL-60 leukemia cells. Furthermore, cotreatment with the proteasome inhibitor <i>N</i>-benzoyloxycarbonyl (<i>Z</i>)-Leu-Leu-leucinal (MG132) blocked SU9516-mediated Mcl-1 down-regulation, implicating proteasomal degradation in diminished expression of this protein. Ectopic expression of Mcl-1 largely blocked SU9516-induced cytochrome <i>c</i> release, Bax translocation, and apoptosis, whereas knockdown of Mcl-1 by small interfering RNA potentiated SU9516 lethality, confirming the functional contribution of Mcl-1 down-regulation to SU9516-induced cell death. It is noteworthy that SU9516 treatment resulted in a marked increase in reactive oxygen species production, which was diminished, along with cell death, by the free radical scavenger <i>N</i>-acetylcysteine (NAC). We were surprised to find that NAC blocked SU9516-mediated inhibition of RNA Pol II CTD phosphorylation on serine 2, reductions in Mcl-1 mRNA levels, and Mcl-1 down-regulation. Together, these findings suggest that SU9516 kills leukemic cells through inhibition of RNA Pol II CTD phosphorylation in association with oxidative damage and down-regulation of Mcl-1 at the transcriptional level, culminating in mitochondrial injury and cell death.

Previously, using primary hepatocytes residing in early G₁ phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21^{Cip-1/WAF1/mda6} (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27^Kip-1 (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2α phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.

Abstract Prior studies have noted that inhibitors of mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2) enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors [PD184352; AZD6244 (ARRY-142886)] interacted in a synergistic manner with geldanamycins [17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin] to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of extracellular signal-regulated kinase 1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was reactive oxygen species dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase-8 or overexpression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase-8 with CD95. Inhibition of p38 MAPK or knockdown of BID, FAS-associated death domain, or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data show that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the apoptotic signal from CD95 to promote cell death. [Mol Cancer Ther 2008;7(9):2633–48]

Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, preferentially induces cell death in neoplastic epithelial cells types while sparing their normal counterparts. The effects of mda-7/IL-24 in acute myeloid leukemia (AML) cells have not been extensively characterized. Treatment with recombinant GST-MDA-7/IL-24 potently induced apoptosis in diverse myeloid leukemia cell types including U937, HL60, MV4-11, EOL-1, and MLL/ENL cells. MDA-7/IL-24 also markedly induced apoptosis in and suppressed the colony-forming capacity of primary AML blasts but exerted minimal toxicity toward normal CD34(+) hematopoietic progenitor cells. MDA-7/IL-24 lethality was associated with pronounced endoplasmic reticulum (ER) stress induction in leukemia cell lines and primary AML blasts, manifested by the accumulation of growth arrest and DNA damage-inducible protein 34 (GADD34), 78-kDa glucose-regulated protein (GRP78)/BiP, inositol-requiring enzyme 1α (IRE1α), and eukaryotic initiation factor 2α phosphorylation. It is noteworthy that short hairpin RNA (shRNA) knockdown of IRE1α, GADD34, or GRP78/BiP significantly enhanced MDA-7/IL-24-mediated apoptosis, indicating a protective role for these molecules against MDA-7/IL-24 lethality. MDA-7/IL-24 also down-regulated the antiapoptotic protein Mcl-1 and sharply increased expression of the proapoptotic proteins Bim and Noxa. Ectopic Mcl-1 expression or shRNA knockdown of Bim or Noxa significantly attenuated MDA-7/IL-24-mediated leukemia cell death. Finally, knockdown of Bax or Bak significantly reduced MDA-7/IL-24 lethality. Together, these findings indicate that MDA-7/IL-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by ER stress induction, Mcl-1 down-regulation, and Bim and Noxa up-regulation. They also suggest that MDA-7/IL-24 warrants further investigation in myeloid leukemia.

Mechanisms underlying interactions between the proteasome inhibitor bortezomib and small molecule Bcl-2 antagonists were examined in GC- and ABC-type human DLBCL (diffuse lymphocytic B-cell lymphoma) cells. Concomitant or sequential exposure to non- or minimally toxic concentrations of bortezomib or other proteasome inhibitors and either HA14-1 or gossypol resulted in a striking increase in Bax/Bak conformational change/translocation, cytochrome c release, caspase activation, and synergistic induction of apoptosis in both GC- and ABC-type cells. These events were associated with a sharp increase in activation of the stress kinase JNK and evidence of ER stress induction (e.g., eIF2α phosphorylation, activation of caspases-2 and - 4, and Grp78 upregulation). Pharmacologic or genetic (e.g., shRNA knockdown ) interruption of JNK signaling attenuated HA14-1/bortezomib lethality and ER stress induction. Genetic disruption of the ER stress pathway (e.g., in cells expressing caspase -4 shRNA or DN-eIF2α) significantly attenuated lethality. The toxicity of this regimen was independent of ROS generation. Finally, HA14-1 significantly increased bortezomib-mediated JNK activation, ER stress induction, and lethality in bortezomib-resistant cells. Collectively these findings indicate that small molecule Bcl-2 antagonists promote bortezomib-mediated mitochondrial injury and lethality in DLBCL cells in association with enhanced JNK activation and ER stress induction. They also raise the possibility that such a strategy may be effective in different DLBCL sub- types (e.g., GC- or ABC), and in bortezomib-resistant disease.

Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38−/CD123+ primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid leukemia.

Acute myeloid leukemia (AML) continues to represent an area of critical unmet need with respect to new and effective targeted therapies. The Bcl-2 family of pro- and antiapoptotic proteins stands at the crossroads of cellular survival and death, and the expression of and interactions between these proteins determine tumor cell fate. Malignant cells, which are often primed for apoptosis, are particularly vulnerable to the simultaneous disruption of cooperative survival signaling pathways. Indeed, the single agent activity of agents such as mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase kinase (MEK) inhibitors in AML has been modest. Much work in recent years has focused on strategies to enhance the therapeutic potential of the bona fide BH3-mimetic, ABT-737, which inhibits B-cell lymphoma 2 (Bcl-2) and Bcl-xL. Most of these strategies target Mcl-1, an antiapoptotic protein not inhibited by ABT-737. The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways are central to the growth, proliferation, and survival of AML cells, and there is much interest currently in pharmacologically interrupting these pathways. Dual inhibitors of PI3K and mTOR overcome some intrinsic disadvantages of rapamycin and its derivatives, which selectively inhibit mTOR. In this review, we discuss why combining dual PI3K/mTOR blockade with inhibition of Bcl-2 and Bcl-xL, by virtue of allowing coordinate inhibition of three mutually synergistic pathways in AML cells, may be a particularly attractive therapeutic strategy in AML, the success of which may be predicted for by basal Akt activation.

Background The coronavirus disease 2019 (COVID-19), a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide, was described to be frequently accompanied by extrapulmonary manifestations, including liver dysfunction. Liver dysfunction and elevated liver enzymes were observed in about 53% of COVID-19 patients. Aim To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction. Methods The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed. Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways. The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis, fibrosis, non-alcoholic fatty liver disease (NAFLD), and hepatitis A/B/C. Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR. Results Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes, DNAJB1, IGF2, EGFR, and HDGF. Concordantly, the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling (liver cirrhosis, Fibrosis, NAFLD, and hepatitis A/B/C). Moreover, we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function, including cytochrome P450 family members, ACAD11, CIDEB, GNMT, and GPAM. Consequently, drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity. In correspondence with the RNA-seq data analysis, we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction. Conclusion Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling, mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.

Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.

Abstract Interactions between the endogenous estradiol metabolite 2-medroxyestradiol (2-ME) and histone deacetylase inhibitors (HDACIs) have been investigated in human leukemia cells. Coadministration of subtoxic or marginally toxic concentrations of 2-ME and SAHA or sodium butyrate in diverse human leukemia-cell types resulted in a marked increase in oxidative damage (eg, generation of reactive oxygen species [ROSs]), mitochondrial injury (eg, cytochrome c release and Bax translocation), caspase activation, and apoptosis. These interactions were also noted in primary human leukemia cells but not in normal bone marrow CD34+ cells. Synergistic interactions between these agents were associated with inactivation of Akt and activation of c-Jun N-terminal kinase (JNK). Essentially all of these events were reversed by free radical scavengers such as the manganese superoxide dismutase (MnSOD) mimetic TBAP and catalase. Notably, treatment with 2-ME/HDACIs resulted in down-regulation of thioredoxin, MnSOD, and glutathione peroxidase. Enforced activation of Akt blocked 2-ME/HDACI-mediated mitochondrial injury, caspase activation, and JNK up-regulation, but not generation of ROSs. Pharmacologic or genetic (siRNA) interruption of the JNK pathway also significantly attenuated the lethality of this regimen. Together, these findings support a model in which antileukemic synergism between 2-ME and HDACIs stems primarily from induction of oxidative damage, leading in turn to Akt inactivation and JNK activation, culminating in mitochondrial injury and apoptosis. They also raise the possibility that these events may preferentially occur in leukemic versus normal hematopoietic cells.

