Mondira Kundu

A molecular mechanism that links the mTOR and autophagy pathways is now revealed. Depending on nutrient availability, the AMPK and mTOR kinases differentially phosphorylate the autophagy-initiating kinase Ulk1 to regulate its activity. Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.

Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X L , BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX- and BAK-independent role for a BCL2-related protein in development.

Production of a red blood cell's hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.

Autophagy is a process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. In yeast, autophagy is activated in response to changes in the extracellular milieu. Depending upon the stimulus, autophagy can degrade cytoplasmic contents nonspecifically or can target the degradation of specific cellular components. Both of these have been adopted in higher eukaryotes and account for the expanding role of autophagy in various cellular processes, as well as contribute to the variation in cellular outcomes after induction of autophagy. In some cases, autophagy appears to be an adaptive response, whereas under other circumstances it is involved in cell death. In mammals, autophagy has been implicated in either the pathogenesis or response to a wide variety of diseases, including neurodegenerative disease, chronic bacterial and viral infections, atherosclerosis, and cancer. As the basic molecular pathways that regulate autophagy are elucidated, the relationship of autophagy to the pathogenesis of various disease states emerges.

Abstract Mitochondrial health is critical for skeletal muscle function and is improved by exercise training through both mitochondrial biogenesis and removal of damaged/dysfunctional mitochondria via mitophagy. The mechanisms underlying exercise-induced mitophagy have not been fully elucidated. Here, we show that acute treadmill running in mice causes mitochondrial oxidative stress at 3–12 h and mitophagy at 6 h post-exercise in skeletal muscle. These changes were monitored using a novel fluorescent reporter gene, pMitoTimer , that allows assessment of mitochondrial oxidative stress and mitophagy in vivo, and were preceded by increased phosphorylation of AMP activated protein kinase (Ampk) at tyrosine 172 and of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555. Using mice expressing dominant negative and constitutively active Ampk in skeletal muscle, we demonstrate that Ulk1 activation is dependent on Ampk. Furthermore, exercise-induced metabolic adaptation requires Ulk1. These findings provide direct evidence of exercise-induced mitophagy and demonstrate the importance of Ampk-Ulk1 signaling in skeletal muscle.

NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2(-/-) and Ripk2(-/-) mice are hypersusceptible to infection with influenza A virus. Ripk2(-/-) cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1(-/-) cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease.

Unc-51-like kinase 1 (Ulk1) plays a central role in autophagy induction. It forms a stable complex with Atg13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200). This complex is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) in a nutrient-dependent way. AMP-activated protein kinase (AMPK), which is activated by LKB1/Strad/Mo25 upon high AMP levels, stimulates autophagy by inhibiting mTORC1. Recently, it has been described that AMPK and Ulk1 interact and that the latter is phosphorylated by AMPK. This phosphorylation leads to the direct activation of Ulk1 by AMPK bypassing mTOR-inhibition. Here we report that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity. Thus, we propose that Ulk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagy. In our model, phosphorylation of AMPK by Ulk1 represents a negative feedback circuit.

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget’s disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.

Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

Abstract Mitochondrial clearance is a well recognized but poorly understood biologic process, and reticulocytes, which undergo programmed mitochondrial clearance, provide a useful model to study this phenomenon. At the ultrastructural level, mitochondrial clearance resembles an autophagy-related process; however, the role of autophagy in mitochondrial clearance has not been established. Here we provide genetic evidence that autophagy pathways, initially identified in yeast, are involved in mitochondrial clearance from reticulocytes. Atg7 is an autophagy protein and an E1-like enzyme, which is required for the activity of dual ubiquitin-like conjugation pathways. Atg7 is required for the conjugation of Atg12 to Atg5, and Atg8 to phosphatidylethanolamine (PE), and is essential for autophagosome formation. In the absence of Atg7, mitochondrial clearance from reticulocytes is diminished but not completely blocked. Mammalian homologs of Atg8 are unmodified in Atg7−/− erythroid cells, indicating that canonical autophagy pathways are inactive. Thus, mitochondrial clearance is regulated by both autophagy-dependent and -independent mechanisms. In addition, mitochondria, which depolarize in wild-type cells before elimination, remain polarized in Atg7−/− reticulocytes in culture. This suggests that mitochondrial depolarization is a consequence rather than a cause of autophagosome formation in reticulocytes.

Young adult childhood cancer survivors are at an increased risk of frailty, a physiologic phenotype typically found among older adults. This phenotype is associated with new-onset chronic health conditions and mortality among both older adults and childhood cancer survivors. Mounting evidence suggests that poor fitness, muscular weakness, and cognitive decline are common among adults treated for childhood malignancies, and that risk factors for these outcomes are not limited to those treated with cranial radiation. Although the pathobiology of this phenotype is not known, early cellular senescence, sterile inflammation, and mitochondrial dysfunction in response to initial cancer or treatment-related insults are hypothesized to play a role. To the authors' knowledge, interventions to prevent or remediate frailty among childhood cancer survivors have not been tested to date. Pharmaceutical, nutraceutical, and lifestyle interventions have demonstrated some promise.

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

The improvement in survival of childhood cancer observed across the past 50 years has resulted in a growing acknowledgment that simply extending the lifespan of survivors is not enough. It is incumbent on both the cancer research and the clinical care communities to also improve the health span of survivors. It is well established that aging adult survivors of childhood cancer are at increased risk of chronic health conditions, relative to the general population. However, as the first generation of survivors age into their 50s and 60s, it has become increasingly evident that this population is also at risk of early onset of physiologic aging. Geriatric measures have uncovered evidence of reduced strength and speed and increased fatigue, all components of frailty, among survivors with a median age of 33 years, which is similar to adults older than 65 years of age in the general population. Furthermore, frailty in survivors independently increased the risk of morbidity and mortality. Although there has been a paucity of research investigating the underlying biologic mechanisms for advanced physiologic age in survivors, results from geriatric populations suggest five biologically plausible mechanisms that may be potentiated by exposure to cancer therapies: increased cellular senescence, reduced telomere length, epigenetic modifications, somatic mutations, and mitochondrial DNA infidelity. There is now a critical need for research to elucidate the biologic mechanisms of premature aging in survivors of childhood cancer. This research could pave the way for new frontiers in the prevention of these life-changing outcomes.

Lactate is used as an energy source by producer cells or shuttled to neighboring cells and tissues. Both glucose and lactate fulfill the bioenergetic demand of neurons, the latter imported from astrocytes. The contribution of astrocytic lactate to neuronal bioenergetics and the mechanisms of astrocytic lactate production are incompletely understood. Through in vivo1H magnetic resonance spectroscopy, 13C glucose mass spectroscopy, and electroencephalographic and molecular studies, here we show that the energy sensor AMP activated protein kinase (AMPK) regulates neuronal survival in a non-cell-autonomous manner. Ampk-null mice are deficient in brain lactate and are seizure prone. Ampk deletion in astroglia, but not neurons, causes neuronal loss in both mammalian and fly brains. Mechanistically, astrocytic AMPK phosphorylated and destabilized thioredoxin-interacting protein (TXNIP), enabling expression and surface translocation of the glucose transporter GLUT1, glucose uptake, and lactate production. Ampk loss in astrocytes causes TXNIP hyperstability, GLUT1 misregulation, inadequate glucose metabolism, and neuronal loss.

Hematopoietic stem and progenitor cell (HSPC) expansion is regulated by intrinsic signaling pathways activated by cytokines. The intracellular kinase JAK2 plays an essential role in cytokine signaling, and activating mutations in JAK2 are found in a number of hematologic malignancies. We previously demonstrated that lymphocyte adaptor protein (Lnk, also known as Sh2b3) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion. Here we show that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced myeloproliferative diseases (MPDs) in mice. Specifically, Lnk deficiency enhanced cytokine-independent JAK/STAT signaling and augmented the ability of oncogenic JAK2 to expand myeloid progenitors in vitro and in vivo. An activated form of JAK2, unable to bind Lnk, caused greater myeloid expansion than activated JAK2 alone and accelerated myelofibrosis, indicating that Lnk directly inhibits oncogenic JAK2 in constraining MPD development. In addition, Lnk deficiency cooperated with the BCR/ABL oncogene, the product of which does not directly interact with or depend on JAK2 or Lnk, in chronic myeloid leukemia (CML) development, suggesting that Lnk also acts through endogenous pathways to constrain HSPCs. Consistent with this idea, aged Lnk-/- mice spontaneously developed a CML-like MPD. Taken together, our data establish Lnk as a bona fide suppressor of MPD in mice and raise the possibility that Lnk dysfunction contributes to the development of hematologic malignancies in humans.

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

Abstract AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the β1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a β1-Ser108 kinase in cells. Cellular β1-Ser108 phosphorylation by ULK1 was dependent on AMPK β-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H2O2). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H2O2-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, Caenorhabditis elegans UNC-51, and Drosophila melanogaster Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. C. elegans UNC-51 and D. melanogaster Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking Ulk1 and Ulk2 in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in ulk1/2 double-knockout mice and central nervous system-specific (Nes-Cre) Ulk1/2-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., Atg7 or Rb1cc1 [RB1-inducible coiled-coil 1]). The brains of Ulk1/2-deficient mice did not show stem cell defects previously attributed to defective autophagy in ambra1 (autophagy/Beclin 1 regulator 1)- and Rb1cc1-deficient mice or accumulation of SQSTM1 (sequestosome 1)+ or ubiquitin+ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.

Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3- to 6-fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1, and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (Ctx) or ischemia-reperfusion (I/R) to induce muscle injury and regeneration and observed increases in Atgs between days 2 and 7 in adult skeletal muscle followed by increased autophagy flux after day 7. Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to Ctx or I/R. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-Ctx or I/R. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-Ctx. These findings suggest that increased autophagy protein and flux occur during muscle regeneration and Ulk1-mediated mitophagy is critical for recovery for the mitochondrial network and hence functional regeneration.

Mammalian Unc-51-like kinases 1 and 2 (ULK1 and ULK2) belong to the ULK/Atg1 family of serine/threonine kinases, which are conserved from yeast to mammals. Although ULK/Atg1 is best known for regulating flux through the autophagy pathway, it has evolutionarily conserved noncanonical functions in protein trafficking that are essential for maintaining cellular homeostasis. As a direct target of energy- and nutrient-sensing kinases, ULK/Atg1 is positioned to regulate the distribution and use of cellular resources in response to metabolic cues. In this review, we provide an overview of the molecular mechanisms through which ULK/Atg1 carries out its canonical and noncanonical functions and the signaling pathways that link its function to metabolism. We also highlight potential contributions of ULK/Atg1 in human diseases, including cancer and neurodegeneration.

Key Points Mitochondrial dysfunction in aged mtDNA-mutator mice is associated with activation of mechanistic target of rapamycin and suppression of autophagy in erythroid cells. Autophagy maintains mitochondrial function in erythroid progenitors of mtDNA-mutator mice, and disrupting it accelerates onset of anemia.

Rapamycin alleviates β-thalassemia by stimulating ULK1-dependent clearance of toxic free α-globin.

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.

Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for 12/15-lipoxygenase (12/15-LOX)-mediated unsaturated fatty acid metabolism in HSC function. HSCs from 12/15-LOX-deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that 12/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in 12/15-LOX-mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical Wnt signaling as measured by nuclear beta-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.

Hematopoietic stem cells (HSCs) are inherently quiescent and self-renewing, yet can differentiate and commit to multiple blood cell types. Intracellular mitochondrial content is dynamic, and there is an increase in mitochondrial content during differentiation and lineage commitment in HSCs. HSCs reside in a hypoxic niche within the bone marrow and rely heavily on glycolysis, while differentiated and committed progenitors rely on oxidative phosphorylation. Increased oxidative phosphorylation during differentiation and commitment is not only due to increased mitochondrial content but also due to changes in mitochondrial cytosolic distribution and efficiency. These changes in the intracellular mitochondrial landscape contribute signals toward regulating differentiation and commitment. Thus, a functional relationship exists between the mitochondria in HSCs and the state of the HSCs (i.e., stemness vs. differentiated). This review focuses on how autophagy-mediated mitochondrial clearance (i.e., mitophagy) may affect HSC mitochondrial content, thereby influencing the fate of HSCs and maintenance of hematopoietic homeostasis.

Key Points Infant acute lymphoblastic leukemia is sensitive to therapeutic targeting by apoptosis, necoptosis, and autophagy activation whether MLL is rearranged or germline. The disease-specific form of triple death mode killing by obatoclax overcomes the intrinsic resistance of MLL-rearranged infant acute lymphoblastic to cell death.

The secretory and autophagy pathways can be thought of as the biosynthetic (i.e., anabolic) and degradative (i.e., catabolic) branches of the endomembrane system. In analogy to anabolic and catabolic pathways in metabolism, there is mounting evidence that the secretory and autophagy pathways are intimately linked and that certain regulatory elements are shared between them. Here we highlight the parallels and points of intersection between these two evolutionarily highly conserved and fundamental endomembrane systems. The intersection of these pathways may play an important role in remodeling membranes during cellular stress.

Membrane and secretory proteins are essential for almost every aspect of cellular function. These proteins are incorporated into ER-derived carriers and transported to the Golgi before being sorted for delivery to their final destination. Although ER-to-Golgi trafficking is highly conserved among eukaryotes, several layers of complexity have been added to meet the increased demands of complex cell types in metazoans. The specialized morphology of neurons and the necessity for precise spatiotemporal control over membrane and secretory protein localization and function make them particularly vulnerable to defects in trafficking. This review summarizes the general mechanisms involved in ER-to-Golgi trafficking and highlights mutations in genes affecting this process, which are associated with neurological diseases in humans.

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.