// Tri Nguyen 1 , Rebecca Parker 1 , Elisa Hawkins 1 , Beata Holkova 1 , Victor Yazbeck 1 , Akhil Kolluri 1 , Maciej Kmieciak 5 , Mohamed Rahmani 1 and Steven Grant 1, 2, 3, 4, 5 1 Division of Hematology/Oncology, Department of Internal Medicine, Virginia Commonwealth University and the Massey Cancer Center, Richmond, VA, USA 2 Departments of Biochemistry, Virginia Commonwealth University, Richmond, VA, USA 3 Departments of Pharmacology, Virginia Commonwealth University, Richmond, VA, USA 4 Virginia Institute for Molecular Medicine, Virginia Commonwealth University, Richmond, VA, USA 5 Massey Cancer Center, Virginia Commonwealth University Health Sciences Center, Richmond, VA, USA Correspondence to: Steven Grant, email: steven.grant@vcuhealth.org Keywords: non-Hodgkin&rsquo;s lymphoma, volasertib, belinostat Received: August 17, 2016&emsp;&emsp;&emsp;&emsp; Accepted: November 22, 2016&emsp;&emsp;&emsp;&emsp; Published: February 23, 2017 ABSTRACT Interactions between the polo-like kinase 1 (PLK1) inhibitor volasertib and the histone deacetylase inhibitor (HDACI) belinostat were examined in diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cells in vitro and in vivo. Exposure of DLBCL cells to very low concentrations of volasertib in combination with belinostat synergistically increased cell death (apoptosis). Similar interactions occurred in GC-, ABC-, double-hit DLBCL cells, MCL cells, bortezomib-resistant cells and primary lymphoma cells. Co-exposure to volasertib/belinostat induced a marked increase in M-phase arrest, phospho-histone H3, mitotic errors, cell death in M-phase, and DNA damage. Belinostat diminished c-Myc mRNA and protein expression, an effect significantly enhanced by volasertib co-exposure. c-Myc knock-down increased DNA damage and cell death in response to volasertib, arguing that c-Myc down-regulation plays a functional role in the lethality of this regimen. Notably, PLK1 knock-down in DLBCL cells significantly increased belinostat-induced M-phase accumulation, phospho-histone H3, &gamma;H2AX, and cell death. Co-administration of volasertib and belinostat dramatically reduced tumor growth in an ABC-DLBCL flank model (U2932) and a systemic double-hit lymphoma model (OCI-Ly18), accompanied by a pronounced increase in survival without significant weight loss or other toxicities. Together, these findings indicate that PLK1/HDAC inhibition warrants attention as a therapeutic strategy in NHL.

Abstract Breast cancer is one of the leading causes of death in females, mainly because of metastasis. Oncometabolites, produced via metabolic reprogramming, can influence metastatic signaling cascades. Accordingly, and based on our previous results, we propose that metabolites from highly metastatic breast cancer cells behave differently from less-metastatic cells and may play a significant role in metastasis. For instance, we aim to identify these metabolites and their role in breast cancer metastasis. Less metastatic cells (MCF-7) were treated with metabolites secreted from highly metastatic cells (MDA-MB-231) and the gene expression of three epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin and vimentin were examined. Some metabolites secreted from MDA-MB-231 cells significantly induced EMT activity. Specifically, hypoxanthine demonstrated a significant EMT effect and increased the migration and invasion effects of MCF-7 cells through a hypoxia-associated mechanism. Hypoxanthine exhibited pro-angiogenic effects via increasing the VEGF and PDGF gene expression and affected lipid metabolism by increasing the gene expression of PCSK-9. Notably, knockdown of purine nucleoside phosphorylase, a gene encoding for an important enzyme in the biosynthesis of hypoxanthine, and inhibition of hypoxanthine uptake caused a significant decrease in hypoxanthine-associated EMT effects. Collectively for the first time, hypoxanthine was identified as a novel metastasis-associated metabolite in breast cancer cells and represents a promising target for diagnosis and therapy.

Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor β (TGF-β)−induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-β treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3′ kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-β-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-β potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-β-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-β treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-β responses. Cross-talk between Smad and mitogen-activated protein kinase pathways has been described recently, and evidence for Smad cooperation with AP-1 is emerging. Here we report that epidermal growth factor (EGF) potentializes transforming growth factor β (TGF-β)−induced Smad3 transactivation in rat hepatocytes, an effect abrogated by TAM-67, a dominant negative mutant of AP-1. Antisense transfection experiments indicated that c-Jun and JunB were involved in the synergistic effect, and endogenous c-Jun physically associated with Smad3 during a combined EGF/TGF-β treatment. We next investigated which signaling pathway transduced by EGF was responsible for the Jun-induced synergism. Whereas inhibition of JNK had no effect, inhibition of the phosphatidylinositol-3′ kinase (PI3-kinase) pathway by LY294002 or by expression of a dominant negative mutant of PI3-kinase reduced EGF/TGF-β-induced Smad3 transcriptional activity. Transfection of an activated Ras with a mutation enabling the activation of the PI3-kinase pathway alone mimicked the EGF/TGF-β potentiation of Smad3 transactivation, and TAM-67 abolished this effect, suggesting that the PI3-kinase pathway stimulates Smad3 via AP-1 stimulation. The EGF/TGF-β-induced activation of Smad3 correlated with PI3-kinase and p38-dependent but not JNK-dependent phosphorylation of c-Jun. Since potentiation of a Smad-binding element-driven gene was also induced by EGF/TGF-β treatment, this novel mechanism of Jun/Smad cooperation might be crucial for diversifying TGF-β responses. transforming growth factor β extracellular signal-regulated kinase mitogen-activated protein kinase c-Jun NH2-terminal kinase/stress-activated protein kinase epidermal growth factor phosphatidylinositol 3′-kinase luciferase galactosidase Smad-binding element TPA-responsive element phosphate-buffered saline electrophoretic mobility shift assay phenylmethylsulfonyl fluoride mitogen-activated protein MAP kinase polyacrylamide gel electrophoresis Transforming growth factor β (TGF-β)1 is a member of a large family of cytokines that includes bone morphogenetic proteins, activins, and several more distantly related factors (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar). TGF-β is central in the regulation of many biological processes, including cell differentiation, growth, adhesion, and apoptosis. TGF-β signals through a system of transmembrane serine/threonine kinase receptors composed of type I and type II receptors (TGF-βRI and TGF-βRII) (see Refs. 2Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 3Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (177) Google Scholar, 4Chen Y.-G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massagué J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (300) Google Scholar and reviewed in Ref. 5Hu P.P.-C. Datto M.B. Wang X.-F. Endocr. Rev. 1998; 19: 349-363Crossref PubMed Google Scholar). Ligand binding to TGF-βRII recruits and activates the TGF-βRI receptor, which phosphorylates Smad2 and Smad3 on their SSXS motif. Smad proteins encompass a conserved amino-terminal domain that binds DNA and a conserved carboxyl-terminal domain that binds receptors and partner Smads. These domains are separated by a less conserved linker region. Phosphorylated Smad2 or Smad3 forms stable complexes with Smad4, which translocate into the nucleus, where they bind the consensus GTCTAGAC sequence found in the promoter of many TGF-β-responsive genes (6Zawel L. Le Dai J. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). Disruption of the Smad pathway or Smad mutations have underscored the functional importance of this signaling pathway in the transcriptional response of target cells to TGF-β (reviewed in Ref. 7Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar).Emerging evidence indicates that TGF-β signaling may also cross-talk with the mitogen-activated protein kinase (MAPK) family of serine/threonine protein kinases. Antagonistic or synergistic interplay between these kinases and Smad signaling has been described. Extracellular signal-regulated kinases (ERK), members of the MAPK, cause a rapid increase in the phosphorylation of Smad2 and Smad3 in their linker region, preventing their translocation into the nucleus and therefore providing a mechanism of repression of TGF-β signaling (8Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar, 9Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (848) Google Scholar). At the opposite, a synergistic mechanism between Smads and MAPK has been proposed, in a kinase downstream of the MAP kinase kinase MEK1-induced Smad2 phosphorylation on the SSXS motif and its nuclear translocation (10De Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar). Recent evidence also indicates that Smad cooperates with AP-1 (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar, 13Dennler S. Prunier C. Ferrand N. Gauthier J.-M. Atfi A. J. Biol. Chem. 2000; 275: 28858-28865Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), a heterodimer of Fos and Jun family members (14Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). Stimulation of AP-1-dependent transcription can be achieved by phosphorylation of the c-Jun transactivation domain by c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK), another member of the MAPK family. Smad and AP-1 response elements are juxtaposed in the promoters of several TGF-β-inducible genes, such as plasminogen activator inhibitor-1 or c-jun, and both sites appear to be critical in the TGF-β response (15Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar, 16Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 17Wong C. Rougier-Chapman E.M. Frederick J.P. Datto M.B. Liberati N.T. Li J.-M. Wang X.-F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (227) Google Scholar).In the present study we addressed the question of whether AP-1/Smad cooperation occurs in normal rat hepatocytes following a combined stimulation with EGF and TGF-β. We show that under these experimental conditions AP-1 induces a strong activation of Smad3 transactivation independent of AP-1 binding to its cognate cis-element. This synergism was mediated by c-Jun and JunB, and a protein-protein interaction between Smad3 and endogenous c-Jun was found during EGF/TGF-β stimulation. Furthermore, we demonstrate that activation of AP-1 via the phosphatidylinositol 3-kinase (PI3-kinase), but not the JNK pathway, is implicated in this functional synergism. Finally, we show that Jun/Smad3 cooperation induced by EGF is effective on a SBE-driven reporter gene. These data suggest that Jun/Smad3 synergism independent of binding to TRE elements might represent another important mechanism of regulation of TGF-β-inducible genes in hepatocytes.DISCUSSIONThere is increasing evidence that several signaling pathways interfere with Smads to regulate TGF-β-responsive genes, resulting in antagonistic as well as synergistic effects. In the present study, we show that EGF alone induces a mild activation of Smad3 transcriptional activity only detectable under conditions of Smad3 overexpression, in agreement with previous reports (10De Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar). More importantly, we show that a combined treatment with EGF and TGF-β strongly activates (about 30-fold) Smad3 transactivation. This synergistic effect is dependent on the presence of AP-1 proteins, since it is prevented by transfection of TAM-67, a dominant negative c-Jun truncated on its transactivating domain acting as an inhibitor of AP-1 function (18Brown P.H. Alani R. Preis L.H. Szabo E. Birrer M.J. Oncogene. 1993; 8: 877-886PubMed Google Scholar, 19Brown P.H. Chen T.K. Birrer M.J. Oncogene. 1994; 9: 791-800PubMed Google Scholar). Recently, cooperation of Smad proteins with the AP-1 proteins Fos and Jun has been documented in mink lung epithelial cells (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar). In vitro binding of Smad3 and Smad4 to all three Jun family members as well as in vivo association between Smads and a TGF-β-phosphorylated form of endogenous c-Jun induced by JNK have been demonstrated in HaCaT cells (12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar). Cooperation of AP-1 proteins with Smad3 and Smad4 occurred via Jun proteins bound to their cognatecis-element, the TRE, or to composite sites containing juxtaposed AP-1 and SBE sites (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar, 17Wong C. Rougier-Chapman E.M. Frederick J.P. Datto M.B. Liberati N.T. Li J.-M. Wang X.-F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (227) Google Scholar), and the interaction was shown to involve 13 carboxyl-terminal amino acids conserved in the three Jun proteins (12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar). The mechanism described herein differs from these previous studies by two major features as follows: 1) by using a Gal reporter system, we could demonstrate that the interaction of Smad3 with c-Jun induced a synergistic effect on Smad3 transactivation independently of binding to the TRE; 2) overexpression of TAM-67, a dominant negative c-Jun which is truncated in its transactivating NH2-terminal domain but still possesses the COOH-terminal domain implicated in the physical interaction with Smad proteins (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar), abrogated the EGF/TGF-β-induced stimulation of Smad3 transactivation, implying that the functional cooperation with Smad3 requires the amino-terminal transactivation domain of Jun proteins. In addition, we also show that the EGF-induced cooperative effect of Jun proteins on Smad3 transactivation was not paralleled by any modification of the binding of Smad proteins to their cognate cis-element, the SBE site, indicating that the increased transcriptional activity of Smad3 proteins is probably due to their association with Jun proteins rather than to the recruitment of additional Smads to the SBE. Despite the fact that a physical association between Jun proteins and Smads has been reported to occur in vitro (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar) and the demonstration that c-Jun coimmunoprecipitates with Smad3 during EGF/TGF-β stimulation (our present result), we failed in detecting c-Jun or other AP-1 proteins bound to the SBE by supershift experiments. This could be due to the lack of sensitivity of the method that is known to produce false negative results (32Osborn M.T. Herrin K. Buzen F.G. Hurlburt B.K. Chambers T.C. BioTechniques. 1999; 27: 887-892Crossref PubMed Scopus (7) Google Scholar).Binding of EGF to its receptor activates the PI3-kinase pathway (28Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (544) Google Scholar, 29Huang C. Ma W.-Y. Dong Z. Mol. Cell. Biol. 1996; 16: 6427-6435Crossref PubMed Scopus (152) Google Scholar, 30Logan S.K. Falasca M. Hu P. Schlessinger J. Mol. Cell. Biol. 1997; 17: 5784-5790Crossref PubMed Scopus (123) Google Scholar), a cascade involved in AP-1 activation (29Huang C. Ma W.-Y. Dong Z. Mol. Cell. Biol. 1996; 16: 6427-6435Crossref PubMed Scopus (152) Google Scholar, 33Huang C. Schmid P.C. Ma W.-Y. Schmid H.H.O. Dong Z. J. Biol. Chem. 1997; 272: 4187-4194Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We show that this pathway is involved in the potentiation of Smad3 transactivation by Jun proteins since inhibition of the PI3-kinase pathway by the highly selective inhibitor LY294002 or by a PI3-kinase dominant negative mutant efficiently blocked the EGF-induced Smad3 transactivation, whereas activation of the PI3-kinase pathway by RasV12C40 reproduced the synergistic effect of EGF stimulation on Smad3 transactivation. The PI3-kinase-activating effect was blunted in cells transfected with TAM-67 which are deficient in AP-1 transactivating function, strongly suggesting that the effect of PI3-kinase is through AP-1 activation rather than the result of direct PI3-kinase-dependent phosphorylation of Smad3. The involvement of PI3-kinase in Smad transactivation has never been demonstrated before. That PI3-kinase activation may be involved in TGF-β signaling had been previously suggested from the observation that wortmannin, another PI3-kinase inhibitor, inhibits TGF-β-stimulated chemotaxis of human neutrophil leukocytes (34Thelen M. Uguccioni M. Bosiger J. Biochem. Biophys. Res. Commun. 1995; 217: 1255-1262Crossref PubMed Scopus (90) Google Scholar). It has also been demonstrated that TGF-β markedly enhanced EGF-induced PI3-kinase activity in human airway smooth muscle cells (35Krymskaya V.P. Hoffman R. Eszterhas A. Ciocca V. Panettieri Jr., R.A. Am. J. Physiol. 1997; 273: L1220-L1227Crossref PubMed Google Scholar). More recently, involvement of PI3-kinase in the inhibitory effect of insulin, EGF, or interleukin-6 on TGF-β-induced apoptosis has been reported (36Roberts R.A. James N.H. Cosulich S.C. Hepatology. 2000; 31: 420-427Crossref PubMed Scopus (90) Google Scholar, 37Chen R.-H. Su Y.-H. Chuang R.L.C. Chang T.-Y. Oncogene. 1998; 17: 1959-1968Crossref PubMed Scopus (177) Google Scholar, 38Chen R.-H. Chang M.-C. Su Y.-H. Tsai S.-T. Kuo M.-L. J. Biol. Chem. 1999; 274: 23013-23019Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), but the relationship with AP-1 function or Smad signaling was not investigated. We show here that one mechanism by which PI3-kinase may cross-talk with Smad signaling is through activation of Jun proteins, which themselves cooperate with Smad3 for transactivation.Another important signaling pathway that originates from the EGF receptor is the MAPK pathway, including the ERK1 and ERK2 pathways, the JNK/SAPK, and the p38 MAPK pathways (Ref. 39Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2408) Google Scholar and for review see Ref.40Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (548) Google Scholar), all implicated in EGF-induced AP-1 activation (14Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). In our study, neither inhibition of the JNK pathway by transfection of a dominant negative mutant (DNMKK4) nor inhibition of the ERK pathway by the specific inhibitor PD98059 decreased the EGF-induced potentialization of Smad3 transactivation, whereas involvement of the p38 pathway was suggested by the inhibitory effect of SB 202190 on EGF/TGF-β-induced Smad3 transactivation. These data confirm and extend the previous demonstration that p38 increases the transcriptional activity of TGF-β-inducible genes (41Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J.-I Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 42Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 43Adachi-Yamada T. Nakamura M. Irie K. Tomoyasu Y. Sano Y. Mori E. Gotoh S. Ueno N. Nishida Y. Matsumoto K. Mol. Cell. Biol. 1999; 19: 2322-2329Crossref PubMed Google Scholar, 44Kimura N. Matsuo R. Nakashima K. Taga T. J. Biol. Chem. 2000; 275: 17647-17652Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In these studies, p38 activation was shown to be induced by TGF-β-activated kinase, a MAP kinase kinase kinase also involved in TGF-β signaling (45Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Marsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1169) Google Scholar, 46Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (516) Google Scholar). According to this model, TGF-β-activated kinase-induced p38 phosphorylation in response to TGF-β triggers the phosphorylation of activating transcription factor 2, a basic leucine zipper protein member of the activating transcription factor/cAMP-response element-binding protein family that shares many structural characteristics with AP-1 proteins. Activated transcription factor 2 forms a complex with Smad4 that is transcriptionally active on Smad-regulated genes (41Hanafusa H. Ninomiya-Tsuji J. Masuyama N. Nishita M. Fujisawa J.-I Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1999; 274: 27161-27167Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 42Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The mechanism of p38 activation found in the present study is clearly different from this model. It is very likely that the activating effect of p38 on Smad3 transactivation detected in our study lies downstream of PI3-kinase activation, since inhibition of the two pathways was not clearly additive. That PI3-kinase can contribute to the activation of protein kinases of the MAPK family, such as ERK, has been previously shown (47Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (301) Google Scholar), but a direct demonstration of p38 activation by the PI3-kinase pathway has never been published. Finally, since our Western blot experiments indicated that EGF induces c-Jun phosphorylation by mechanisms dependent of PI3-kinase and p38, it is very likely, but not proven, that these phosphorylation events are implicated in the potentiation of Smad3 transactivation by EGF. In sharp contrast with our results describing a stimulating effect of Jun/Smad3 cooperation for transactivation in the context of PI3-kinase-induced Jun activation, two recent examples of interplay between Jun proteins and Smad3 leading to transcriptional repression of Smad3 have recently been published (13Dennler S. Prunier C. Ferrand N. Gauthier J.-M. Atfi A. J. Biol. Chem. 2000; 275: 28858-28865Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 48Verrecchia F. Pessah M. Atfi A. Mauviel A. J. Biol. Chem. 2000; 275: 30226-30231Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). In one model, Jun activated via a TGF-β-induced JNK binds to Smad3 on an SBE element and inhibits Smad3 transcriptional activity (13Dennler S. Prunier C. Ferrand N. Gauthier J.-M. Atfi A. J. Biol. Chem. 2000; 275: 28858-28865Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In a second model, c-Jun activated by tumor necrosis factor α binds to Smad3 and prevents its binding to DNA, therefore acting again as a transcriptional repressor (48Verrecchia F. Pessah M. Atfi A. Mauviel A. J. Biol. Chem. 2000; 275: 30226-30231Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Collectively, these data and ours suggest that the functional interplay between Jun and Smads is far more complicated than previously thought and might vary according to the mechanisms of Jun activation.In summary, we have described another novel mechanism allowing TGF-β to integrate with regulatory networks of the cell. Whether this pathway is specific to the hepatocyte environment remains to be determined. We show that such a mechanism operates on an SBE-driven gene, although with a lower magnitude, and therefore represents a potential mechanism of regulation of TGF-β-inducible genes. In normal hepatocytes, simultaneous treatment with EGF and TGF-β induces a proliferation arrest (49Nakamura T. Tomita Y. Hirai R. Yamaoka K. Kaji K. Ichihara A. Biochem. Biophys. Res. Commun. 1985; 133: 1042-1050Crossref PubMed Scopus (300) Google Scholar, 50Carr B.I. Hayashi I. Branum E.L. Moses H.L. Cancer Res. 1986; 46: 2330-2334PubMed Google Scholar, 51Mc Mahon J.B. Richards W.L. DelCampo A.A. Song M.K. Thorgeirsson S.S. Cancer Res. 1986; 46: 4665-4671PubMed Google Scholar). Whether Jun/Smad3 synergism is at work on the promoter of Smad-responsive gene inhibitors of the cell cycle will be the subject of our next investigations. Transforming growth factor β (TGF-β)1 is a member of a large family of cytokines that includes bone morphogenetic proteins, activins, and several more distantly related factors (1Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar). TGF-β is central in the regulation of many biological processes, including cell differentiation, growth, adhesion, and apoptosis. TGF-β signals through a system of transmembrane serine/threonine kinase receptors composed of type I and type II receptors (TGF-βRI and TGF-βRII) (see Refs. 2Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar, 3Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (177) Google Scholar, 4Chen Y.-G. Hata A. Lo R.S. Wotton D. Shi Y. Pavletich N. Massagué J. Genes Dev. 1998; 12: 2144-2152Crossref PubMed Scopus (300) Google Scholar and reviewed in Ref. 5Hu P.P.-C. Datto M.B. Wang X.-F. Endocr. Rev. 1998; 19: 349-363Crossref PubMed Google Scholar). Ligand binding to TGF-βRII recruits and activates the TGF-βRI receptor, which phosphorylates Smad2 and Smad3 on their SSXS motif. Smad proteins encompass a conserved amino-terminal domain that binds DNA and a conserved carboxyl-terminal domain that binds receptors and partner Smads. These domains are separated by a less conserved linker region. Phosphorylated Smad2 or Smad3 forms stable complexes with Smad4, which translocate into the nucleus, where they bind the consensus GTCTAGAC sequence found in the promoter of many TGF-β-responsive genes (6Zawel L. Le Dai J. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). Disruption of the Smad pathway or Smad mutations have underscored the functional importance of this signaling pathway in the transcriptional response of target cells to TGF-β (reviewed in Ref. 7Massagué J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Emerging evidence indicates that TGF-β signaling may also cross-talk with the mitogen-activated protein kinase (MAPK) family of serine/threonine protein kinases. Antagonistic or synergistic interplay between these kinases and Smad signaling has been described. Extracellular signal-regulated kinases (ERK), members of the MAPK, cause a rapid increase in the phosphorylation of Smad2 and Smad3 in their linker region, preventing their translocation into the nucleus and therefore providing a mechanism of repression of TGF-β signaling (8Kretzschmar M. Doody J. Massagué J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (766) Google Scholar, 9Kretzschmar M. Doody J. Timokhina I. Massagué J. Genes Dev. 1999; 13: 804-816Crossref PubMed Scopus (848) Google Scholar). At the opposite, a synergistic mechanism between Smads and MAPK has been proposed, in a kinase downstream of the MAP kinase kinase MEK1-induced Smad2 phosphorylation on the SSXS motif and its nuclear translocation (10De Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (252) Google Scholar). Recent evidence also indicates that Smad cooperates with AP-1 (11Zhang Y. Feng X.-H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (679) Google Scholar, 12Liberati N.T. Datto M.B. Frederick J.P. Shen X. Wong C. Rougier-Chapman E.M. Wang X.-F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4844-4849Crossref PubMed Scopus (271) Google Scholar, 13Dennler S. Prunier C. Ferrand N. Gauthier J.-M. Atfi A. J. Biol. Chem. 2000; 275: 28858-28865Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), a heterodimer of Fos and Jun family members (14Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2245) Google Scholar). Stimulation of AP-1-dependent transcription can be achieved by phosphorylation of the c-Jun transactivation domain by c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK), another member of the MAPK family. Smad and AP-1 response elements are juxtaposed in the promoters of several TGF-β-inducible genes, such as plasminogen activator inhibitor-1 or c-jun, and both sites appear to be critical in the TGF-β response (15Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar, 16Yingling J.M. Datto M.B. Wong C. Frederick J.P. Liberati N.T. Wang X.-F. Mol. Cell. Biol. 1997; 17: 7019-7028Crossref PubMed Google Scholar, 17Wong C. Rougier-Chapman E.M. Frederick J.P. Datto M.B. Liberati N.T. Li J.-M. Wang X.-F. Mol. Cell. Biol. 1999; 19: 1821-1830Crossref PubMed Scopus (227) Google Scholar). In the present study we addressed the question of whether AP-1/Smad cooperation occurs in normal rat hepatocytes following a combined stimulation with EGF and TGF-β. We show that under these experimental conditions AP-1 induces a strong activation of Smad3 transactivation independent of AP-1 binding to its cognate cis-element. This synergism was mediated by c-Jun and JunB, and a protein-protein interaction between Smad3 and endogenous c-Jun was found during EGF/TGF-β stimulation. Furthermore, we demonstrate that activation of AP-1 via the phosphatidylinositol 3-kinase (PI3-kinase), but not the JNK pathway, is implicated in this functional synergism. Finally, we show that Jun/Smad3 cooperation induced by EGF is effective on a SBE-driven reporter gene. These data suggest that Jun/Smad3 synergism independent of binding to TRE elements might represent another important mechanism of regulation of TGF-β-inducible genes in hepatocytes. DISCUSSIONThere is increasing evidence that several signaling pathways interfere with Smads to regulate TGF-β-responsive genes, resulting in antagonistic as well as synergistic effects. In the present study, we show that EGF alone induces a mild activation of Smad3 transcriptional activity only detectable under conditions of Smad3 overexpression, in agreement with previous reports (10De Caestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B

The role of NFkappaB in regulating G1 arrest and maturation induced by the histone deacetylase inhibitor sodium butyrate (NaB) was examined in human myelomonocytic leukemia cells (U937). Cells stably transfected with an IkappaBalpha "super-repressor" lacking phosphorylation sites necessary for proteasomal degradation exhibited diminished IkBa phosphorylation and NF-kappaB DNA binding upon exposure to TNFalpha When exposed to NaB (1 mM; 48 hr) or PMA (5 nM; 24 hr), IkappaBalphaM cells displayed a marked reduction in G1 arrest compared to Neo controls. In each case, this was accompanied by a significant reduction in the percentage of cells expressing the differentiation markers CD11a, CD11b, and CD18. The impairment in NaB-induced maturation in mutant cells was associated with a reciprocal increase in apoptosis. In contrast to impairment in NaB- or PMA-induced NF-kappaB DNA binding, stable expression of the IkappaBalphaM did not modify DNA binding of SP1 or AP2 transcription factors. IkappaBalphaM cells also displayed impairment in NaB- and PMA-mediated induction of p21CIP1 and phosphorylation (inactivation) of p34cdc2, as well as diminished levels of pRb-bound E2F1. Finally, the NF-kappaB inhibitor CAPE antagonized NaB- and PMA-related NF-kappaB DNA binding as well as induction of p21CIP1. Together, these findings suggest that NF-kappaB plays an important functional role in mediating NaB-induced p21CIP1 induction, G1 arrest, and maturation in human myelomonocytic leukemia cells, and that disruption of the NF-kappaB pathway causes cells to engage an alternative, apoptotic program.

Abstract Purpose: The purpose of this study was to characterize interactions between the farnesyltransferase inhibitor L744832 and the checkpoint abrogator UCN-01 in drug-sensitive and drug-resistant human myeloma cell lines and primary CD138+ multiple myeloma cells. Experimental Design: Wild-type and drug-resistant myeloma cell lines were exposed to UCN-01 ± L744832 for 24 hours, after which mitochondrial injury, caspase activation, apoptosis, and various perturbations in signaling and survival pathways were monitored. Results: Simultaneous exposure of myeloma cells to marginally toxic concentrations of L744832 and UCN-01 resulted in a synergistic induction of mitochondrial damage, caspase activation, and apoptosis, associated with activation of p34cdc2 and c-Jun-NH2-kinase and inactivation of extracellular signal-regulated kinase, Akt, GSK-3, p70S6K, and signal transducers and activators of transcription 3 (STAT3). Enhanced lethality for the combination was also observed in primary CD138+ myeloma cells, but not in their CD138− counterparts. L744832/UCN-01–mediated lethality was not attenuated by conventional resistance mechanisms to cytotoxic drugs (e.g., melphalan or dexamethasone), addition of exogenous interleukin-6 or insulin-like growth factor-I, or the presence of stromal cells. In contrast, enforced activation of STAT3 significantly protected myeloma cells from L744832/UCN-01–induced apoptosis. Conclusions: Coadministration of the farnesyltransferase inhibitor L744832 promotes UCN-01–induced apoptosis in human multiple myeloma cells through a process that may involve perturbations in various survival signaling pathways, including extracellular signal-regulated kinase, Akt, and STAT3, and through a process capable of circumventing conventional modes of myeloma cell resistance, including growth factor– and stromal cell–related mechanisms. They also raise the possibility that combined treatment with farnesyltransferase inhibitors and UCN-01 could represent a novel therapeutic strategy in multiple myeloma.