UNC-51-like kinases 1 and 2 (ULK1/2) are serine/threonine kinases that are best known for their evolutionarily conserved role in the autophagy pathway. Upon sensing the nutrient status of a cell, ULK1/2 integrate signals from upstream cellular energy sensors such as mTOR and AMPK and relay them to the downstream components of the autophagy machinery. ULK1/2 also play indispensable roles in the selective autophagy pathway, removing damaged mitochondria, invading pathogens, and toxic protein aggregates. Additional functions of ULK1/2 have emerged beyond autophagy, including roles in protein trafficking, RNP granule dynamics, and signaling events impacting innate immunity, axon guidance, cellular homeostasis, and cell fate. Therefore, it is no surprise that alterations in ULK1/2 expression and activity have been linked with pathophysiological processes, including cancer, neurological disorders, and cardiovascular diseases. Growing evidence suggests that ULK1/2 function as biological rheostats, tuning cellular functions to intra and extra-cellular cues. Given their broad physiological relevance, ULK1/2 are candidate targets for small molecule activators or inhibitors that may pave the way for the development of therapeutics for the treatment of diseases in humans.

SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A-D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals.

Core-binding factor β (CBFβ) and CBFα2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFβ or CBFα2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFβ and CBFα2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion ofCBFB with MYH11, the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for aCbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb−/−embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfβ in hematopoietic cells. In midgestation embryos, Cbfβ was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and thatCbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfβ was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfβ was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.

<h2>Summary</h2> Dominant RUNX1 inhibition has been proposed as a common pathway for CBF leukemia. CBFβ-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBFβ-SMMHC fusion in AML patients lacks HABD. Here, we report that the type I CBFβ-SMMHC protein binds RUNX1 inefficiently. Knockin mice expressing CBFβ-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia-initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBFβ-SMMHC.

A fundamental function of autophagy conserved from yeast to mammals is mobilization of macromolecules during times of limited nutrient availability, permitting organisms to survive under starvation conditions. In yeast, autophagy is initiated following nitrogen or carbon deprivation, and autophagy mutants die rapidly under these conditions. Similarly, in mammals, autophagy is upregulated in most organs following initiation of starvation, and is critical for survival in the perinatal period following abrupt termination of the placental nutrient supply. The nutrient-sensing kinase, mammalian target of rapamycin, coordinates cellular proliferation and growth with nutrient availability, at least in part by regulating protein synthesis and autophagy-mediated degradation. This review focusses on the regulation of autophagy by Tor, a mammalian target of rapamycin, and Ulk1, a mammalian homolog of Atg1, in response to changes in nutrient availability. Given the importance of mitochondria in maintaining bioenergetic homestasis, and potentially as a source of membrane for autophagosomes during starvation, possible roles for mitochondria in this process are also discussed.