Abstract Purpose: Acute myelogenous leukemia (AML) is an aggressive disease with a poor outcome. We investigated mechanisms by which the anti-AML activity of ABT-199 (venetoclax) could be potentiated by dual mTORC1/TORC2 inhibition. Experimental Design: Venetoclax/INK128 synergism was assessed in various AML cell lines and primary patient AML samples in vitro. AML cells overexpressing MCL-1, constitutively active AKT, BAK, and/or BAX knockout, and acquired venetoclax resistance were investigated to define mechanisms underlying interactions. The antileukemic efficacy of this regimen was also examined in xenograft and patient-derived xenograft (PDX) models. Results: Combination treatment with venetoclax and INK128 (but not the mTORC1 inhibitor rapamycin) dramatically enhanced cell death in AML cell lines. Synergism was associated with p-AKT and p-4EBP1 downregulation and dependent upon MCL-1 downregulation and BAK/BAX upregulation as MCL-1 overexpression and BAX/BAK knockout abrogated cell death. Constitutive AKT activation opposed synergism between venetoclax and PI3K or AKT inhibitors, but not INK128. Combination treatment also synergistically induced cell death in venetoclax-resistant AML cells. Similar events occurred in primary patient-derived leukemia samples but not normal CD34+ cells. Finally, venetoclax and INK128 co-treatment displayed increased antileukemia effects in in vivo xenograft and PDX models. Conclusions: The venetoclax/INK128 regimen exerts significant antileukemic activity in various preclinical models through mechanisms involving MCL-1 downregulation and BAK/BAX activation, and offers potential advantages over PI3K or AKT inhibitors in cells with constitutive AKT activation. This regimen is active against primary and venetoclax-resistant AML cells, and in in vivo AML models. Further investigation of this strategy appears warranted.

To characterize interactions between the heat shock protein 90 antagonist 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 inhibitor PD184352 in Bcr/abl(+) leukemia cells sensitive and resistant to imatinib mesylate.K562 and LAMA 84 cells were exposed to varying concentrations of DMAG and PD184352 for 48 hours; after which, mitochondrial integrity, caspase activation, and apoptosis were monitored. Parallel studies were done in imatinib mesylate-resistant cells, including BaF3 cells transfected with plasmids encoding clinically relevant Bcr/abl mutations conferring imatinib mesylate resistance (e.g., E255K, M351T, and T315I) and primary CD34(+) bone marrow cells from patients refractory to imatinib mesylate.Cotreatment of Bcr/abl(+) cells with minimally toxic concentrations of DMAG and PD184352 resulted in synergistic induction of mitochondrial injury (cytochrome c release and Bax conformational change), events associated with the pronounced and sustained inactivation of ERK1/2 accompanied by down-regulation of Bcl-x(L). Conversely, cells ectopically expressing Bcl-x(L) displayed significant protection against PD184352/DMAG-mediated lethality. This regimen effectively induced apoptosis in K562 cells overexpressing Bcr/abl, in BaF3 cells expressing various clinically relevant Bcr/abl mutations, and in primary CD34(+) cells from patients resistant to imatinib mesylate, but was relatively sparing of normal CD34(+) bone marrow cells.A regimen combining the heat shock protein 90 antagonist DMAG and the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor potently induces apoptosis in Bcr/abl(+) cells, including those resistant to imatinib mesylate through various mechanisms including Bcr/abl kinase mutations, through a process that may involve sustained ERK1/2 inactivation and Bcl-x(L) down-regulation. This strategy warrants further attention in Bcr/abl(+) hematopoietic malignancies, particularly those resistant to Bcr/abl kinase inhibitors.

The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca(2+) and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B). Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML. We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D). To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, p&lt;0.001 (Fig. 1E). In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth in vitro and in vivo. The clinical trial examining the efficacy of this combination is ongoing (Calithera, NCT02071927). Disclosures Lorenzi: Erytech Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties: NIH-held patent related to L-asparaginase. DiNardo:Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

&lt;div&gt;Abstract&lt;p&gt;Inhibitors targeting BCL-2 apoptotic proteins have significant potential for the treatment of acute myeloid leukemia (AML); however, complete responses are observed in only 20% of patients, suggesting that targeting BCL-2 alone is insufficient to yield durable responses. Here, we assessed the efficacy of coadministration of the PI3K/mTOR inhibitor GDC-0980 or the p110β-sparing PI3K inhibitor taselisib with the selective BCL-2 antagonist venetoclax in AML cells. Tetracycline-inducible downregulation of BCL-2 significantly sensitized MV4-11 and MOLM-13 AML cells to PI3K inhibition. Venetoclax/GDC-0980 coadministration induced rapid and pronounced BAX mitochondrial translocation, cytochrome c release, and apoptosis in various AML cell lines in association with AKT/mTOR inactivation and MCL-1 downregulation; ectopic expression of MCL-1 significantly protected cells from this regimen. Combined treatment was also effective against primary AML blasts from 17 patients, including those bearing various genetic abnormalities. Venetoclax/GDC-0980 markedly induced apoptosis in primitive CD34&lt;sup&gt;+&lt;/sup&gt;/38&lt;sup&gt;−&lt;/sup&gt;/123&lt;sup&gt;+&lt;/sup&gt; AML cell populations but not in normal hematopoietic progenitor CD34&lt;sup&gt;+&lt;/sup&gt; cells. The regimen was also active against AML cells displaying intrinsic or acquired venetoclax resistance or tumor microenvironment–associated resistance. Either combinatorial treatment markedly reduced AML growth and prolonged survival in a systemic AML xenograft mouse model and diminished AML growth in two patient-derived xenograft models. Venetoclax/GDC-0980 activity was partially diminished in BAK&lt;sup&gt;−/−&lt;/sup&gt; cells and failed to induce apoptosis in BAX&lt;sup&gt;−/−&lt;/sup&gt; and BAX&lt;sup&gt;−/−&lt;/sup&gt;BAK&lt;sup&gt;−/−&lt;/sup&gt; cells, whereas BIM&lt;sup&gt;−/−&lt;/sup&gt; cells were fully sensitive. Similar results were observed with venetoclax alone in &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; systemic xenograft models. Collectively, these studies demonstrate that venetoclax/GDC-0980 exhibits potent anti-AML activity primarily through BAX and, to a lesser extent, BAK. These findings argue that dual BCL-2 and PI3K inhibition warrants further evaluation in AML.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; Combined treatment with clinically relevant PI3K and BCL-2 inhibitors may prove effective in the treatment of acute myeloid leukemia. &lt;i&gt;Cancer Res; 78(11); 3075–86. ©2018 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

The 14-kilodalton human growth hormone (14 kDa hGH) N-terminal fragment derived from the proteolytic cleavage of its full-length counterpart has been shown to sustain antiangiogenic potentials. This study investigated the antitumoral and antimetastatic effects of 14 kDa hGH on B16-F10 murine melanoma cells. B16-F10 murine melanoma cells transfected with 14 kDa hGH expression vectors showed a significant reduction in cellular proliferation and migration associated with an increase in cell apoptosis in vitro. In vivo, 14 kDa hGH mitigated tumor growth and metastasis of B16-F10 cells and was associated with a significant reduction in tumor angiogenesis. Similarly, 14 kDa hGH expression reduced human brain microvascular endothelial (HBME) cell proliferation, migration, and tube formation abilities and triggered apoptosis in vitro. The antiangiogenic effects of 14 kDa hGH on HBME cells were abolished when we stably downregulated plasminogen activator inhibitor-1 (PAI-1) expression in vitro. In this study, we showed the potential anticancer role of 14 kDa hGH, its ability to inhibit primary tumor growth and metastasis establishment, and the possible involvement of PAI-1 in promoting its antiangiogenic effects. Therefore, these results suggest that the 14 kDa hGH fragment can be used as a therapeutic molecule to inhibit angiogenesis and cancer progression.

Activation of the transcriptional regulator AP-1, a dimeric complex formed of various combinations of Fos and Jun proteins, is a key step in the cellular response to mitogens. Because different dimers are believed to display different regulatory functions, we hypothesized that transformed cells that lack normal growth constraints might display AP-1 dimers that are different from those of normal cells. We therefore compared in primary and transformed rat hepatocytes (1) the composition of AP-1 dimers under basal conditions and (2) AP-1 induction by epidermal growth factor (EGF). Under basal conditions, AP-1 contained predominantly Jun homodimers in both cell types. However, whereas normal hepatocytes contained only JunD, both JunD and JunB were present in the AP-1 complex of 7777 cells. EGF treatment triggered almost identical programs of fos and jun gene activation at the messenger RNA (mRNA) level in both cell types, with an early accumulation of c-fos, c-jun, and junB mRNAs, but no change in junD mRNA levels. In both cell types, c-Fos and Fra-1 proteins increased after EGF treatment, but differences in the induction of Jun proteins were noted, with an increase of c-Jun in hepatocytes and an increase of JunB in 7777 cells. In both cell types, activation of AP-1 DNA binding activity by EGF was accompanied by the recruitment of Fra-1 into AP-1, detected earlier in 7777 cells than in hepatocytes, and with the transient appearance of c-Fos in 7777 cells only. Finally, EGF activated AP-1-dependent transcription in 7777 cells but not in hepatocytes. These data indicate important differences in the functional activity of AP-1 in transformed hepatocytes.