The NF-κB/Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression. Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1. Earlier studies indicated that E2F-1, a transcription factor that stimulates expression of S-phase-specific genes, is able to repress transcription directed by the human immunodeficiency virus (HIV-1) type-1 promoter in a variety of cells, including those of glial and lymphocytic origin. Here, we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-κB enhancer region and to interact with the NF-κB subunit, p50. Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-κB elements. Gel retardation/immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins. Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences. In vitro transcription assay demonstrates that E2F-1 represses NF-κB mediated transcription in a cell-free system. Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-κB binding sites in E2F-1 mediated repression of HIV-1 promoter, in vivo. The results of this study suggest that NF-κB activity may be regulated by its interaction with the cell cycle regulatory protein, E2F-1. The NF-κB/Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression. Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1. Earlier studies indicated that E2F-1, a transcription factor that stimulates expression of S-phase-specific genes, is able to repress transcription directed by the human immunodeficiency virus (HIV-1) type-1 promoter in a variety of cells, including those of glial and lymphocytic origin. Here, we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-κB enhancer region and to interact with the NF-κB subunit, p50. Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-κB elements. Gel retardation/immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins. Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences. In vitro transcription assay demonstrates that E2F-1 represses NF-κB mediated transcription in a cell-free system. Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-κB binding sites in E2F-1 mediated repression of HIV-1 promoter, in vivo. The results of this study suggest that NF-κB activity may be regulated by its interaction with the cell cycle regulatory protein, E2F-1. Infection with the human immunodeficiency virus type-1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type-1; LTR, long terminal repeat; GST, glutathione S-transferase; DHFR, dihydrofolate reductase; PAGE, polyacrylamide gel electrophoresis; GR, gel retardation; PCNA, proliferating cell nuclear antigen. gives rise to a progressive disease which can be separated into three clinical phases. The acute phase of infection occurs within 1–3 weeks after exposure and is characterized by mononucleosis-like symptoms and plasma viremia (1Clark S.J. Saag M.S. Decker W.D. Campbell-Hill S. Roberson J.L. Veldkamp P.J. Kappes J.C. Hahn B.H. Shaw G.M. N. Engl. J. Med. 1991; 324: 954-960Crossref PubMed Scopus (567) Google Scholar, 2Cooper D.A. Gold J. Maclean P. Donovan B. Finlayson R. Barnes T.G. Michelmore H.M. Brooke P. Penny R. Lancet. 1985; 1: 537-540Abstract PubMed Scopus (524) Google Scholar, 3Daar E.S. Moudgil T. Meyer R.D. Ho D.D. N. Engl. J. Med. 1991; 324: 961-964Crossref PubMed Scopus (721) Google Scholar). This initial phase is followed by an extended asymptomatic phase during which there is often a slow, but progressive decline in immune function that leads to the final stage, acquired immunodeficiency syndrome (AIDS). During the stage of clinical latency, the viral burden in the peripheral bloodstream is markedly diminished, but viral replication persists in the lymph nodes (4Embretson J. Zupancic M. Beneke J. Till M. Wolinsky S. Ribas J.L. Burke A. Haase A.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 357-361Crossref PubMed Scopus (167) Google Scholar, 5Embretson J. Zupancic M. Ribas J.L. Burke A. Racz P. Tenner-Racz K. Haase A.T. Nature. 1993; 362: 359-362Crossref PubMed Scopus (1251) Google Scholar, 6Ho D.D. Moudgil T. Alam M. N. Engl. J. Med. 1989; 321: 1621-1625Crossref PubMed Scopus (906) Google Scholar, 7Pantaleo G. Graziosi C. Demarest J.F Butini L. Montroni M. Fox C.H. Orenstein J.M. Kotler D.P. Fauci A.S. Nature. 1993; 362: 355-358Crossref PubMed Scopus (1573) Google Scholar). Although there is no direct correlation between clinical and viral latency, the discrepancy between the number of cells harboring viral DNA and the small number of cells expressing viral RNA during this phase, suggests that factors which regulate HIV-1 gene expression may play a role in delaying the onset of AIDS (4Embretson J. Zupancic M. Beneke J. Till M. Wolinsky S. Ribas J.L. Burke A. Haase A.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 357-361Crossref PubMed Scopus (167) Google Scholar, 5Embretson J. Zupancic M. Ribas J.L. Burke A. Racz P. Tenner-Racz K. Haase A.T. Nature. 1993; 362: 359-362Crossref PubMed Scopus (1251) Google Scholar). Cellular proliferation and differentiation are among the many parameters involved in determining the extent of HIV-1 replication in different host cells. For example, mitogenic activation of peripheral blood lymphocytes and stimulation of peripheral blood monocytes and promonocytic cell lines to differentiate are required for productive replication of HIV-1 (8Margolick J.B. Volkman D.J. Folks T.M. Fauci A.S. J. Immunol. 1987; 138: 1719-1723PubMed Google Scholar, 9Schuitemaker H. Kootstra N.A. Koppelman M.H. Bruisten S.M. Huisman H.G. Tersmette M. Miedema F. J. Clin. Invest. 1992; 89: 1154-1160Crossref PubMed Scopus (156) Google Scholar, 10Stevenson M. Stanwick T.L. Dempsey M.P. Lamonica C.A. EMBO J. 1990; 9: 1551-1560Crossref PubMed Scopus (645) Google Scholar). Quiescent (G0) T lymphocytes and monocytes and γ-irradiated (G1 arrested) monocyte-derived macrophages are not permissive to HIV replication because of their inability to complete the reverse transcription process (11He J. Choe S. Walker R. Di Marzio P. Morgan D.O. Landau N.R. J. Virol. 1995; 69: 6705-6711Crossref PubMed Scopus (0) Google Scholar, 12Schuitemaker H. Res. Immunol. 1994; 145: 588-592Crossref PubMed Scopus (10) Google Scholar, 13Schuitemaker H. Kootstra N.A. Fouchier R.A. Hooibrink B. Miedema F. EMBO J. 1994; 13: 5929-5936Crossref PubMed Scopus (78) Google Scholar). However, many agents that promote the arrest of proliferating T lymphocytes and monocytes in G1 (UV irradiation, transforming growth factor-β, and tumor necrosis factor-α, sodium butyrate, etc.) stimulate viral replication by facilitating later stages in the viral life cycle including transcription (14Ahuja S.S. Paliogianni F. Yamada H. Balow J.E. Boumpas D.T. J. Immunol. 1993; 150: 3109-3118PubMed Google Scholar, 15Haapajarvi T. Kivinen L. Pitkanen K. Laiho M. Oncogene. 1995; 11: 151-159PubMed Google Scholar, 16Stenstrand K. Larsen J.K. Wulf H.C. Lange G. Wantzin G. Photodermatol. 1987; 4: 36-42PubMed Google Scholar, 17Vicenzi E. Poli G. Chem. Biol. Interact. 1994; 91: 101-109Crossref PubMed Scopus (20) Google Scholar, 18Vilcek J. Lee T.H. J. Biol. Chem. 1991; 266: 7313-7316Abstract Full Text PDF PubMed Google Scholar). These observations suggest that although the macromolecular environment provided by cells during the G0and early G1 phase is not suitable for early steps in the viral life cycle, the repertoire of cellular transcription factors in the G1 phase can increase levels of HIV-1 transcription. Normal progression of cells from the G1 to S phase is regulated in part by E2F-1. E2F-1 belongs to family of proteins including E2F-1 to -5 which are thought to play an important role in both promoting and restricting cell cycle progression (19La Thangue N.B. Biochem. Soc. Trans. 1996; 24: 54-59Crossref PubMed Scopus (63) Google Scholar, 20Slansky J.E. Farnhamm P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar). Overexpression of E2F-1 in tissue culture cells stimulates them to enter S-phase by activating transcription of a subset of S-phase genes. These genes contain the consensus E2F-binding site (5′-TTTRRCGC-3′) in their promoters (20Slansky J.E. Farnhamm P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar). During early G1, the retinoblastoma protein, Rb p105, binds to E2F-1. This interaction not only prevents the latter from activating transcription, but also promotes active repression of some promoters by the complex (21Hsiao K. McMahon S. Farnham P. Genes Dev. 1994; 8: 1526-1537Crossref PubMed Scopus (221) Google Scholar, 22Johnson D. Ohtani K. Nevins J. Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (453) Google Scholar, 23Lam E.W. Watson R.J. EMBO J. 1993; 12: 2705-2713Crossref PubMed Scopus (317) Google Scholar, 24Weintraub S. Prater C. Dean D. Nature. 1992; 358: 259-261Crossref PubMed Scopus (560) Google Scholar). Phosphorylation of Rb later in G1 abolishes the interaction between Rb and E2F-1 (25Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (883) Google Scholar). Subsequent completion of S-phase requires inactivation of E2F-1 DNA binding activity (26Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin Jr., W.G. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 27Krek W. Xu G. Livingston D.M. Cell. 1995; 83: 1149-1158Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 28Xu M. Sheppard K. Peng C. Yee A. Piwnica-Worms H. Mol. Cell. Biol. 1994; 14: 8420-8431Crossref PubMed Scopus (259) Google Scholar). Recently, E2F-1 knockout mice showed a high incidence of unusual tumors and abnormalities in T cell maturation suggesting that in vivo, E2F-1 may also play an important role in restraining cell growth (29Field S.J. Tsai F.-Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar, 30Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). Previously, we demonstrated that E2F-1 represses HIV-1 transcription through the NF-κB enhancer region of the HIV-1 LTR (31Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). The nuclear factor-κB (NF-κB)/Rel family of transcription factors regulate HIV-1 transcription primarily through two tandem consensus binding sites in the HIV-LTR (32Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1453) Google Scholar). Several observations suggest that increased NF-κB binding during early G1 may be critical for HIV transcription and therefore replication. First, NF-κB/Rel family members are activated by a wide variety of stimuli, including tumor necrosis factor-α, sodium butyrate, retinoic acid, and UV irradiation that stimulate HIV-1 replication and promote G1 arrest (14Ahuja S.S. Paliogianni F. Yamada H. Balow J.E. Boumpas D.T. J. Immunol. 1993; 150: 3109-3118PubMed Google Scholar, 15Haapajarvi T. Kivinen L. Pitkanen K. Laiho M. Oncogene. 1995; 11: 151-159PubMed Google Scholar, 16Stenstrand K. Larsen J.K. Wulf H.C. Lange G. Wantzin G. Photodermatol. 1987; 4: 36-42PubMed Google Scholar, 17Vicenzi E. Poli G. Chem. Biol. Interact. 1994; 91: 101-109Crossref PubMed Scopus (20) Google Scholar, 18Vilcek J. Lee T.H. J. Biol. Chem. 1991; 266: 7313-7316Abstract Full Text PDF PubMed Google Scholar). Second, studies in mouse fibroblasts have shown increased NF-κB binding activity during the G0-G1transition (33Baldwin Jr., A. Azizkhan J.C. Jensen D.E. Beg A.A. Coodly L.R. Mol. Cell. Biol. 1991; 11: 4943-4951Crossref PubMed Google Scholar). Since the G0-G1 transition is traversed during both activation and differentiation; it is not surprising that NF-κB binding activity increases during mitogenic activation of peripheral blood lymphocytes and differentiation of monocytes, correlating with the increase in HIV-replication observed in these cells (8Margolick J.B. Volkman D.J. Folks T.M. Fauci A.S. J. Immunol. 1987; 138: 1719-1723PubMed Google Scholar, 9Schuitemaker H. Kootstra N.A. Koppelman M.H. Bruisten S.M. Huisman H.G. Tersmette M. Miedema F. J. Clin. Invest. 1992; 89: 1154-1160Crossref PubMed Scopus (156) Google Scholar, 34Folks T. Kelly J. Benn S. Kinter A. Justement J. Gold J. Redfield R. Sell K.W. Fauci A.S. J. Immunol. 1986; 136: 4049-4053PubMed Google Scholar). Finally, recent studies have demonstrated that the 65-kDa NF-κB subunit (p65) associates with G1cyclin-cdk complexes and that NF-κB mediated HIV-1 transcription is inhibited by their kinase activity (35Perkins N.D. Felzien L.K. Betts J.C. Leung K.Y. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). In this study, we provide further evidence that the activity of NF-κB on the HIV-1 LTR may be regulated in a cell cycle-dependent fashion. We demonstrate that the cell-cycle regulatory protein, E2F-1, suppresses the transcriptional activity of the HIV-1 LTR by interfering with NF-κB function. We present a model whereby E2F-1 represses HIV-LTR-driven transcription by interacting with the 50-kDa NF-κB subunit (p50), both on and off the DNA. Gel retardation assays were performed as described previously (36Mudryj M. Hiebert S.W. Nevins J.R. EMBO J. 1990; 9: 2179-2184Crossref PubMed Scopus (172) Google Scholar). Glutathione S-transferase (GST)-E2F-1, GST-E2F-1-(88–437), GST-E2F-1-(88–241), and GST-E2F-1-(241–437) expression plasmids were kindly provided by David Hall (Thomas Jefferson University). GST fusion proteins were prepared according to standard protocol with minor modifications. Bacterial pellets were resuspended in lysis buffer containing 500 mmKCl, 20 mm Tris (pH 7.5), 0.2 mm EDTA, 0.1% Nonidet P-40, 20% glycerol, and 100 μg/ml lysozyme; and incubated for 15 min on ice. Cells were sonicated three times (10-s pulses) and centrifuged for 30 min at 18,000 rpm. GST fusion proteins were purified by affinity chromatography using a glutathione-Sepharose column (Sigma). Purified baculovirus-produced NF-κB subunits, p50 and p65, were kindly provided by Timothy Coleman (Human Genome Sciences). The following double-stranded oligonucleotides were used in gel retardation assays: 2κB, 2κBmutant, 1κB, 1κBmutant, and DHFR E2F. The 2κB oligonucleotide spans the HIV-1 enhancer sequence and is depicted in Fig. 1, panel D. The 2κBmutant oligonucleotide is identical to 2κB, except for a 2-base pair substitution in the E2F-1-binding site (5′-TTTCCGC-3′ to 5′-TTTCCTA-3′). The 1κB oligonucleotide contains HIV-1 LTR sequences from −110 to −91 relative to the +1 transcription start site and includes the 5′ NF-κB-binding site (5′-ACAAGGGACTTTCCGCTGGG-3′). 1κBmutant is identical to 1κB, except for a 3-base pair substitution (GGG to CTC) which abolishes NF-κB binding activity (32Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1453) Google Scholar). The DHFR E2F oligonucleotide contains one E2F-binding site from the DHFR promoter (5′-ATCTTTCGCGCGCGCTTTCTA-3′). Methylation interference assay was performed as described previously (37Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Chanda V. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1996Google Scholar). Briefly, individual strands of the 2κB probe were end-labeled and hybridized to the unlabeled complementary strand. The probe was methylated by incubation with dimethyl sulfate and then incubated with 250 ng of GST-E2F-1. Bands corresponding to the E2F-DNA complex and the free probe were cut out after separation by nondenaturing polyacrylamide gel electrophoresis. The DNA was retrieved by electroelution onto DEAE membrane followed by high salt extraction and then cleaved by incubation with piperidine. After removal of the piperidine by lyophilization, the cleaved DNA was run on a 6% denaturing (urea) polyacrylamide gel. Gel retardation assay was performed as described previously (36Mudryj M. Hiebert S.W. Nevins J.R. EMBO J. 1990; 9: 2179-2184Crossref PubMed Scopus (172) Google Scholar). Bands were excised, soaked in SDS sample buffer for 1 h, and loaded onto a 10% denaturing (SDS) polyacrylamide (29:1) gel. Samples were resolved by SDS-PAGE and analyzed by Western blot (37Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Chanda V. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1996Google Scholar). Nuclear extracts were prepared as described previously (37Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Chanda V. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1996Google Scholar). Subsequent steps were also performed at 4 °C. 300 μg of precleared nuclear extract were incubated with primary antibodies overnight. Immune complexes were precipitated with Sepharose A. The pellets were washed three times with TNN buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40, 0.2 mm phenylmethylsulfonyl fluoride) and resuspended in SDS sample buffer. Proteins were resolved by SDS-PAGE and analyzed by Western blot (37Ausubel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Chanda V. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1996Google Scholar). GST pull-down experiments were performed in GST binding buffer (50 mm Tris, pH 7.5, 150 mmNaCl, 0.1% Nonidet P-40, 5 mm EDTA, 50 mmsodium fluoride). GST fusion proteins were prepared as described above, and kept on the glutathione beads. 200 μg of bacterial lysate were included in reactions using recombinant NF-κB subunits to eliminate nonspecific interactions. Pellets were washed 3 times in GST binding buffer and resuspended in SDS sample buffer. KH95 (Santa Cruz) is a mouse monoclonal specific for human and mouse E2F-1. PC10 (Santa Cruz) is a mouse monoclonal antibody that recognizes mouse and human PCNA. BF683 (Santa Cruz) is a mouse monoclonal antibody specific for human cyclin A. Sc-114 (Santa Cruz) is a rabbit polyclonal antibody raised against the nuclear localization signal of p50. NR1157 is a rabbit polyclonal antibody which recognizes p50 and was kindly provided by Dr. Nancy Rice (National Cancer Institute, Frederick, MD). Jurkat cells were transfected by electroporation using the conditions specified by the manufacturer (Bio-Rad). 1 × 107 cells were cotransfected with a total of 20 μg of DNA. Whole cell extracts were prepared by freeze-thawing cells harvested 48 h after transfection. Chlorampheicol acetyltransferase assay was performed as described previously (38Taylor J.P. Pomerantz R.J. Bagasra O. Chowdhury M. Rappaport J. Khalili K. Amini S. EMBO J. 1992; 11: 3395-3403Crossref PubMed Scopus (99) Google Scholar). The following chloramphenicol acetyltransferase reporter plasmids were used in transient transfection assay: −117/+3, mtE2F, mtκB1, mtκB2. The plasmid −117/+3 has been described previously (38Taylor J.P. Pomerantz R.J. Bagasra O. Chowdhury M. Rappaport J. Khalili K. Amini S. EMBO J. 1992; 11: 3395-3403Crossref PubMed Scopus (99) Google Scholar). The name delineates the portion of the HIV-1 LTR included in each construct; nucleotide positions are described with respect to the +1 transcription start site. Polymerase chain reaction-mediated site-directed mutagenesis was used to introduce point mutations in the −117/+3 CAT reporter construct (39Rashtchian A. Curr. Opin. Biotechnol. 1995; 6: 30-36Crossref PubMed Scopus (38) Google Scholar). The mtE2F construct contains CC to AT substitution in the putative E2F-binding site (5′-TTTCCGC-3′) and the mtκB1 construct contains a GGG to CTC substitution in the first κB site (GGGACTTTCC). The plasmid, mtκB2, was constructed by isolating the TaqI-BglII fragment from the HIV-CATΔκB construct which contains a GGG to TCT substitution in both NF-κB-binding sites. TheTaqI-BglII fragment spans from nucleotides −117 to +24 of the HIV-1 LTR. The CMV-neo-E2F-1 expression plasmid has been described previously (36Mudryj M. Hiebert S.W. Nevins J.R. EMBO J. 1990; 9: 2179-2184Crossref PubMed Scopus (172) Google Scholar). In vitrotranscription using a highly purified reconstituted system was performed as described previously (40Chiang C.M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar, 41Segil N. Guermah M. Hoffmann A. Roeder R.G. Heintz N. Genes Dev. 1996; 10: 2389-2400Crossref PubMed Scopus (161) Google Scholar). The reactions contained purified general transcription factors, the co-activator fraction (USA) and flag-tagged TFIID. The HIV-1 LTR (pMHIV wt) and the adenovirus major late promoter (pMLΔ53)-driven constructs served as templates (42Kretzschmar M. Meisterernst M. Scheidereit C. Li G. Roeder R.G. Genes Dev. 1992; 6: 761-774Crossref PubMed Scopus (97) Google Scholar). We previously demonstrated that a functional E2F-1 responsive element lies within the enhancer region of the HIV-1 LTR (31Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). The ability of E2F-1 to bind to this element in vitrowas demonstrated with gel retardation (GR) and methylation interference assays. GR analysis using recombinant GST-E2F-1 and a 37-base pair oligonucleotide probe containing the minimal responsive region (nucleotides −117 to −80 with respect to the transcription start site, +1) showed that E2F-1 is able to interact with this LTR sequence (Fig. 1 A, lanes 1–3). The E2F element from the DHFR promoter served as a positive control demonstrating the fidelity of the recombinant GST-E2F-1 (Fig. 1 A, lanes 4–6). The GST component of the fusion protein had no binding activity for either the HIV-LTR or DHFR E2F elements (data not shown). Specificity of E2F-1 binding to the HIV-LTR was verified by competition analysis utilizing a variety of unlabeled competitor oligonucleotides. The wild type HIV-1 sequence containing two copies of the κB motif (2κB) and the DHFR E2F sequences competed for E2F-binding (Fig. 1 B, lanes 1–3, 8, and 9), while the oligonucleotide containing one copy of κB sequence (1κB) and its mutant variant (1κBmt) were less effective (Fig. 1 B, lanes 4–5 and 6–7). The addition of an E2F-1 specific monoclonal antibody (KH95, Santa Cruz) to the binding reaction decreased the intensity of the major complex and induced the formation of a novel band with slower electrophoretic mobility, demonstrating the presence of E2F-1 in the original complex (Fig. 1 B, lane 10). Methylation interference assay using purified components revealed that GST-E2F-1 interacts with the guanine residues on both strands of the sequence (Fig. 1, C and D). Mutation of two critical guanine and cytosine residues of the coding strand completely abrogated the association of E2F-1 with the 2κB probe (Fig. 1 E, compare lanes 1 and 2to 3 and 4). Gel retardation analysis using various E2F-1 deletion constructs demonstrated that the basic helix-loop-helix domain of E2F-1 which is responsible for binding to the consensus E2F-1 site also mediates its interaction with the site in the HIV-1 LTR (data not shown). Since the binding site for E2F-1 partially overlaps the cis-elements recognized by the NF-κB subunits, we speculated that E2F-1 may prevent association of NF-κB with their targets in the LTR. Results from the gel retardation assay indicated that the addition of increasing amounts of E2F-1 to binding reactions containing p50 homodimers alters the electrophoretic mobility of the p50 complex and leads to the formation of novel bands (Fig. 2 A). Two-dimensional gel electrophoresis where the individual bands from the GR assay were analyzed for their protein content by Western blot analysis indicated that the intermediate complexes contain both E2F-1 and p50 (Fig. 2 B). Novel complexes containing E2F-1, p50, and p65 were formed when E2F-1 and baculovirus-produced p50/p65 heterodimers were used in an identical assay (data not shown). The E2F-1 deletions containing amino acids 88–241 or amino acids 241–437 were used to further analyze the binding of E2F-1 and p50 in GR assay (Fig. 3 A). The appearance of distinct novel complexes containing both the DNA-binding domain of E2F-1 (88–241) and p50 (Fig. 3, A, lanes 1–5; B, lanes a-d) demonstrated that both proteins can bind simultaneously to the HIV-1 LTR. This suggests that the novel complexes seen in Fig.2 A may represent displacement of p50 homodimers by E2F-1. However, the E2F-1 mutant (241–437) that does not interact with DNA participates in a DNA-binding complex when incubated with p50 (Fig. 3,A, lanes 6–10; B, lanes e-g). This indicated that in addition to displacing p50, E2F-1 may interact directly with p50.Figure 3Formation of novel complexes containing E2F-mutants and p50 in vitro. A, gel retardation assay was performed using 250 ng of recombinant p50 and 250 or 500 ng of the indicated GST-E2F-1 fusion protein. The E2F-1 deletion mutants containing amino acids 88–241 and amino acids 241–437 were used because the former binds to the 2κB probe, while the latter does not.Lanes 1–10 represent a 2-h exposure of the film.Lanes *4 and *5 are identical to lanes 4 and 5, but are derived from an overnight exposure of the same gel. B, the complexes labeled a-g inpanel A were excised from the gel, soaked in SDS sample buffer for 1 h at room temperature, and analyzed by SDS-PAGE and Western blot analysis. Membranes were incubated first with an anti-GST rabbit polyclonal antibody to detect the E2F-1 fusion proteins, and then with NR1157 to detect p50. The asterisks (*) indicate a degradation product of E2F-1 (amino acids 241–437).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunoprecipitation assay was performed to detect the presence of endogenous p50/E2F-1 heterodimers. Since deletion of the first 88 amino acids of E2F-1 did not alter its ability to repress the HIV-1 LTR when used in transient transfection assays (data not shown), nuclear extract was prepared from NIH3T3 cells that constitutively expressed detectable levels of a truncated version of human E2F-1 containing amino acids 88–437. Immunoprecipitation with an antibody specific for E2F-1 resulted in the precipitation of p50 (Fig.4 A, lane 1) and E2F-1 (88–437) (data not shown). The control antibodies against PCNA and human cyclin A were unable to precipitate significant amounts of p50, demonstrating the specificity of the reaction (Fig. 4 A, lanes 2 and 3). The small amount of p50 in lane 2is likely to result from an indirect interaction, as the truncated ectopically expressed E2F-1 was immunoprecipitated with the antibody against PCNA (data not shown). However, we cannot exclude the possibility of a weak interaction between PCNA and p50. GST pull-down assays demonstrated that GST-E2F-1 binds directly to the baculovirus-produced p50 (Fig. 4 B, lanes 1 and2), but interacts to a lesser extent when p50 is in the form of a complex with its cellular partner, p65 (Fig. 4 B, lanes 3 and 4). This suggests that the ratio between E2F-1, p50, and p65 may dictate the extent of their interactions and consequent modulation of transcription. GST pull-down assay using various E2F-1 deletions indicated that the C-terminal region of E2F-1 (amino acids 241–437) contains the p50-binding domain (Fig.4 C). Furthermore, since deletion of amino acids 1–88 reduced the amount of p50 pulled down (Fig. 4 C, comparelanes 2 and 3), a region within the first 88 amino acids may be an important contributor to the interaction between p50 and E2F-1 in vitro. However, it is important to note that this region does not appear to have a critical functional role with respect to HIV-1 transcription (as discussed above). To assess the relative contributions of NF-κB and E2F-1 to the observed regulatory eventin vivo, transient transfection studies were performed. Since treatment of cells with phorbol myristate acetate induces nuclear translocation of p50 and p65 and increases NF-κB-mediated transcription of HIV-1 promoter (32Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1453) Google Scholar), it was of interest to examine the effect of E2F-1 overexpression on HIV-1 transcription in phorbol myristate acetate-treated T-lymphocytic cells. As shown in Fig.5 A, E2F-1 was able to repress phorbol myristate acetate-stimulated levels of HIV-1 LTR-directed transcription. This observation confirms that E2F-1 suppresses κB-mediated activation. In the next series of experiments, several mutant reporter constructs containing nucleotide substitutions in the κB and E2F-binding sites were creat

Abstract Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act dominant-negatively to suppress the normal function of the Runt-related transcription factor 1 (RUNX)/core binding factor β (CBFβ) complexes. In addition, loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with the familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1lacZ/lacZ embryonic stem (ES) cells that have homozygous disruption of the Runx1 gene with N-ethyl-N-nitrosourea (ENU). We observed an increased incidence of T-lymphoblastic lymphoma in Runx1lacZ/lacZ compared with wild-type chimeras and confirmed that the tumors were of ES-cell origin. Our results therefore suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.

(Molecular Cell 62, 491–506; May 19, 2016) The authors regret to report a minor error in the motif scoring equation presented in the Supplemental Experimental Procedures. An extra minus sign was inadvertently included in the listed formula. The actual formula should be:score(i)=∑i=1nlog(Pm(i)Pbackground(i))(corrected) Instead ofscore(i)=∑i=1n−log(Pm(i)Pbackground(i))(original) The correct equation was used to generate the PSSM scores listed in Table S3. Accordingly, the highest-ranking motifs are associated with the most positive values in Table S3. The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular HomeostasisJoo et al.Molecular CellMay 19, 2016In BriefJoo et al. demonstrate that the autophagy-inducing kinases ULK1 and ULK2 regulate ER-to-Golgi trafficking of specific cargo by phosphorylating the COPII scaffold SEC16A. This non-canonical (ATG13-independent) function of the ULKs is essential for maintaining cellular homeostasis under basal physiologic conditions. Full-Text PDF Open Archive

In addition to their roles in cellular metabolism and apoptosis, mitochondria function as signaling platforms in the innate immune response. In Nature, West et al. (2015) demonstrate that mitochondrial stress triggers a type I interferon response and confers viral resistance via release of mtDNA and activation of the cGAS-STING pathway.

Protein-protein interaction can play an important role in the control of several biological events including gene transcription, replication and cell proliferation. E2F-1 is a DNA-binding transcription factor which, upon interaction with its target DNA sequence, induces expression of several S phase specific genes allowing progression of the cell cycle. Evidently, the activity of this protein is modulated by its cellular partner, pRb, which in the hypophosphorylated form, binds to E2F-1 and inactivates its transcriptional ability. In this study, we have demonstrated that expression of a sequence-specific single-stranded DNA binding protein, Pur alpha, in cells decreases the ability of E2F-1 to exert its transcriptional activity upon the responsive promoter derived from DHFR. Results from band shift experiments revealed that while Pur alpha does not recognize the double-stranded DNA fragment containing the E2F-1 binding site, it has the ability to inhibit E2F-1 interaction with its target DNA sequence. Results from GST pull-down assays and the combined immunoprecipitation/Western blot analysis of nuclear extracts revealed a direct association of E2F-1 with Pur alpha in the absence of the DNA molecule containing the E2F-1 binding site. The association of Pur alpha with E2F-1 may increase the stability of E2F-1, as a higher level of E2F-1 was detected in cells coexpressing Pur alpha and E2F-1. The importance of these observations with respect to the role of Pur alpha in the control of cell cycle progression is discussed.

Mitochondria are the site of oxidative phosphorylation in animal cells and a primary target of reactive oxygen species‐mediated damage. To prevent the accumulation of damaged mitochondria, mammalian cells have evolved strategies for their elimination. Autophagy is one means for the controlled elimination of mitochondria; however, although there has been considerable progress in defining the requirements for nonselective autophagy, relatively little is known about the genes that regulate selective autophagy of organelles. To improve our understanding of mitochondrial autophagy in mammals, we have undertaken a genetic analysis of mitochondrial clearance in murine reticulocytes. Reticulocytes provide an ideal model to study this process, because mitochondria are rapidly cleared from reticulocytes during normal development through an autophagy‐related process. Here we describe several methods for monitoring mitochondrial clearance and autophagy in reticulocytes, and show that in reticulocytes these processes require genes involved in both nonselective and selective autophagy.

Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.

R-DXd is an antibody–drug conjugate comprised of a humanised IgG1 antibody against cadherin 6 (CDH6), a stable linker selectively cleaved within tumour cells, and a membrane permeable topoisomerase I inhibitor. Overexpression of CDH6, a cell adhesion protein, occurs in the majority of OVC cases and is associated with poor prognosis. In an ongoing phase 1 trial (NCT04707248), R-DXd demonstrated an acceptable safety profile and an early efficacy signal. All patients received prior taxane and platinum therapies and were unselected for CDH6 tumour expression. Part A (dose escalation) assessed the tolerability of R-DXd at 1.6 to 9.6 mg/kg, and 8.0 mg/kg was determined to be the maximum tolerated dose. Doses of 4.8 to 8.0 mg/kg were expanded in Part B. A subgroup analysis of patients with OVC who received R-DXd at 4.8 to 8.0 mg/kg is reported here. As of 03 March 2023, 42 patients with OVC had received R-DXd at 4.8 (n = 7), 6.4 (n = 20), and 8.0 (n = 15) mg/kg: 40 (95%) had platinum-resistant disease, 29 (69%) had received prior bevacizumab, and 26 (62%) had received prior PARP inhibitors. The median prior lines of therapy was 4.0 (range 1 – 13). Twenty-one patients (50%) were still receiving study treatment. The median treatment duration was 18.1 wks (range, 3.0–93.9). Any-grade treatment-emergent adverse events (TEAEs) were experienced by 37 patients (88%), and grade ≥ 3 TEAEs were observed in 21 (50%). The most common all-grade TEAEs were nausea (55%), fatigue (40%), vomiting (38%), and diarrhoea (33%). AEs led to R-DXd discontinuation in 14% of patients. The confirmed overall response rate (RECIST v1.1) in patients with measurable disease was 38% (13 of 34): 67% (95% CI, 22.3 – 95.7; 4/6, including 1 CR) at 4.8 mg/kg, 33% (95% CI, 11.8 – 61.6; 5/15) at 6.4 mg/kg, and 31% (95% CI, 9.1 – 6.4; 4/13) at 8.0 mg/kg. Two patients with unconfirmed partial responses were still on treatment. Eleven of 21 GCIG-evaluable patients (52%) had a CA-125 response. In heavily pretreated OVC patients without CDH6 preselection, R-DXd demonstrated acceptable safety and encouraging preliminary efficacy, which supports further clinical development in OVC.

<h2>Abstract</h2> Autophagy – a highly regulated intracellular degradation process – is pivotal in maintaining cellular homeostasis. Liquid–liquid phase separation (LLPS) is a fundamental mechanism regulating the formation and function of membrane-less compartments. Recent research has unveiled connections between LLPS and autophagy, suggesting that phase separation events may orchestrate the spatiotemporal organization of autophagic machinery and cargo sequestration. The Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) family of proteins is best known for its regulatory role in initiating autophagy, but there is growing evidence that the functional spectrum of ULK/Atg1 extends beyond autophagy regulation. In this review, we explore the spatial and temporal regulation of the ULK/Atg1 family of kinases, focusing on their recruitment to LLPS-driven compartments, and highlighting their multifaceted functions beyond their traditional role.

Inv(16)(p13q22) is associated with acute myeloid leukemia subtype M4Eo, which is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This chromosomal rearrangement results in the fusion of CBFB and MYH11 genes. Mouse models indicate that the fusion gene, Cbfb-MYH11, inhibits differentiation of hematopoietic cells. Although expression of Cbfb-MYH11 is not sufficient for leukemogenesis, a combination of Cbfb-MYH11 and additional mutations can lead specifically to the development of myeloid leukemia. Normally, CBFbeta interacts with CBFalpha to form a transcriptionally active nuclear complex. In vitro studies indicate that expression of CBFB-MYH11 leads to sequestration of CBFalpha2 in the cytoplasm. It also has been shown to inhibit CBF-mediated transactivation, slow cell cycle progression, delay the apoptotic response to DNA damaging agents, and protect CBFalpha2 from degradation. The importance of these functions in vivo remains to be determined.