The B-cell lymphoma-2 (Bcl-2) family of pro- and anti-apoptotic proteins controls the mitochondrial pathway of apoptosis. Their central role in mediating the final common pathway of apoptosis in response to internal and external stressors makes them attractive therapeutic targets in neoplastic cells, which are often “primed” for apoptosis. The major anti-apoptotic proteins are Bcl-2, Bcl-xL and myeloid cell leukemia-1 (Mcl-1). These promote cellular survival by sequestering pro-apoptotic proteins which function as apoptosis “sensitizers” (e.g., Bim, Bad) or “effectors” (Bax, Bak). Multiple studies have implicated Bcl-2 family proteins in AML pathogenesis, prognosis and resistance to chemotherapy. The discovery of the “BH3-mimetic” ABT-737, a specific inhibitor of the anti-apoptotic actions of Bcl-2 and Bcl-xL, demonstrated for the first time that specific protein-protein interactions could be disrupted using a small molecule [1]. Several groups [2, 3] have shown that Mcl-1, which is not inhibited by ABT-737, is the primary determinant of resistance to this agent. This, along with recent evidence that Mcl-1 may be more critical to the development and maintenance of AML than Bcl-2 or Bcl-xL [4], has prompted interest in combining agents that down-regulate Mcl-1 with ABT-737 in AML to simultaneously disable multiple arms of the mitochondrial apoptotic regulatory machinery. Thus, cyclin-dependent kinase inhibitors [3] and sorafenib [5] synergize with ABT-737 in pre-clinical studies of AML. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the most frequently dysregulated survival pathways in human malignancies, including AML, leading to its emergence as a therapeutic target in this disease [6]. For example, internal tandem duplication mutations in the FLT3 gene, affecting approximately 25% of patients with AML, lead to constitutive FLT3 signaling which activates Akt and confers a poor prognosis. Similarly, Ras and KIT mutations, common in core-binding factor AML, activate the PI3K/Akt/mTOR pathway. In many cases, the basis for addiction to this pathway, manifested by basal Akt activation, remains unknown. While mTOR inhibitors are approved for advanced renal cell carcinoma and the PI3K delta inhibitor GS-1101 (CAL-101) appears promising in CLL, a new class of agents that inhibit both PI3K and mTOR signaling (e.g., NVP-BEZ235, GDC0980) may offer additional therapeutic advantages in AML [7]. In light of the above considerations, and the known ability of PI3K inhibitors to down-regulate Mcl-1 and up-regulate/activate the pro-apoptotic proteins Bim, Bad, Bax and Bak, the effects of dual PI3K/mTOR and Bcl-2/Bcl-xL inhibition in AML cell lines, patient-derived leukemic blasts, and xenograft models of AML were recently examined [8]. Notably, tet-inducible short hairpin RNA constructs directed against Akt or Bcl-2 and Bcl-xL individually or together, and studies employing pharmacologic inhibitors (e.g., NVP-BEZ235/PI-103 and ABT-737), revealed that the PI3K/Akt/mTOR pathway and Bcl-2/Bcl-xL play important coordinate roles in protecting leukemia cells from lethality. Specifically, genetic or pharmacologic interventions simultaneously inhibiting the PI3K/Akt/mTOR pathway and disabling Bcl-2/Bcl-xL dramatically enhanced leukemic cell death, with minimal toxicity toward normal CD34+ hematopoietic progenitors. These findings were recapitulated in vivo in an AML xenograft model with little toxicity, likely reflecting both a dependence of leukemic cells on the PI3K/Akt/mTOR pathway for survival, and relative sparing of normal cells by BH3-mimetic treatment. Mcl-1 down-regulation resulted from PI3K/mTOR inhibition, at least in part through GSK3 activation, which promotes the proteasomal degradation of Mcl-1. Although BEZ235 alone down-regulated Mcl-1, it did not significantly trigger cell death, underscoring the importance of concomitant Bcl-2/Bcl-xL inhibition, as all three major anti-apoptotic proteins sequester the apoptosis effectors Bax and Bak. Significantly, release of Bax and Bak from Mcl-1, Bcl-2 and Bcl-xL was observed. Another novel finding was a marked increase in Bim binding to Bcl-2 and Bcl-xL accompanying PI3K/mTOR inhibition, a phenomenon abolished by ABT-737. Presumably Bim, liberated from these two anti-apoptotic proteins, as well as Mcl-1 (due to down-regulation), triggered apoptosis by activating Bax and Bak. A summary of these interactions is presented in Figure ​Figure1.1. A particularly intriguing observation was an apparent correlation between responses to combined treatment with PI3K/mTOR inhibitors and ABT-737 and baseline Akt activation in primary AML blasts, raising the possibility that such cells may be addicted to this pathway and particularly vulnerable to its interruption. In fact, it is conceivable that assessment of Akt pathway activation may be a more reliable predictor of susceptibility to a dual PI3K/mTOR blockade and BH3-mimetic strategy than mutations in related proteins. Clearly, definitive answers to this question will require analysis of a considerably larger number of specimens. In any case, a major thrust of recent therapeutic approaches to AML has been to tailor drug therapy to specific genetic mutations, e.g., those in FLT3 or KIT. However, the success of this approach is uncertain due to the upstream location of these mutated oncoproteins and the marked redundancy of signal transduction pathways. Pending more definitive validation, the present findings suggest novel candidate biomarkers (e.g., basal pathway activation) that might better help realize the goal of personalized therapy for AML. Figure 1 Model of interactions between PI3K/AKT/mTOR pathway inhibitors and Bcl-2 antagonists

Aim: Colorectal cancer (CRC) is one of the leading cancers in the world. Even though its mortality and pathophysiology are well documented in the US and the European countries, it is seldom studied in North African population. Recent studies have shown link of HER2 overexpression in oesophageal and gastric cancers. The aim of this study is to assess the HER2 protein and mRNA expression and its correlation with tumor pathogenesis in Libyan CRC patients.Methodology: A total of 17 FFPE tissue blocks were collected from patients with primary CRC. The HER2 protein expression was assessed by immunohistochemistry and the mRNA expression was assessed using qRT-PCR. Survival analysis of the role of HER2 overexpression on rectal adenocarcinoma was carried out on additional 165 patients.Results: From the CRC cohort, adenocarcinoma was found to be more frequent accounting for 88.2%, and 11.8% for mucinous adenocarcinomas. Almost 47% of the cases were positive for HER2 (score ≥ 2+) and about 50% adenocarcinoma cases with tumor grade II were positive for HER2. Moreover, 57.4% adenocarcinoma patients with grade-II tumor had undergone right hemicolectomy. Furthermore, significant correlation (p = 0.03) between the HER2 mRNA expression with the tumor grade was observed. In addition, poor overall all survival was observed with high HER2 expression in rectum adenocarcinoma.Conclusion: To our knowledge, this is the first study that HER2 overexpression correlates with more aggressive colorectal cancer in North African population. Our study shows that HER2 overexpression associates with right colon surgeries. Also, the correlation of mRNA and protein expression could warrant the implementation of a nationwide screening program for HER2 positivity in CRC patients. Taken together, stratifying patients according to HER2 expression can help in the diagnosis and prognosis of CRC patients from North African origin.

Immunomodulation and chronic inflammation are important mechanisms utilized by cancer cells to evade the immune defense and promote tumor progression. Therefore, various efforts were focused on the development of approaches to reprogram the immune response to increase the immune detection of cancer cells and enhance patient response to various types of therapy. A number of regulatory proteins were investigated and proposed as potential targets for immunomodulatory therapeutic approaches including p53 and Snail. In this study, we investigated the immunomodulatory effect of disrupting Snail-p53 binding induced by the oncogenic KRAS to suppress p53 signaling. We analyzed the transcriptomic profile mediated by Snail-p53 binding inhibitor GN25 in non-small cell lung cancer cells (A549) using Next generation whole RNA-sequencing. Notably, we observed a significant enrichment in transcripts involved in immune response pathways especially those contributing to neutrophil (IL8) and T-cell mediated immunity (BCL6, and CD81). Moreover, transcripts associated with NF-κB signaling were also enriched which may play an important role in the immunomodulatory effect of Snail-p53 binding. Further analysis revealed that the immune expression signature of GN25 overlaps with the signature of other therapeutic compounds known to exhibit immunomodulatory effects validating the immunomodulatory potential of targeting Snail-p53 binding. The effects of GN25 on the immune response pathways suggest that targeting Snail-p53 binding might be a potentially effective therapeutic strategy.

The human T cell repertoire is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T cell receptor (TCR) loci. To determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T cell receptor alpha (TRA) and beta (TRB) loci were arranged in a self-similar manner when the spacing between adjacent segments was considered as a function of the size of the neighboring gene segment. The ratio of genomic distance between either the J (in TRA) or D (in TRB) segments and successive V segments on these loci declined logarithmically. Accounting for the gene segments occurring on helical DNA molecules, in a logarithmic distribution, sine and cosine functions of the log transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression across the locus, supporting a log-periodic organization. T cell clonal frequencies, based on V and J segment usage, from three normal stem cell donors plotted against the respective segment locations on TRB locus demonstrated a periodic variation. We hypothesize that this quasi-periodic variation in T cell clonal repertoire may be influenced by the location of the gene segments on the logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices, impacting probability of DNA recombination.

Background/Aims: Stimulation of activator protein-1 (AP-1), a Fos/Jun complex, is a key event in the cell response to growth factors. We have investigated whether hepatocyte growth factor (HGF) induces differential AP-1 responses in normal and transformed rat hepatocytes, the 7777 cells. Methods: Primocultures of isolated hepatocytes or 7777 cells were stimulated with HGF. Gene expression was evaluated by ribonuclease protection assay and Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. Identification of the proteins bound to the probes was made by supershift assays with specific antibodies. Cells were electroporated with plasmids containing an AP-1-dependent chloramphenicol acetyl transferase (CAT) gene, and CAT activity was measured 24 h after treatment with medium alone or HGF. Results: In both cell types, HGF triggered the same program of jun family mRNA activation, but distinct Fos/Jun proteins accumulated in the nucleus. HGF increased DNA-binding activity to the phorbol 12-O-tetradecanoate-13-acetate responsive element (TRE) in both cell types, but distinct TRE-binding proteins were recruited in the AP-1 dimers. HGF also increased consistently binding to a cAMP responsive element (CRE) in hepatocytes only. Finally, HGF triggered TRE- and CRE-dependent gene activations in hepatocytes but TRE-dependent gene activation alone in 7777 cells. Conclusions: HGF-induced AP-1 activation leads to the formation of distinct dimers with different functional capacities in normal and transformed hepatocytes. These data suggest the importance of qualitative abnormalities of the AP-1 complex for the establishment or maintainance of a transformed phenotype.

Previously interferon (IFN)gamma-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were demonstrated. The aim of this study was to further investigate the mechanisms of IFNgamma-, Fas-, and cycloheximide (CHX)-induced programmed cell death, with special attention to the role of transcriptional factors NF-kappaB and STAT1.In a human conjunctival cell line (Chang conjunctival cells) apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab) alone (24 or 48 hours) or, as previously reported, with 300 U/ml of human recombinant IFNgamma alone (48 hours). To study the role of IFNgamma on Fas-induced apoptosis, cells were treated first with IFNgamma at 30 U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied for 24 hours. Moreover, to study the influence of CHX on Fas- and IFNgamma-induced apoptosis, cells were treated for 24 hours with 300 U/ml IFNgamma together with a nontoxic concentration (1 microg/ml) of CHX, or with 500 ng/ml anti-Fas ab together with 1 microg/ml CHX (24 hours). After treatment, cell viability (neutral red assay), mitochondrial membrane potential (rhodamine 123 assay), chromatin condensation (Hoechst 33342 assay), and the index Hoechst/neutral red were studied by cold light microplate cytometry. The apoptotic process was sought for by contrast phase microscopy and DAPI staining and was confirmed by immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 were investigated by Western blot analysis. NF-kappaB and STAT DNA-binding activities were studied by electrophoretic mobility shift assays (EMSA).After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20% and 30%, respectively, of apoptotic cells were observed. When anti-Fas sera were applied after IFNgamma pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by an index Hoechst/neutral red greater than one. All these modifications were preceded by a decrease in mitochondrial membrane potential. EMSA revealed that NF-kappaB was activated after IFNgamma and anti-Fas ab treatments and inhibited after CHX treatment. STAT1 was strongly activated after IFNgamma treatment and only in a minor degree after anti-Fas ab treatment. STAT1-binding activity persisted after CHX treatment.The relative resistance of Chang cells toward Fas-induced apoptosis could be related to the activation of NF-kappaB. IFNgamma-induced programmed cell death preferentially involves the activation of STAT1 that counterbalances NF-kappaB antiapoptotic effects. In fact, Fas-induced apoptosis was potentiated by IFNgamma or CHX treatments. These results suggest that NF-kappaB activation could maintain cell viability as well as participate in IFNgamma-induced inflammatory modifications, whereas STAT1 activation could provide, in this model, a proapoptotic signal.

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21CIP1 induction, p27KIP1 cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21CIP1 mRNA antisense construct, suggesting that p21CIP1 expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program. Key Words:PMA, UCN-01, Differentiation, Apoptosis

The newly identified melanoma-associated adaptor ShcD was found to translocate to the nucleus upon hydrogen peroxide treatment. Therefore, the aim of this study was to identify the ShcD network in melanoma cells under oxidative stress. LC-MS/MS and GFP-trap were performed to study the ShcD phosphorylation status during acute severe oxidative stress. ShcD was found to be phosphorylated at threonine-159 (Thr159) in response to 5 mM H2O2 treatment. The GPS 2.1 phosphorylation prediction program predicted that the Thr159Pro motif, housed in the N-terminus of the ShcD-CH2 domain, is a potential phosphorylation site for MAPKs (ERK, JNK or p38). Co-immunoprecipitation experiments revealed that ShcD mainly interacts with ERK in B16 and MM138 melanoma cells under both hydrogen peroxide-untreated and -treated conditions. Moreover, ShcD interacts with both phosphorylated and un-phosphorylated ERK, although the interaction between ShcD and phospho-ERK was primarily observed after H2O2 treatment. A MEK inhibitor (U0126) enhanced the interaction between ShcD and unphosphorylated ERK under oxidative stress conditions. Furthermore, Thr159 was mutated to either alanine (A) or glutamic acid (E) to study whether the threonine phosphorylation state influences the ShcD/ERK interaction. Introducing the T159E mutation obliterated the ShcD/ERK interaction. To identify the functional impact of the ShcD/ERK interaction on cell survival signalling under oxidative stress conditions, caspase 3/7 assays and 7AAD cell death assays were used. The ShcD/ERK interaction promoted anti-survival signalling upon exposure to hydrogen peroxide, while U0126 treatment reduced death signalling. Our data also showed that the death signalling initiated by the ShcD/ERK interaction was accompanied by p21 phosphorylation. In summary, these data identified ShcD, via its interaction with ERK, as a proapoptotic protein under oxidative stress conditions.

To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases.

Abstract Dysregulation of the PI3K/AKT/mTOR pathway and histone deacetylases (HDACs) has been described in diffuse large B-cell lymphoma (DLBCL). Previous studies demonstrated that combined treatment with PI3K/AKT pathway signaling inhibitors (e.g., LY294002, or perifosine) and histone deacetylase inhibitors (HDACis, e.g., sodium butyrate, vorinostat) resulted in a dramatic induction of apoptosis in human myeloid leukemia cells (Rahmani et al., Oncogene 22:6231-42, 2003; Rahmani et al., Cancer Res 65:2422-32, 2005). Here we examined interactions between the clinically relevant dual PI3K/mTOR inhibitor BEZ235 and the pan-HDACi panobinostat (both Novartis) in DLBCL cells. Notably, combined treatment with BEZ235 (25-200 nM) and panobinostat (5-15 nM) resulted in sharp decreases in cell growth and viability and profound induction of mitochondrial dysfunction and apoptosis. These events were observed in various DLBCL cell lines including both GC-DLBCL (SUDHL4, SUDHL16, OCI-LY7) and ABC-DLBCL (HBL-1, TMD8) as well as in mantle cell lymphoma cells (JeKo-1). Enhanced lethality of this regimen was accompanied by a marked increase in cytochrome c and AIF release into the cytosol, caspase-3 activation, and PARP cleavage. It was also associated with down-regulation of Mcl-1, a pronounced increase in H3 and H4 acetylation, and up-regulation of phospho- gH2AX, an indicator of DNA damage (e.g., DNA double-strand breaks). In addition, panobinostat robustly induced p21CIP1 accumulation in DLBCL cells, an event that was largely abrogated by BEZ235. Of note, treatment with BEZ235 alone or in combination with panobinostat triggered a decrease in GSK3 phosphorylation and levels of its downstream target, b-catenin consistent with GSK3 activation. Interestingly, inhibition of GSK3 by CHIR-98014 or the GSK3 inhibitor IX (BIO), but not its inactive analogue MeBIO, significantly diminished BEZ235/panobinostat lethality, arguing that GSK3 activation plays a significant functional role in lethality. Immunoprecipitation studies revealed that down-regulation of Mcl-1 was associated with enhanced binding of Bim to Bcl-xL and Bcl-2 and a marked decrease in Bak binding to Bcl-xL. In addition, knockdown of Bak also markedly diminished BEZ235/panobinostat-mediated lethality, as assessed by Annexin V/PI positivity. Together, these findings suggest that Bak plays a key functional role in the pronounced activity of BEZ235/panobinostat toward DLBCL cells. They also raise the possibility that BEZ235 may enhance panobinostat lethality by increasing Bim binding to Bcl-xL/Bcl-2, leading to the release of Bak/Bax from Bcl-2/Bcl-xL, culminating in apoptosis. Significantly, HS-5 stromal cell-conditioned media failed to protect DLBCL cells from combined panobinostat/BEZ235 treatment, suggesting that this strategy may be effective in circumventing microenvironmental forms of resistance. Combined treatment also exhibited robust activity against primary DLBCL cells, whereas exposure to the same regimens did not significantly reduce the viability of normal CD34+ progenitor cells nor did it reduce their clonogenic potential. Finally, in vivo studies utilizing a murine xenograft model bearing SUDHL4 cells revealed that co-treatment with BEZ235 and panobinostat markedly reduced in vivo tumor growth, whereas agents administered individually exhibited only modest effects. In addition, Kaplan-Meier analysis revealed that combined treatment significantly prolonged the survival of mice; in contrast, single agents were ineffective in increasing survival. Together, these findings suggest that the anti-DLBCL activities of combined BEZ235 and panobinostat treatment may involve multiple mechanisms, including Mcl-1 down-regulation, increased Bim binding to Bcl-2/Bcl-xL, release of Bak from Bcl-xL, abrogation of p21CIP1 accumulation, induction of DNA damage, and GSK3 activation, culminating in Bax/Bak activation and apoptosis. These findings raise the possibility that combining PI3K/AKT/mTOR inhibitors (e.g., BEZ235) and HDACis (e.g., panobinostat), may represent a novel and effective strategy against various DLBCL subtypes and possibly other hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu). CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells.