Human immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-κB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex. Human immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-κB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex. Neuropathological features of HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. infection include reactive astrogliosis, neuronal loss, widespread myelin pallor, subtle alteration of neocortical dendritic processes, and formation of multinucleated giant cells (1Everall I.P. Luthert P.J. Lantos P.L. Lancet. 1991; 337: 1119-1121Abstract PubMed Scopus (436) Google Scholar, 2Ketzler S. Weis S. Haug H. Budka H. Acta Neuropathol. 1990; 80: 92-94Crossref PubMed Scopus (343) Google Scholar, 3Wiley C.A. Masliah E. Morey M. Lemere C. De Teresa R. Grafe M. Hansen L. Terry R. Ann. Neurol. 1991; 29: 651-657Crossref PubMed Scopus (432) Google Scholar). The magnitude of the clinical dysfunction and CNS pathology associated with HIV-1 infection is difficult to reconcile with the small number of HIV-1-infected macrophages and microglia in the brain (4Merrill J.E. Chen C.I. FASEB J. 1991; 5: 2391-2397Crossref PubMed Scopus (332) Google Scholar, 5Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. Science. 1988; 239: 586-592Crossref PubMed Scopus (1082) Google Scholar, 6Vazeux R. Lacroix-Ciaudo C. Blanche S. Clemont M.C. Henin D. Gray F. Boccon-Gibod L. Tardieu M. Am. J. Pathol. 1992; 140: 137-144PubMed Google Scholar). This apparent paradox has led to the hypothesis that indirect effects of HIV-1 infection including the release of neurotoxic viral proteins and cytokines may mediate some of the pathobiological alterations observed in CNS cells. Tat, a viral regulatory protein, may be produced by HIV-1-infected macrophages and resident microglia, as well as infected astrocytes in the brain (7Budka H. Acta Neuropathol. 1990; 76: 611-619Crossref Scopus (86) Google Scholar, 8Gyorkey F. Melnick J.L. Gyorky P.J. J. Infect. Dis. 1987; 155: 870-876Crossref PubMed Scopus (125) Google Scholar, 9Koenig S. Gendelman H.E. Orenstein J.M. DalCanto M.C. Pezeshkpour G.H. Yungbluth M. Janotta F. Aksamit A. Martin M.A. Fauci A.S. Science. 1986; 233: 1089-1093Crossref PubMed Scopus (1348) Google Scholar, 10Sharer L.R. Cho E.S. Epstein L.G. Hum. Pathol. 1985; 16: 760-765Crossref PubMed Scopus (182) Google Scholar, 11Tornatore C. Nath A. Amemiya K. Major E.O. J. Virol. 1991; 65: 6094-6100Crossref PubMed Google Scholar, 12Wiley C. Shrier R.D. Nelson J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1053) Google Scholar). Earlier studies indicated that Tat may be released from infected cells, be taken up by uninfected neighboring cells, and exert its regulatory action (13Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2286) Google Scholar, 14Mann, D. A., and Frankel, A. D. (1991) 10,1733–1739.Google Scholar). Tat is an accessory protein that stimulates HIV-1 expression and has a pleiotropic effect on cells, ranging from stimulating cell proliferation to inducing apoptosis, depending on the cell type (14Mann, D. A., and Frankel, A. D. (1991) 10,1733–1739.Google Scholar, 15Benjouad A. Mabrouk K. Moulard M. Gluckman J.C. Rochat H. Van Rietschoten J. Sabatier J.M. FEBS Lett. 1993; 319: 119-124Crossref PubMed Scopus (21) Google Scholar, 16Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Crossref PubMed Google Scholar, 17Gibellini D. Caputo A. Celeghini C. Bassini A. La Placa M. Capitani S. Zauli G. Br. J. Haematol. 1995; 89: 24-33Crossref PubMed Scopus (52) Google Scholar, 18Lachgar A. Bernard J. Bizzini B. Astgen A. Le Coq H. Fouchard M. Chams V. Feldman M. Burny A. Zagury J.F. Biomed. Pharmacother. 1996; 50: 13-18Crossref PubMed Scopus (6) Google Scholar, 19Li C.J. Friedman D.J. Wang C. Metelev V. Pardee A.B. Science. 1995; 268: 429-431Crossref PubMed Scopus (546) Google Scholar, 20Patki A.H. Lederman M.M. Cell. Immunol. 1996; 169: 40-46Crossref PubMed Scopus (31) Google Scholar, 21Puri R.K. Leland P. Aggarwal B.B. Aids Res. Hum. Retroviruses. 1995; 11: 31-40Crossref PubMed Scopus (35) Google Scholar, 22Purvis S.F. Jacobberger J.W. Sramkoski R.M. Patki A.H. Lederman M.M. AIDS Res. Hum. Retroviruses. 1995; 11: 443-450Crossref PubMed Scopus (82) Google Scholar, 23Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (317) Google Scholar, 24Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar, 25Zauli G. Furlini G. Re M.C. Milani D. Capitani S. La Placa M. New Microbiol. 1993; 16: 115-120PubMed Google Scholar). Astrocytes secrete many supporting growth factors and are involved in neurotransmitter uptake and in maintaining the integrity of the blood-brain barrier. Thus, biological agents that alter the proliferation and activation state of these cells may lead to a broad spectrum of abnormalities and dysfunction (26Oldstone M.B.A. Sinha Y.N. Blount P. Tishon A. Rodriguez M. von Wedel R. Lampert P.W. Science. 1982; 218: 1125-1127Crossref PubMed Scopus (146) Google Scholar). Earlier observations showed that overexpression of Tat in astrocytic cells and treatment of cells with extracellular Tat can stimulate expression of several important cellular genes, including cytokines and extracellular matrix proteins (27Cupp C. Taylor J.P. Khalili K. Amini S. Oncogene. 1993; 8: 2231-2236PubMed Google Scholar, 28da Cunha A. Jackson R.W. Vitkovic L. J. Neuroimmunol. 1995; 60: 125-133Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 29Rasty S. Thatikunta P. Gordon J. Khalili K. Amini S. Glorioso J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6073-6078Crossref PubMed Scopus (37) Google Scholar, 30Taylor J.P. Cupp C. Diaz A. Chowdhury M. Khalili K. Jimenez S.A. Amini S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9617-9621Crossref PubMed Scopus (89) Google Scholar). These observations led us to the hypothesis that Tat may alter the activation and proliferation state of astrocytes and contribute to the pathogenesis of AIDS-associated dementia.The reciprocal nature of the interaction between virus and host is expected, since HIV-1 is susceptible to regulation by cellular factors and therefore by the state of the host cell. There is evidence to suggest that cellular factors, including B-myb, E2F-1, and p53, which are involved in the control of cellular proliferation, may play a role in modulation of HIV-1 gene expression (31Duan L. Ozaki I. Oakes J.W. Taylor J.P. Khalili K. Pomerantz R.J. J. Virol. 1994; 68: 4302-4313Crossref PubMed Google Scholar, 32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar, 33Sala A. Kundu M. Casela I. Engelhard A. Calabretta B. Grasso L. Paggi M.G. Giordano A. Watson R.J. Khalili K. Peschle C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 532-536Crossref PubMed Scopus (91) Google Scholar). Normal cellular proliferation occurs through an orderly progression of positive and negative regulatory events and is orchestrated by the activity of complexes consisting of cyclins and their associated catalytic partners, the cyclin-dependent kinases (34Hunt T. Semin. Cell. Biol. 1991; 2: 213-222PubMed Google Scholar, 35Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 36Pines J. Trends Biochem. Sci. 1993; 18: 195-197Abstract Full Text PDF PubMed Scopus (405) Google Scholar, 37Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1986) Google Scholar). During the G0/G1 phase, the decision of cells to commit to the cell cycle is partly dependent on the appropriate activation of G1 cyclin-Cdk complexes by extracellular stimuli. One of the most well characterized targets of these G1 cyclin-Cdk complexes is the retinoblastoma protein, Rb (38Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (915) Google Scholar, 39Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (221) Google Scholar, 40Resnitzky D. Reed S.I. Mol. Cell. Biol. 1995; 15: 3463-3469Crossref PubMed Scopus (435) Google Scholar). Early in the G1 phase, Rb exists primarily in an underphosphorylated state, at which time it interacts with the transcription factor, E2F-1, and inhibits its growth-promoting function (41Buchkovich K. Duffy L.A. Harlow E. Cell. 1989; 58: 1097-1105Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 42Flemington E.K. Speck S.H. Kaelin Jr., W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6914-6918Crossref PubMed Scopus (285) Google Scholar, 43Hiebert S.W. Chellappan S.P. Horowitz J.M. Nevins J.R. Genes Dev. 1992; 6: 177-185Crossref PubMed Scopus (466) Google Scholar, 44Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Phosphorylation of Rb in late G1 by the G1 cyclin-Cdk complexes results in the release of free E2F-1, which activates transcription of several genes involved in S phase (45Slansky J.E. Farnham P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar).In this study, we sought to further examine the interplay between virus and host cell cycle progression. We demonstrate that the viral transactivator protein, Tat, is able to arrest human astrocytic cells, U-87MG, in the G1 phase of the cell cycle by dysregulating the expression and activity of cyclin E and Cdk2. This results in accumulation of Rb in its underphosphorylated form, which in turn augments transcription directed by the HIV-1 LTR. We propose that in addition to its ability to directly transactivate the HIV-1 LTR, Tat alters the proliferation state of astrocytes and facilitates expression and replication of the HIV-1 genome.DISCUSSIONAIDS dementia complex is one of the most prevalent neurological complications of HIV-1 infection of the central nervous system. ADC affects almost 10% of AIDS patients (73Bacellar H. Munoz A. Miller E.N. Cohen B.A. Besley D. Selnes O.A. Becker J.T. McArthur J.C. Neurology. 1994; 44: 1892-1900Crossref PubMed Google Scholar). Productive infection in the CNS occurs primarily in macrophages and resident microglia (74Lipton S.A. Gendelman H.E. N. Engl. J. Med. 1995; 332: 934-940Crossref PubMed Scopus (441) Google Scholar, 75Simpson D.M. Tagliati M. Ann. Intern. Med. 1994; 121 (Correction (1995) Ann. Intern. Med. 122, 317): 769-785Crossref PubMed Scopus (195) Google Scholar). However, there is evidence of a “restricted” infection in astrocytes (76Blumberg B.M. Gelbard H.A. Epstein L.G. Virus Res. 1994; 32: 253-267Crossref PubMed Scopus (80) Google Scholar). The term “restricted” is used to describe the restriction of viral gene expression to the regulatory proteins, Tat, Rev, and Nef, which are derived from the multiply spliced viral mRNA species commonly found in infected astrocytes (62Tornatore C. Meyers K. Atwood W. Conant K. Major E.O. J. Virol. 1994; 68: 93-102Crossref PubMed Google Scholar). To account for the discrepancy between the small infected cell population and the widespread pathology, the current models regarding the neuropathogenesis of ADC propose a major role for indirect effects of HIV-1 infection (77Dewhurst S. Gelbard H.A. Fine S.M. Mol. Med. Today. 1996; 2: 16-23Abstract Full Text PDF PubMed Scopus (39) Google Scholar). In this respect, the infection of astrocytes may play a central role in the pathogenesis of ADC. Infected astrocytes and microglial cells can release the viral protein Tat. Tat can be taken up by neighboring cells in a biologically active form that can stimulate the expression of cytokines, including IL-1, IL-6, tumor necrosis factor-α, and transforming growth factor-β and several extracellular matrix proteins in the CNS (25Zauli G. Furlini G. Re M.C. Milani D. Capitani S. La Placa M. New Microbiol. 1993; 16: 115-120PubMed Google Scholar, 27Cupp C. Taylor J.P. Khalili K. Amini S. Oncogene. 1993; 8: 2231-2236PubMed Google Scholar, 29Rasty S. Thatikunta P. Gordon J. Khalili K. Amini S. Glorioso J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6073-6078Crossref PubMed Scopus (37) Google Scholar, 53Buonaguro L. Barillari G. Chang H.K. Bohan C.A. Kao V. Morgan R. Gallo R.C. Ensoli B. J. Virol. 1992; 66: 7159-7167Crossref PubMed Google Scholar, 54Gibellini D. Zauli G. Re M.C. Milani D. Furlini G. Caramelli E. Capitani S. La Placa M. Br. J. Haematol. 1994; 88: 261-267Crossref PubMed Scopus (73) Google Scholar, 55Hofman F.M. Wright A.D. Dohadwala M.M. Wong-Staal F. Walker S.M. Blood. 1993; 82: 2774-2780Crossref PubMed Google Scholar, 56Hofman F.M. Dohadwala M.M. Wright A.D. Hinton D.R. Walker S.M. J. Neuroimmunol. 1994; 54: 19-28Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 57Nabell L.M. Raja R.H. Sayeski P.P. Paterson A.J. Kudlow J.E. Cell Growth Differ. 1994; 5: 87-93PubMed Google Scholar, 58Rautonen J. Rautonen N. Martin N.L. Wara D.W. AIDS Res. Hum. Retroviruses. 1994; 10: 781-785Crossref PubMed Scopus (41) Google Scholar, 59Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Crossref PubMed Scopus (232) Google Scholar, 78Zauli G. La Placa M. Vignoli M. Re M.C. Gibellini D. Furlini G. Milani D. Marchisio M. Mazzoni M. Capitani S. J. Acquired Immune Defic. Syndrome Hum. Retrovirol. 1995; 10: 306-316Crossref PubMed Scopus (68) Google Scholar). Tat has also been shown to induce neuronal death in culture (79Nath A. Psooy K. Martin C. Knudsen B. Magnuson D.S. Haughey N. Geiger J.D. J. Virol. 1996; 70: 1475-1480Crossref PubMed Google Scholar, 80Strijbos P.J. Zamani M.R. Rothwell N.J. Arbuthnott G. Harkiss G. Neurosci Lett. 1995; 197: 215-218Crossref PubMed Scopus (24) Google Scholar, 81Weeks B.S. Lieberman D.M. Johnson B. Roque E. Green M. Loewenstein P. Oldfield E.H. Kleinman H.K. J. Neurosci. Res. 1995; 42: 34-40Crossref PubMed Scopus (67) Google Scholar). When injected into the brains of mice, Tat can stimulate edema and gliosis (82Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-529Crossref PubMed Scopus (133) Google Scholar). Here, we provide the first evidence that Tat inhibits the proliferation of glioblastoma cells, which are similar in many respects to activated astrocytes. By altering the cellular pathways involved in regulating astrocyte proliferation, Tat has the potential to disrupt the supportive function of astrocytes and contribute to the neuronal loss associated with ADC.Cellular proliferation is regulated by a series of positive and negative phosphorylation events, many of which involve cyclins and cyclin-dependent kinases. The progression of cells from G1 to S phase relies primarily on the cyclin D-Cdk4, cyclin D-Cdk6, and cyclin E-Cdk2 complex subunits (34Hunt T. Semin. Cell. Biol. 1991; 2: 213-222PubMed Google Scholar, 35Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 36Pines J. Trends Biochem. Sci. 1993; 18: 195-197Abstract Full Text PDF PubMed Scopus (405) Google Scholar, 37Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1986) Google Scholar). In this study, we demonstrate that Tat may block cells in the G1 phase by inhibiting the kinase activity of Cdk2 in astrocytic cells. The dissociation between the cyclin E- and Cdk2-associated kinase activity may also contribute to the disruption in the cell cycle. Other groups have demonstrated that Tat may inhibit the proliferative response of T-lymphocytes to antigenic and mitogenic stimuli (16Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Crossref PubMed Google Scholar, 18Lachgar A. Bernard J. Bizzini B. Astgen A. Le Coq H. Fouchard M. Chams V. Feldman M. Burny A. Zagury J.F. Biomed. Pharmacother. 1996; 50: 13-18Crossref PubMed Scopus (6) Google Scholar, 20Patki A.H. Lederman M.M. Cell. Immunol. 1996; 169: 40-46Crossref PubMed Scopus (31) Google Scholar, 23Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (317) Google Scholar). This negative response is associated with decreased IL-2 production (21Puri R.K. Leland P. Aggarwal B.B. Aids Res. Hum. Retroviruses. 1995; 11: 31-40Crossref PubMed Scopus (35) Google Scholar). IL-2 decreases the expression of the Cdk inhibitor p27 that inactivates the kinase activity of the cyclin E-Cdk2 complex (83Nourse J. Firpo E. Flanagan W.M. Coats S. Polyak K. Lee M.H. Massague J. Crabtree G.R. Roberts J.M. Nature. 1994; 372: 570-573Crossref PubMed Scopus (903) Google Scholar). Thus, it appears that Tat targets the same cellular pathway in cells of astrocytic and lymphocytic origin to inhibit cell growth. It should be noted that Tat has been associated with increased cellular proliferation in T cells (78Zauli G. La Placa M. Vignoli M. Re M.C. Gibellini D. Furlini G. Milani D. Marchisio M. Mazzoni M. Capitani S. J. Acquired Immune Defic. Syndrome Hum. Retrovirol. 1995; 10: 306-316Crossref PubMed Scopus (68) Google Scholar). Although the reasons for this discrepancy have not been elucidated, it is possible that it may depend on the culture conditions and amount of Tat used in the assays.The retinoblastoma susceptibility gene product is one of the targets of the cyclin E-Cdk2 complex (39Hatakeyama M. Brill J.A. Fink G.R. Weinberg R.A. Genes Dev. 1994; 8: 1759-1771Crossref PubMed Scopus (221) Google Scholar). The decrease in phosphorylated Rb detected in Tat-treated glioblastoma cells is therefore likely to be associated with the diminished cyclin E-Cdk2 kinase activity in these cells. The decreased phosphorylation of Rb in Tat-treated cells has implications for HIV-1 gene expression, since the underphosphorylated form of Rb stimulates HIV-1 transcription. When Rb exists in this form, it can interact with E2F-1 and prevent it from modulating transcription (41Buchkovich K. Duffy L.A. Harlow E. Cell. 1989; 58: 1097-1105Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 42Flemington E.K. Speck S.H. Kaelin Jr., W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6914-6918Crossref PubMed Scopus (285) Google Scholar, 43Hiebert S.W. Chellappan S.P. Horowitz J.M. Nevins J.R. Genes Dev. 1992; 6: 177-185Crossref PubMed Scopus (466) Google Scholar, 44Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Earlier studies demonstrated that the cell cycle regulatory protein, E2F-1, represses the activity of the HIV-1 promoter by binding to a site within the HIV-1 enhancer region and interacting with the 50-kDa subunit of NF-κB (p50) (32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). 4Kundu, M., Guermah, M., Roeder, R. G., Amini, S., and Khalili, K. (1997) J. Biol. Chem. 27229468–29474. Since the same region of the promoter is targeted by Rb, it is possible that Rb modulates HIV-1 transcription by binding to E2F-1 and inhibiting its repressive activity. It appears that by arresting cells in the G1 phase, Tat is able to maintain cells in a state that is favorable for HIV-1 transcription (Fig. 6).The observations presented in this study provide the first evidence that HIV-1, akin to the DNA tumor viruses, encodes regulatory proteins that manipulate host cell proliferation to promote viral advantage. Both HIV-1 and the DNA tumor viruses encode proteins that target the activity of the retinoblastoma protein, although to different ends. By preventing Rb phosphorylation, Tat blocks cellular proliferation at the G1 phase. By contrast, E1A, T-antigen, and E7 bind Rb and disrupt its interaction with E2F-1, which promotes entry of cells into the S phase (84Levine A.J. Annu. Rev. Biochem. 1993; 62: 623-651Crossref PubMed Scopus (475) Google Scholar). The phase of the cell cycle favored by HIV-1versus the DNA tumor viruses reflects one of the fundamental differences between these viruses: the nature of the genome. HIV-1 relies on the host cell transcription machinery for replication, because its genome consists of RNA. The genomes of the adenovirus, Simian virus 40, and human papilloma virus consist of DNA, so these viruses rely on the host cell DNA synthesis machinery for replication. Another interesting parallel is that both HIV-1 and adenovirus modulate the function of E2F-1 on their respective promoters to promote viral transcription. E1A promotes the release of E2F-1, which activates transcription of the adenovirus E2 promoter (44Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar, 85Cress W.D. Nevins J.R. Curr. Top. Microbiol. Immunol. 1996; 208: 63-78Crossref PubMed Scopus (55) Google Scholar). Tat, on the other hand, promotes sequestration of E2F-1, a negative regulator of HIV-1 transcription (32Kundu M. Srinivasan A. Pomerantz R.J. Khalili K. J. Virol. 1995; 69: 6940-6946Crossref PubMed Google Scholar). 4Kundu, M., Guermah, M., Roeder, R. G., Amini, S., and Khalili, K. (1997) J. Biol. Chem. 27229468–29474.Taken together, our data suggest that the complex interplay between virus and host, with respect to host cell cycle and HIV-1 replication, may promote HIV-1 gene expression in astrocytic cells. Furthermore, these interactions, by altering the state of astrocytes and stimulating the production of neurotoxic factors, could contribute to the pathogenesis of ADC. Neuropathological features of HIV-1 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. 1The abbreviations used are: HIV, human immunodeficiency virus; CNS, central nervous system; LTR, long terminal repeat; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; IL, interleukin; ADC, AIDS dementia complex; pRb, hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. infection include reactive astrogliosis, neuronal loss, widespread myelin pallor, subtle alteration of neocortical dendritic processes, and formation of multinucleated giant cells (1Everall I.P. Luthert P.J. Lantos P.L. Lancet. 1991; 337: 1119-1121Abstract PubMed Scopus (436) Google Scholar, 2Ketzler S. Weis S. Haug H. Budka H. Acta Neuropathol. 1990; 80: 92-94Crossref PubMed Scopus (343) Google Scholar, 3Wiley C.A. Masliah E. Morey M. Lemere C. De Teresa R. Grafe M. Hansen L. Terry R. Ann. Neurol. 1991; 29: 651-657Crossref PubMed Scopus (432) Google Scholar). The magnitude of the clinical dysfunction and CNS pathology associated with HIV-1 infection is difficult to reconcile with the small number of HIV-1-infected macrophages and microglia in the brain (4Merrill J.E. Chen C.I. FASEB J. 1991; 5: 2391-2397Crossref PubMed Scopus (332) Google Scholar, 5Price R.W. Brew B. Sidtis J. Rosenblum M. Scheck A.C. Cleary P. Science. 1988; 239: 586-592Crossref PubMed Scopus (1082) Google Scholar, 6Vazeux R. Lacroix-Ciaudo C. Blanche S. Clemont M.C. Henin D. Gray F. Boccon-Gibod L. Tardieu M. Am. J. Pathol. 1992; 140: 137-144PubMed Google Scholar). This apparent paradox has led to the hypothesis that indirect effects of HIV-1 infection including the release of neurotoxic viral proteins and cytokines may mediate some of the pathobiological alterations observed in CNS cells. Tat, a viral regulatory protein, may be produced by HIV-1-infected macrophages and resident microglia, as well as infected astrocytes in the brain (7Budka H. Acta Neuropathol. 1990; 76: 611-619Crossref Scopus (86) Google Scholar, 8Gyorkey F. Melnick J.L. Gyorky P.J. J. Infect. Dis. 1987; 155: 870-876Crossref PubMed Scopus (125) Google Scholar, 9Koenig S. Gendelman H.E. Orenstein J.M. DalCanto M.C. Pezeshkpour G.H. Yungbluth M. Janotta F. Aksamit A. Martin M.A. Fauci A.S. Science. 1986; 233: 1089-1093Crossref PubMed Scopus (1348) Google Scholar, 10Sharer L.R. Cho E.S. Epstein L.G. Hum. Pathol. 1985; 16: 760-765Crossref PubMed Scopus (182) Google Scholar, 11Tornatore C. Nath A. Amemiya K. Major E.O. J. Virol. 1991; 65: 6094-6100Crossref PubMed Google Scholar, 12Wiley C. Shrier R.D. Nelson J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1053) Google Scholar). Earlier studies indicated that Tat may be released from infected cells, be taken up by uninfected neighboring cells, and exert its regulatory action (13Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2286) Google Scholar, 14Mann, D. A., and Frankel, A. D. (1991) 10,1733–1739.Google Scholar). Tat is an accessory protein that stimulates HIV-1 expression and has a pleiotropic effect on cells, ranging from stimulating cell proliferation to inducing apoptosis, depending on the cell type (14Mann, D. A., and Frankel, A. D. (1991) 10,1733–1739.Google Scholar, 15Benjouad A. Mabrouk K. Moulard M. Gluckman J.C. Rochat H. Van Rietschoten J. Sabatier J.M. FEBS Lett. 1993; 319: 119-124Crossref PubMed Scopus (21) Google Scholar, 16Chirmule N. Than S. Khan S.A. Pahwa S. J. Virol. 1995; 69: 492-498Crossref PubMed Google Scholar, 17Gibellini D. Caputo A. Celeghini C. Bassini A. La Placa M. Capitani S. Zauli G. Br. J. Haematol. 1995; 89: 24-33Crossref PubMed Scopus (52) Google Scholar, 18Lachgar A. Bernard J. Bizzini B. Astgen A. Le Coq H. Fouchard M. Chams V. Feldman M. Burny A. Zagury J.F. Biomed. Pharmacother. 1996; 50: 13-18Crossref PubMed Scopus (6) Google Scholar, 19Li C.J. Friedman D.J. Wang C. Metelev V. Pardee A.B. Science. 1995; 268: 429-431Crossref PubMed Scopus (546) Google Scholar, 20Patki A.H. Lederman M.M. Cell. Immunol. 1996; 169: 40-46Crossref PubMed Scopus (31) Google Scholar, 21Puri R.K. Leland P. Aggarwal B.B. Aids Res. Hum. Retroviruses. 1995; 11: 31-40Crossref PubMed Scopus (35) Google Scholar, 22Purvis S.F. Jacobberger J.W. Sramkoski R.M. Patki A.H. Lederman M.M. AIDS Res. Hum. Retroviruses. 1995; 11: 443-450Crossref PubMed Scopus (82) Google Scholar, 23Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (317) Google Scholar, 24Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar, 25Zauli G. Furlini G. Re M.C. Milani D. Capitani S. La Placa M. New Microbiol. 1993; 16: 115-120PubMed Google Scholar). Astrocytes secrete many supporting growth factors and are involved in neurotransmitter uptake and in maintaining the integrity of the blood-brain barrier. Thus, biological agents that alter the proliferation and activation state of these cells may lead to a broad spectrum of abnormalities and dysfunction (26Oldstone M.B.A. Sinha