Abstract Introduction There are roughly 75,000 new cases of Non-Hodgkin's Lymphoma every year, representing almost 5% of all new cancer diagnoses. Indolent B-Cell Non-Hodgkin's Lymphoma (i-NHL) represents a heterogeneous group of lymphoproliferative malignancies, encompassing 40% of NHL, that remains largely incurable. Follicular lymphoma and marginal zone lymphoma (MZL) are the two most common subtypes of i-NHL. The B-cell receptor signaling pathway is activated in B-cell malignancy and mediates its activity mainly through the Phosphoinositide 3-kinase (PI3K) pathway. Furthermore, novel PI3K inhibitors, such as idelalisib and copanlisib, have shown impressive clinical activity in several indolent lymphomas including MZL. This further supports the important role of the PI3K pathway in these tumors. Therefore, we hypothesized that the PI3K-mTOR (mammalian target of rapamycin) pathway is sufficient for driving the pathogenesis of marginal zone lymphomas. Methods In order to test our hypothesis, we generated a genetically engineered mouse model carrying heterozygous knockout alleles of both the tumor suppressor genes Phosphatase and Tensin Homolog (PTEN) and Liver Kinase B1 (LKB1), leading to over-activation of the PI3K-mTOR pathway in all mouse tissues. We closely monitored these mice for tumor formation by at least weekly physical examinations for several months. Upon tumor detection, tumor size was recorded weekly using calipers, with an experimental endpoint of 15-20mm in any dimension. Upon reaching this endpoint, or if the surrounding area became necrotic/ulcerated, if the mouse's physical/behavioral condition deteriorated, or if there were any adverse conditions warranting mouse sacrifice, the mouse was euthanized and any existing tumors and any other tissues of interest were harvested. One half of the tumor was immediately preserved in a 4% paraformaldehyde solution and prepared for sectioning, H&amp;E and immunohistochemical staining, with CD3 (T-cell marker), and PAX-5 (B-cell marker). The 2nd half of the tumor was processed using a "stomacher" machine to dissociate the tumor cells, which were then counted and frozen in accordance with cryopreservation guidelines suggested by the American Type Culture Collection (ATCC). Results We generated an initial cohort of 49 Pten+/-/Lkb1+/- mice. Among all mice, the average survival time was 6 months. Thirty mice died or were sacrificed due to disease progression, defined as either lymph node enlargement and/or splenomegaly. All mice showed either lymphadenopathy or splenomegaly (Figure 1). By Kaplan-Meier analysis, we see a steady decrease in both tumor-free and overall survival after 3 months of age. Utilizing the product limit method, the median survival time was 6 months (95% CI: 6, 8). A total of 51 lymph nodes were sent for IHC and pathological identification. Of the 51 nodes, 61.5% (N=32) showed indolent Non-Hodgkin's Lymphoma, 25% (N=13) were atypical, and 11.5% (N=6) were reactive. All lymph nodes with indolent NHL were Marginal Zone subtype (Figure 2), except one case that was suspicious for follicular lymphoma. We have not identified any large cell lymphoma or development of other malignancies. Discussion Marginal zone lymphoma remains an incurable indolent lymphoma that lacks preclinical models. As novel agents become available, it is important to have a better understanding of the underlying pathogenesis of this malignancy and be able to model it in a immunocompetent mouse with a preserved microenvironment. Our data provides, for the first time, a proof of concept on the role of the PI3K-mTOR pathway in the pathogenesis of marginal zone lymphoma and paves the way for future studies understanding the biology of this disease, and developing rational therapies for this incurable malignancy. Disclosures No relevant conflicts of interest to declare.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that uses the proteasome ubiquitin system to target proteins of interest and promote their degradation with remarkable selectivity. Importantly, unlike conventional small molecule inhibitors, PROTACs have proven highly effective in targeting undruggable proteins and those bearing mutations. Because of these considerations, PROTACs have increasingly become an emerging technology for the development of novel targeted anticancer therapeutics. Interestingly, many PROTACs have demonstrated a great potency and specificity in degrading several oncogenic drivers. Many of these, following extensive preclinical evaluation, have reached advanced stages of clinical testing in various cancers including hematologic malignancies. In this review, we provide a comprehensive summary of the recent advances in the development of PROTACs as therapeutic strategies in diverse hematological malignancies. A particular attention has been given to clinically relevant PROTACs and those targeting oncogenic mutants that drive resistance to therapies. We also discus limitations, and various considerations to optimize the design for effective PROTACs.

&lt;div&gt;Abstract&lt;p&gt;The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; levels and reduced GRP78/BiP expression in a Ca&lt;sup&gt;2+&lt;/sup&gt;-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca&lt;sup&gt;2+&lt;/sup&gt; or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca&lt;sup&gt;2+&lt;/sup&gt; quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca&lt;sup&gt;2+&lt;/sup&gt; levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca&lt;sup&gt;2+&lt;/sup&gt; and loss of GRP78/BiP function, leading to &lt;i&gt;de novo&lt;/i&gt; ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation. Mol Cancer Ther; 9(5); 1378–95. ©2010 AACR.&lt;/p&gt;&lt;/div&gt;

Supplementary Data from 17-Allylamino-17-Demethoxygeldanamycin and MEK1/2 Inhibitors Kill GI Tumor Cells via Ca&lt;sup&gt;2+&lt;/sup&gt;-Dependent Suppression of GRP78/BiP and Induction of Ceramide and Reactive Oxygen Species

&lt;div&gt;Abstract&lt;p&gt;The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; levels and reduced GRP78/BiP expression in a Ca&lt;sup&gt;2+&lt;/sup&gt;-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca&lt;sup&gt;2+&lt;/sup&gt; or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca&lt;sup&gt;2+&lt;/sup&gt; quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca&lt;sup&gt;2+&lt;/sup&gt; levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca&lt;sup&gt;2+&lt;/sup&gt; and loss of GRP78/BiP function, leading to &lt;i&gt;de novo&lt;/i&gt; ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation. Mol Cancer Ther; 9(5); 1378–95. ©2010 AACR.&lt;/p&gt;&lt;/div&gt;

Supplementary Data from 17-Allylamino-17-Demethoxygeldanamycin and MEK1/2 Inhibitors Kill GI Tumor Cells via Ca&lt;sup&gt;2+&lt;/sup&gt;-Dependent Suppression of GRP78/BiP and Induction of Ceramide and Reactive Oxygen Species

&lt;div&gt;Abstract&lt;p&gt;Melanoma differentiation-associated gene-7/interleukin-24 (&lt;i&gt;mda&lt;/i&gt;-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the &lt;i&gt;mda&lt;/i&gt;-7/IL-24–induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked &lt;i&gt;mda&lt;/i&gt;-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to &lt;i&gt;mda&lt;/i&gt;-7/IL-24. Mcl-1 downregulation by &lt;i&gt;mda&lt;/i&gt;-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress–induced cancer-selective apoptosis by &lt;i&gt;mda&lt;/i&gt;-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, &lt;i&gt;mda&lt;/i&gt;-7/IL-24 might provide an effective therapeutic for this disease. Cancer Res; 70(12); 5034–45. ©2010 AACR.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Effects of concomitant inhibition of the PI3K/AKT/mTOR pathway and Bcl-2/Bcl-xL (BCL2L1) were examined in human myeloid leukemia cells. Tetracycline-inducible Bcl-2 and Bcl-xL dual knockdown sharply increased PI3K/AKT/mTOR inhibitor lethality. Conversely, inducible knockdown or dominant-negative AKT increased, whereas constitutively active AKT reduced lethality of the Bcl-2/Bcl-xL inhibitor ABT-737. Furthermore, PI3K/mTOR inhibitors (e.g., BEZ235 and PI-103) synergistically increased ABT-737–mediated cell death in multiple leukemia cell lines and reduced colony formation in leukemic, but not normal, CD34+ cells. Notably, increased lethality was observed in four of six primary acute myelogenous leukemia (AML) specimens. Responding, but not nonresponding, samples exhibited basal AKT phosphorylation. PI3K/mTOR inhibitors markedly downregulated Mcl-1 but increased Bim binding to Bcl-2/Bcl-xL; the latter effect was abrogated by ABT-737. Combined treatment also markedly diminished Bax/Bak binding to Mcl-1, Bcl-2, or Bcl-xL. Bax, Bak, or Bim (BCL2L11) knockdown or Mcl-1 overexpression significantly diminished regimen-induced apoptosis. Interestingly, pharmacologic inhibition or short hairpin RNA knockdown of GSK3α/β significantly attenuated Mcl-1 downregulation and decreased apoptosis. In a systemic AML xenograft model, dual tetracycline-inducible knockdown of Bcl-2/Bcl-xL sharply increased BEZ235 antileukemic effects. In a subcutaneous xenograft model, BEZ235 and ABT-737 coadministration significantly diminished tumor growth, downregulated Mcl-1, activated caspases, and prolonged survival. Together, these findings suggest that antileukemic synergism between PI3K/AKT/mTOR inhibitors and BH3 mimetics involves multiple mechanisms, including Mcl-1 downregulation, release of Bim from Bcl-2/Bcl-xL as well as Bak and Bax from Mcl-1/Bcl-2/Bcl-xL, and GSK3α/β, culminating in Bax/Bak activation and apoptosis. They also argue that combining PI3K/AKT/mTOR inhibitors with BH3 mimetics warrants attention in AML, particularly in the setting of basal AKT activation and/or addiction. &lt;i&gt;Cancer Res; 73(4); 1340–51. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 64K, Primary antibodies and cell lines generation&lt;/p&gt;