Abstract Background Adult childhood cancer survivors are at risk for frailty, including low muscle mass and weakness (sarcopenia). Using peripheral blood mitochondrial DNA copy number (mtDNAcn) as a proxy for functional mitochondria, this study describes cross-sectional associations between mtDNAcn and sarcopenia among survivors. Methods Among 1762 adult childhood cancer survivors (51.6% male; median age = 29.4 years, interquartile range [IQR] = 23.3-36.8), with a median of 20.6 years from diagnosis (IQR = 15.2-28.2), mtDNAcn estimates were derived from whole-genome sequencing. A subset was validated by quantitative polymerase chain reaction and evaluated cross-sectionally using multivariable logistic regression for their association with sarcopenia, defined by race-, age-, and sex-specific low lean muscle mass or weak grip strength. All statistical tests were 2-sided. Results The prevalence of sarcopenia was 27.0%, higher among female than male survivors (31.5% vs 22.9%; P &amp;lt; .001) and associated with age at diagnosis; 51.7% of survivors with sarcopenia were diagnosed ages 4-13 years (P = .01). Sarcopenia was most prevalent (39.0%) among central nervous system tumor survivors. Cranial radiation (odds ratio [OR] = 1.84, 95% confidence interval [CI] = 1.32 to 2.59) and alkylating agents (OR = 1.34, 95% CI = 1.04 to 1.72) increased, whereas glucocorticoids decreased odds (OR = 0.72, 95% CI = 0.56 to 0.93) of sarcopenia. mtDNAcn decreased with age (β = −0.81, P = .002) and was higher among female survivors (β = 9.23, P = .01) and among survivors with a C allele at mt.204 (β = −17.9, P = .02). In adjusted models, every standard deviation decrease in mtDNAcn increased the odds of sarcopenia 20% (OR = 1.20, 95% CI = 1.07 to 1.34). Conclusions A growing body of evidence supports peripheral blood mtDNAcn as a biomarker for adverse health outcomes; however, this study is the first to report an association between mtDNAcn and sarcopenia among childhood cancer survivors.

The Tat protein of HIV-1 is a potent activator of transcription directed by the viral long terminal repeat. In most cell types this activation requires a specific interaction between Tat and an RNA target termed TAR in the 5'-leader sequence of HIV-1 mRNAs. We have previously reported that in astrocytic cells Tat is capable of activating transcription in a TAR-independent manner through an alternative Tat-responsive element in the LTR (J. P. Taylor et al., EMBO J. 11(9), 3395-3403, 1992). In this report we demonstrate that TAR-independent activation by Tat can effectively bypass competition by decoy TAR RNA molecules. Studies with site-directed mutants demonstrate that the RNA-binding domain of Tat is dispensable for TAR-independent activation of HIV-1. In contrast, the requirement for specific components of the Tat activation domain suggests that common targets exist for this viral trans-activator to exert its activity in TAR-independent and TAR-dependent transactivation pathways of HIV-1 transcriptional activation.

The transcription factor Cbf beta forms a heterodimeric complex with members of the Runx family of proteins. Together, Cbf beta and Runx1 play a critical role in the establishment of definitive hematopoiesis in mouse embryos. Previously, we used a Cbfb-GFP "knock-in" mouse model to demonstrate that Cbf beta is expressed in hematopoietic stem cells of the mouse fetal liver and aorta-gonad-mesonephros (Blood 100 (2002), 2449). We also examined the expression pattern of Cbf beta in different lineages of adult hematopoietic cells and found that it is expressed uniformly in all lineages except B lymphocytes and erythroid cells. Cbf beta expression decreases during maturation of B cells in the adult bone marrow, and is not expressed in nucleated erythroid precursors. Here, we examine the expression of Cbf beta in various hematopoietic lineages, including myeloid, lymphoid, and erythroid during late stages of embryonic development, and compare it to the pattern observed in adults. We find that there are subtle differences in expression of Cbf beta-GFP in embryonic hematopoietic cells compared to their adult counterparts, but that the overall pattern is the same. Our data complement recently published data on hematopoetic defects observed in transgenic Cbfb-null mouse embryos partially rescued by ectopic expression of Cbfb (Nature Genet. 32 (2002), 633; Nature Genet. 32 (2002), 645). and supports the emerging view that Cbf beta and Runx proteins are required for normal maturation of hematopoietic cells as well as establishment of definitive hematopoiesis.

In this issue, Roberts et al. (2014) describe how hexokinase and mTORC1 cooperate to sense disequilibrium between glucose uptake and utilization and direct the balance of anabolism and catabolism to ensure the appropriate use of cellular resources.

Abstract The remodeling and stiffening of the extracellular matrix (ECM) associated with breast cancers is a well-recognized modulator of disease progression. However, how changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remains poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We demonstrate that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs), and as a consequence impedes cell contraction and migration. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn impedes the mechanotransduction of breast cancer cells. ULK1/2 and the well characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Thus, our study reveals ULK1/2 as important regulators of PXN-dependent mechanotransduction. Highlights ULK1/2 interact with PXN and phosphorylate PXN at S32 and S119 in response to mechanical stimuli ULK1/2-mediated phosphorylation of PXN regulates mechanotransduction and migration of breast cancer cells ULK1/2 modulate the biomaterial properties of focal adhesions through PXN phosphorylation ULK1/2 and FAK/Src act antagonistically in mechanotransduction through competitive phosphorylation of PXN

Over 50% of childhood cancer survivors are exercise intolerant, with maximal aerobic capacities comparable to individuals decades older, suggesting early physiologic ageing. In addition, 36% of survivors are obese. Optimal exercise capacity provides a foundation to support daily function and healthy body habitus and is associated with benefits to cognition, cardiovascular health, and longevity. Cellular senescence and inflammation are key mechanisms that drive age-related disease, quantifiable as biomarkers in peripheral blood.This study aimed to evaluate associations between p16INKa, a biomarker of cellular senescence, and inflammation and exercise capacity among adult survivors of childhood cancer.Eligible survivors were recruited from the St. Jude Lifetime (SJLIFE) Cohort Study. Exercise capacity was assessed by maximal oxygen uptake (VO2, ml/kg/min) obtained via cardiopulmonary exercise testing using a modified Bruce protocol. Body fat (%) was determined from dual energy x-ray absorptiometry (DEXA). Peripheral blood samples were used to evaluate log2 p16INK4a mRNA expression, a biomarker of cellular senescence, and inflammation with high sensitivity C-reactive protein (hs-CRP) levels. Multivariable regression evaluated associations between p16INK4a, hs-CRP, body fat, and exercise capacity.Participants included 185 five-year childhood cancer survivors (mean age 36.6 [range 20.1 - 55.7] years, 44% male, 77% non-Hispanic white, 53% leukemia/lymphoma). Compared to males, females had lower peak VO2 (mean ± SD, 22.5 ± 8.2 vs. 28.8 ± 7.7 ml/kg/min, p<0.01), higher p16INK4a expression (9.6 ± 1.2 vs. 9.2 ± 1.2 fold, p=0.02), and hs-CRP concentration (5.9 ± 8.4 vs. 3.3 ± 3.9 mg/L, p=0.01). Among females (n=103), hs-CRP concentration (β -0.2, 95% CI -0.34 to -0.05, p=0.01) and p16INK4a expression (β-5.32, 95% CI 10.42 to -0.22, p=0.04) were inversely associated and statistically significant with peak exercise capacity, with a significant interaction between p16INK4a expression and body fat (β 0.15, 95% CI 0.02 to 0.28, p=0.03). Among males (n=82), p16INK4a expression (β -1.01, 95% CI -2.14 to 0.12, p=0.08), and body fat (β -0.54, 95% CI -0.70 to -0.38, p<0.01) were inversely associated with peak exercise capacity.Inflammation and p16INK4a expression, a biomarker of cellular senescence, are associated with lower exercise capacity in childhood cancer survivors, suggesting potential targets or outcome measures for interventions designed to prevent or remediate accelerated physiologic ageing in this population.

Proliferation of eukaryotic cells is orchestrated by a series of cellular proteins which participate in various stages of the cell cycle to guide the cell through mitosis. Some of these proteins, including E2F1, play a critical role in G1 and S phases by coordinately regulating expression of several important cell cycle-associated genes. On the basis of recent observations indicating a block in human immunodeficiency virus type 1 (HIV-1) replication in cells arrested in G1/S phase of the cell cycle, we sought to evaluate the regulatory action of E2F1 on transcription from the HIV-1 long terminal repeat (LTR). Results from transient transfection of cells with an E2F1 expression plasmid indicated that E2F1 has the ability to suppress basal transcriptional activity of the LTR and to diminish the extent of the Tat-induced activation of the viral promoter. Deletion analysis of the HIV-1 LTR in transfection studies revealed the presence of two major elements responsive to E2F1 repression located distally (-454 to -381) and proximally (-117 to -80) with respect to the +1 transcription start site. E2F1-mediated suppression of LTR activity was observed in a wide range of human cell lines. Expression of E2F1 by a transgene showed an inhibitory effect on the levels of reverse transcriptase activity obtained upon introduction of the proviral genome into cells. The data presented in this study suggest that cellular regulatory proteins involved in the progression of cells through the mitotic cycle could play crucial roles in determining the efficiency of HIV-1 replication during the various stages of infection. The possible roles of these factors in viral latency and activation are discussed.

Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific rb1cc1- and atg14-conditional knockout (cKO) mice by using Ckm/Ckmm2-Cre and compared their phenotypes to those of ulk1 ulk2-conditional double-knockout (cDKO) mice. atg14-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both atg14-cKO and rb1cc1-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin+ SQSTM1+ deposits, but only those of rb1cc1-cKO mice showed TARDBP/TDP-43+ pathology and other features of the inclusion body myopathy-like disease we previously described in ulk1 ulk2-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP+ pathology.ABBREVIATIONS:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100.

(Neuron 78, 65–80; April 10, 2013) In the original online version of this publication, the labels “Presynaptic NMJ” and “Postsynaptic NMJ” were reversed in Figure 1E. This has been corrected for the online and the print versions. VCP Is Essential for Mitochondrial Quality Control by PINK1/Parkin and this Function Is Impaired by VCP MutationsKim et al.NeuronMarch 14, 2013In BriefMutations in VCP cause diseases in the nervous system, muscle, and/or bone through an unknown mechanism. Kim et al. demonstrate that VCP is an essential component of the PINK1/Parkin pathway of mitochondrial quality control, and disease mutations impair this function. Full-Text PDF Open Archive

Unc51 like autophagy activating kinase 1 (Ulk1), the primary autophagy regulator, has been linked to metabolic adaptation in skeletal muscle to exercise training. Here we compared the roles of Ulk1 and homologous Ulk2 in skeletal muscle insulin action following exercise training to gain more mechanistic insights. Inducible, skeletal muscle-specific Ulk1 knock-out (Ulk1-iMKO) mice and global Ulk2 knock-out (Ulk2 –/– ) mice were subjected to voluntary wheel running for 6 weeks followed by assessment of exercise capacity, glucose tolerance, and insulin signaling in skeletal muscle after a bolus injection of insulin. Both Ulk1-iMKO and Ulk2 –/– mice had improved endurance exercise capacity post-exercise. Ulk1-iMKO did not improve glucose clearance during glucose tolerance test, while Ulk2 –/– had only marginal improvement. However, exercise training-induced improvement of insulin action in skeletal muscle, indicated by Akt-S473 phosphorylation, was only impaired in Ulk1-iMKO. These data suggest that Ulk1, but not Ulk2, is required for exercise training-induced improvement of insulin action in skeletal muscle, implicating crosstalk between catabolic and anabolic signaling as integral to metabolic adaptation to energetic stress.

Autophagy is a process by which cytoplasmic components are sequestered in double membrane vesicles and degraded upon fusion with lysosomal compartments. In yeast, autophagy is activated in response to changes in the extracellular milieu. Depending upon the stimulus, autophagy can degrade cytoplasmic contents nonspecifically or can target the degradation of specific cellular components. Both of these have been adopted in higher eukaryotes and account for the expanding role of autophagy in various cellular processes, as well as contribute to the variation in cellular outcomes after induction of autophagy. In some cases, autophagy appears to be an adaptive response, whereas under other circumstances it is involved in cell death. In mammals, autophagy has been implicated in either the pathogenesis or response to a wide variety of diseases, including neurodegenerative disease, chronic bacterial and viral infections, atherosclerosis, and cancer. As the basic molecular pathways that regulate autophagy are elucidated, the relationship of autophagy to the pathogenesis of various disease states emerges.

Abstract Cell death pathways are desired targets of small molecule inhibitors since their deregulation plays an important role in chemotherapy resistance. Obatoclax binds to the BH3 pocket of anti-apoptotic BCL-2 family proteins, inhibiting their interactions with pro-apoptotic BCL-2 family members. Before we found potent obatoclax activity in MLL/AF4+ cell lines and 6 MLL rearranged (MLL+) leukemias. In apoptosis assays of the cell lines obatoclax increases TUNEL staining but minimally activates caspase 3 (Rege ASH 2005; Zhang AACR 2007). In this study, we tested the cytotoxicity of obatoclax in additional MLL+ leukemias and investigated its mechanism of action with a focus on autophagy (ATG), since ATG proteins such as beclin 1 also interact with anti-apoptotic BCL-2 family members. Methods: MLL partner genes were determined by molecular/cytogenetic methods. 17 primary MLL+ leukemias including the original 6 (12 ALL/11 infants, 1 child; 3 AML/1 each infant, child, adolescent; 2 bilineal/2 infants) were tested in MTT assays after 72 h obatoclax exposures. MTT assays on cytotoxic drug-obatoclax combinations were performed in a primary MLL/AF4+ ALL and interactions were studied by response surface modeling. MCL-1/BAK complex inhibition was tested in this ALL by co-IP/immunoblot analysis. Cell death and ATG were studied in obatoclax treated RS4:11 and/or SEM-K2 cells by PI flow, LC3 and p62 Western blot analysis and EM, using doxorubicin as a control for apoptosis. Gene expression changes after vehicle treatment vs. obatoclax treatment at the IC50 and IC90 for 6 h were studied using Affymetrix HG_U133 Plus2.0 arrays. Differentially expressed genes overall and those specifically associated with ATG were queried by ANOVA (p&amp;lt;0.01, ≥50% change in mean expression considered as significant). Q-RT PCR analysis of basal expression levels of select ATG genes (BECN1, WIPI1, MAP1LC3B) was performed in 10 of the 17 primary cases and correlations with the IC50 values were determined using Pearson correlation coefficients and their levels of significance. Changes in the expression patterns of these genes after vehicle treatment vs. treatment with obatoclax at the IC50 for 6 h and 48 h were compared in 6 cases by Q-RT PCR and cluster analysis. Results: MLL partner genes were AF4, ENL and other in 6, 5 and 1 ALL, respectively; AF9 in 2 and AF6 in 1 AML; and AF4 and ENL in 1 each bilineal leukemia. The single agent IC50’s of obatoclax suggested greater sensitivity in ALL (13–834 nM; median 104 nM) than AML (243–488nM; median 341 nM), and were 79 nM and 508 nM in the bilineal leukemias. In addition to synergy with ARAC, ADR, VP16 and DEX (Zhang AACR 2007) there was synergy with LASP and VCR in the primary MLL/AF4+ ALL. In the same ALL obatoclax decreased MCL-1/BAK dimers, suggesting interaction with the MCL-1 target, and obatoclax increased high molecular weight MCL-1/BAK complexes and decreased MCL-1, the latter of which would also decrease MCL-1/BAK dimers. Obatoclax treatment of RS4:11 and SEM-K2 cells increased PI staining and LC-3I to LC-3II conversion; EM analysis of SEM-K2 cells revealed phagophores, autophagosomes and autophagolysosomes indicative of ATG induction. Lack of p62 accumulation showed that ATG was not blocked. EM findings of apoptosis occurred in SEM-K2 cells after doxorubicin exposure. ATG was not the most affected pathway in microarray analyses of obatoclax treated SEM-K2 and RS4:11 cells, but specific analysis of ATG-related genes showed WIPI1 and MAP1LC3B upregulation. Moreover, the basal expression of BECN1 was positively correlated with obatoclax activity in the 10 primary cases (r =0.659; p=0.038), which is consistent with importance of this pathway in the drug response. ATG gene expression analysis in obatoclax treated primary MLL+ cases identified 2 patient clusters; in one cluster obatoclax decreased BECN1 and increased WIPI1 expression; in the other ATG gene expression changes were more variable. Conclusions: Obatoclax induces cell death in MLL+ leukemias via the ATG pathway even though they are apoptosis competent. This is distinct from the apoptosis activation in other cancer cell types and indicates that the targets of obatoclax are disease-specific. The activity in a broad spectrum of MLL+ leukemias indicates that obatoclax is a promising molecularly targeted agent for this population.

Evaluating the differential expression of a set of genes belonging to a common biological process or ontology has proven to be a very useful tool for biological discovery. However, existing gene-set association methods are limited to applications that evaluate differential expression across k⩾2 treatment groups or biological categories. This limitation precludes researchers from most effectively evaluating the association with other phenotypes that may be more clinically meaningful, such as quantitative variables or censored survival time variables. Projection onto the Orthogonal Space Testing (POST) is proposed as a general procedure that can robustly evaluate the association of a gene-set with several different types of phenotypic data (categorical, ordinal, continuous, or censored). For each gene-set, POST transforms the gene profiles into a set of eigenvectors and then uses statistical modeling to compute a set of z-statistics that measure the association of each eigenvector with the phenotype. The overall gene-set statistic is the sum of squared z-statistics weighted by the corresponding eigenvalues. Finally, bootstrapping is used to compute a p-value. POST may evaluate associations with or without adjustment for covariates. In simulation studies, it is shown that the performance of POST in evaluating the association with a categorical phenotype is similar to or exceeds that of existing methods. In evaluating the association of 875 biological processes with the time to relapse of pediatric acute myeloid leukemia, POST identified the well-known oncogenic WNT signaling pathway as its top hit. These results indicate that POST can be a very useful tool for evaluating the association of a gene-set with a variety of different phenotypes. We have developed an R package named POST which is freely available in Bioconductor.

Abstract Studies on the etiology of MDS have been hampered by the lack of relevant animal model systems. Numerous murine models of MDS/MPD overlap syndromes have been described, but MDS/MPD overlap syndromes are now felt to be clinically distinct from MDS. Likewise, a number of murine models of myeloid leukemia have been developed in which dysplastic features arise during conversion to frank leukemia. Here we describe the hematopoietic phenotype of the PolgD257A mitochondrial mutator mouse and demonstrate that this model system fulfills the myeloid dysplasia criteria outlined in the Bethesda Proposals (Blood, 100, 238, 2002). The PolgD257A allele eliminates the proofreading activity of Polg, the sole mitochondrial DNA (mtDNA) polymerase, and PolgA/A mice rapidly accumulate mtDNA mutations. Starting at 9 months of age, PolgA/A mice display multiple features of accelerated aging and develop a progressive and ultimately lethal anemia (Science, 309, 481, 2005). In data obtained at time of necropsy for other studies, the hemoglobin of young PolgA/A mice is identical to that of controls but by 10 months of age begins to fall below 10 g/dL. Overall, while Polg+/+ mice maintain a hemoglobin of greater than 10 g/dL through 30 months of age, the hemoglobin of PolgA/A drops below 5 g/dL as the mice succumb to their anemia at 13–16 months of age. As the hemoglobin of aging PolgA/A mice falls the MCV demonstrates a striking increase, from 53.6 fL to 69.8 fL. In order to confirm these intial findings, complete blood counts have been assessed prospectively in mice of all three genotypes (Polg+/+, Polg+/A, and PolgA/A; 5 mice per genotype) every other month up to 8 months of age and monthly thereafter. Full data up to 11 months of age, as well as 12 month data from all 5 PolgA/A mice, will be presented. As expected, up to eight months of age no significant difference in hemoglobin levels are apparent, but by eight months PolgA/A mice demonstrate a significant increase in MCV (51.5 fL) compared to either Polg+/A (44.9, p=0.0015) or Polg+/+ (44.5, p=0.0010) littermate controls. The disparity in MCV between PolgA/A mice and the other genotypes is progressive, with the MCV at 9 months increasing to 53.4 fL in PolgA/A mice (N=5), compared to 44.8 (p&lt;0.0001) in the available 9 month data from the other two genotypes (N=4). In order to determine if this macrocytic anemia is associated with defects in hematopoietic maturation, morphologic examinations were performed on the marrow of old (12–13 month old) mice of each genotype. Despite the profound anemia of PolgA/A mice, their marrow is hypercellular (95–100%) compared to littermate Polg+/+ mice (85–90%), displaying megakaryocyte dysplasia (paucity of cytoplasm and abnormal nuclear lobation) and an increase in immature MPO+ myeloid precursors. In sum, these feature fulfill the diagnostic criteria for a myeloid dysplasia without evidence of myeloproliferation. In order to determine if the macrocytic anemia with myelodysplastic bone marrow morphology seen in PolgA/A mice is the result of cell-intrinsic effects at the level of the hematopoietic stem cell, as opposed to systemic effects of mtDNA mutations, both competitive and non-competitive marrow transplants have been performed. Data at 16-weeks post-transplant will be presented.

Abstract Abstract 987 Autophagy plays an important role in maintaining mitochondrial integrity, directing lysosome-mediated destruction of cellular cargo, including damaged or dysfunctional mitochondria. Flux through the autophagy pathway is rapidly induced to promote survival in response to metabolic or proteotoxic stress resulting from exposure to noxious environmental cues, such as starvation, hypoxia or heat stress and likely contributes to the increase in mitochondrial turnover observed under each of these conditions. Dysregulation of this process has been linked to the pathogenesis of diseases, including anemia, diabetes, neurodegeneration and cancer. Atg1 is a serine-threonine kinase that directs the autophagy machinery to appropriate cargo in responses to changes in the availability of carbon and nitrogen in yeast. Ulk1, one of the mammalian homologues of Atg1, is required for starvation-induced autophagy and clearance of mitochondria in terminally differentiating erythroid cells. The function of Ulk1 is also regulated by AMP dependent Kinase (AMPK)-mediated phosphorylation, however, the precise molecular consequence of this post-translational modification has not been explored. Our preliminary findings indicate that AMPK phosphorylates Ulk1 during red blood cell maturation and in response to mitochondrial uncoupling, and that this phosphorylation is critical for mitochondrial clearance. Therefore, we sought to use these systems to explore the mechanism by which AMPK phosphorylation regulates Ulk1 function. We previously demonstrated that the stability and kinase activity of Ulk1 depends on its physical interaction with Hsp90 and the kinase-specific co-chaperone, Cdc37. Hsp90 is an abundant chaperone that directs the maturation and activation of a restricted group of metastable proteins, typically kinases and signaling molecules, and orchestrates a broad response to cellular stress. Here, we demonstrate that AMPK phosphorylation of Ulk1 does not affect Ulk1 kinase activity, but instead promotes its release from Hsp90 and its localization to damaged mitochondria. Preliminary studies indicate that the serine-proline rich domain of Ulk1, which contains at least 4 residues that are phosphorylated by AMPK, is an intrinsically disordered domain. We hypothesize that phosphorylation of Ulk1 by AMPK stabilizes a predicted alpha-helical structure within this domain and contributes to release of Hsp90. These findings are important because they provide significant insight into the regulation and function of Ulk1, a protein involved in mitochondrial turnover during red blood cell maturation and in proliferating cells. Disclosures: No relevant conflicts of interest to declare.

Abstract Although myelodysplastic syndrome (MDS) is uncommon in children, with an annual estimated incidence of 0.5 to 4 cases per million children compared to 20 to 40 cases per million in adults, the incidence of MDS appears to be increasing in the United States with the improvements in survival rates of childhood cancers, at least in part because the intensive chemo- and radiotherapy regimens that cure pediatric patients with cancer may also cause therapy-related MDS. Unlike in other blood-related cancers, the disease-causing mutations in pediatric MDS and t-MDS/AML are unknown. Nevertheless, recent studies have suggested that the mitochondrial dysfunction associated with MDS results from the inability of hematopoietic stem/progenitor cells to appropriately cope with mitochondrial damage andmitochondrial dysfunction appears to be an early event in the development of t-MDS/AML. Autophagy is a catabolic process involved in the sequestration and breakdown of cellular components, including organelles such as mitochondria and long-lived or misfolded proteins. By sequestering and degrading depolarized fragments of mitochondria that result from cycles of mitochondrial fusion and fission, autophagy plays a critical role in the maintenance of healthy pools of mitochondria. Given the importance of autophagy in mitochondrial quality control and increasing evidence that mitochondrial dysfunction plays a role in the pathogenesis of MDS, we sought to explore the role of autophagy in the pathogenesis of MDS associated with mitochondrial dysfunction using the previously established PolgAmt/mt mouse model (a.k.a. mtDNA mutator mice). The mtDNA mutator mice express a proof-reading-deficient mutant of mtDNA polymerase (PolgA-D257A), which results in increased mitochondrial mutation frequencies in multiple tissues, and a decline in respiratory function of mitochondria-encoded complexes. The delayed onset of an MDS-like phenotype in these mice provides a window of time to interrogate the role of autophagy in the progression of disease symptoms. Disruption of autophagy in erythroid progenitors did not cause a significant decrease in hemoglobin or red blood cell count in PolgAwt/wt mice, however, it accelerated the onset of anemia and morbidity significantly in PolgAmt/mt mice, suggesting that autophagy delays the onset of symptoms associated with pathologic mtDNA mutations. Flow cytometric evaluation of peripheral blood of PolgAmt/mt mice and wild-type littermates revealed an unexpected defect in RBC maturation in PolgAmt/mt animals, which became apparent with age or following serial phlebotomy. The defect was similar to that observed in autophagy-defective Ulk1-deficient mice suggesting that pathogenic mtDNA mutations may cause an age-dependent delay in autophagy. Transformed PolgAmt/mt murine embryonic fibroblasts (MEFs) with mitochondrial dysfunction also showed a defect in basal LC3 conversion and p62 accumulation, consistent with impaired flux through the autophagy pathway. Together, our data indicates that although autophagy initially combats accumulation of dysfunctional mitochondria resulting from an increased mtDNA mutation burden, the latter eventually leads to a decline in basal autophagy and impaired clearance of mitochondria, which contributes to the decline in mitochondrial function and progression of MDS-like disease in mice. Disclosures: No relevant conflicts of interest to declare.

Autophagy is an intracellular bulk degradation process in which cytosolic proteins/organelles are sequestered into double-membrane vesicles termed autophagosomes to be fused with lysosomes for degradation. Recent evidence suggests that an alternative form of autophagy, which utilizes distinct sources of membrane for autophagosome formation, may exist. Whether alternative autophagy exists in the heart is unknown. Cardiac-specific Atg7 -KO ( Atg7 -CKO) and systemic Ulk1 -KO mice were subjected to starvation for 48 hours. LC3-II expression and p62 degradation were increased in wild-type (WT) mice after starvation (ST), indicating increased autophagy. In Atg7 -CKO mice, LC3-II expression was reduced and p62 was markedly increased at baseline and in response to ST, indicating inhibition of LC3-dependent autophagy. In contrast, in Ulk1 -KO mice, neither LC3-II nor p62 was affected at baseline or in response to ST. However, autolysosome formation evaluated by Lamp2/Rab9 staining, indicators of unconventional autophagy, was abolished. Furthermore, the level of polyubiquitinated protein was increased after ST. Echocardiographic analyses showed that fractional shortening (FS) was maintained after ST in WT mice (38%). However, in Atg7 -CKO mice, FS deteriorated at baseline (22%) but was not further affected by ST (20%). In contrast, in Ulk1 -KO mice, FS was relatively preserved at baseline (32%) but remarkably decreased during ST (19%). After glucose deprivation (GD) GFP-LC3 was co-localized with calnexin (ER marker) in Ad-sh-Control- and Ad-sh-Ulk1-transduced, but not in Ad-sh-Atg7-transduced, cultured cardiomyocytes (CMs) in vitro. In contrast, Lamp2 was co-localized with TGN46 (Golgi marker) in Ad-sh-Control and Ad-sh-Atg7-transduced, but not in Ad-sh-Ulk1-transduced, CMs after GD. These results suggest that the autophagosomes are derived from ER in LC3-dependent autophagy and Golgi in Rab9-dependent autophagy in CMs. Atg7 is required for maintaining cardiac function at baseline in an LC3-dependent manner, whereas Ulk1 is required for preserving cardiac function during ST, possibly through stimulation of Rab9-dependent autophagy. These results support the presence of alternative autophagy in the heart.

Autophagy plays an important role in mediating protein quality control (QC) in cells. Inhibition of autophagy below physiological levels induces protein aggregation and cellular dysfunction. On the...

Evaluating the differential expression of a set of genes belonging to a common biological process or ontology has proven to be a very useful tool for biological discovery. However, existing gene-set association methods are limited to applications that evaluate differential expression across k ≥ 2 treatment groups or biological categories. This limitation precludes researchers from most effectively evaluating the association with other phenotypes that may be more clinically meaningful, such as quantitative variables or censored survival time variables. Projection onto the Orthogonal Space Testing (POST) is proposed as a general procedure that can robustly evaluate the association of a gene-set with several different types of phenotypic data (categorical, ordinal, continuous, or censored). For each gene-set, POST transforms the gene profiles into a set of eigenvectors and then uses statistical modeling to compute a set of z-statistics that measure the association of each eigenvector with the phenotype. The overall gene-set statistic is the sum of squared z-statistics weighted by the corresponding eigenvalues. Finally, bootstrapping is used to compute a p-value. POST may evaluate associations with or without adjustment for covariates. In simulation studies, it is shown that the performance of POST in evaluating the association with a categorical phenotype is similar to or exceeds that of existing methods. In evaluating the association of 875 biological processes with the time to relapse of pediatric acute myeloid leukemia, POST identified the well-known oncogenic WNT signaling pathway as its top hit. These results indicate that POST can be a very useful tool for evaluating the association of a gene-set with a variety of different phenotypes.

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, <i>Caenorhabditis elegans</i> UNC-51, and <i>Drosophila melanogaster</i> Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. <i>C. elegans</i> UNC-51 and <i>D. melanogaster</i> Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking <i>Ulk1</i> and <i>Ulk2</i> in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in <i>ulk1/2</i> double-knockout mice and central nervous system-specific (<i>Nes-Cre</i>) <i>Ulk1/2</i>-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., <i>Atg7</i> or <i>Rb1cc1</i> [RB1-inducible coiled-coil 1]). The brains of <i>Ulk1/2</i>-deficient mice did not show stem cell defects previously attributed to defective autophagy in <i>ambra1</i> (autophagy/Beclin 1 regulator 1)- and <i>Rb1cc1</i>-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.

Abstract Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.

The development of red blood cells involves distinct stages of maturation that result in the loss of intracellular organelles, including mitochondria, to form biconcave erythrocytes. FOXO3A is a transcription factor that is known to regulate the response to reactive oxygen-species (ROS) in differentiating red blood cells through upregulating the transcription of ROS scavenging enzymes. However, the impact of FOXO3A on the differentiation of human erythroid has not been well characterized. Recently, we found in human CD34+ cells that inactivation of FOXO3A impairs multiple aspects of terminal erythroid maturation, including the clearance of mitochondria. Our results suggest that FOXO3A suppresses the expression of genes involved in cell cycle progression and mitochondrial translation and upregulation of those involved in autophagy. One of the genes that FOXO3A upregulates is the autophagy-inducing kinase, ULK1. Knockdown of ULK1 in human cells impairs mitochondrial clearance, overexpression of ULK1 fails to rescue the defect in erythroid maturation observed in FOXO3A deficient cells. Together, this data suggests that FOXO3A regulates mitochondrial content during terminal stages of differentiation through autophagy dependent and independent mechanisms.

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, <i>Caenorhabditis elegans</i> UNC-51, and <i>Drosophila melanogaster</i> Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. <i>C. elegans</i> UNC-51 and <i>D. melanogaster</i> Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking <i>Ulk1</i> and <i>Ulk2</i> in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in <i>ulk1/2</i> double-knockout mice and central nervous system-specific (<i>Nes-Cre</i>) <i>Ulk1/2</i>-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., <i>Atg7</i> or <i>Rb1cc1</i> [RB1-inducible coiled-coil 1]). The brains of <i>Ulk1/2</i>-deficient mice did not show stem cell defects previously attributed to defective autophagy in <i>ambra1</i> (autophagy/Beclin 1 regulator 1)- and <i>Rb1cc1</i>-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.

Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific <i>rb1cc1-</i> and <i>atg14-</i>conditional knockout (cKO) mice by using <i>Ckm/Ckmm2-Cre</i> and compared their phenotypes to those of <i>ulk1 ulk2</i>-conditional double-knockout (cDKO) mice. <i>atg14</i>-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both <i>atg14</i>-cKO and <i>rb1cc1</i>-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin⁺ SQSTM1⁺ deposits, but only those of <i>rb1cc1</i>-cKO mice showed TARDBP/TDP-43⁺ pathology and other features of the inclusion body myopathy–like disease we previously described in <i>ulk1 ulk2</i>-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP⁺ pathology. <b>ABBREVIATIONS</b>:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&amp;E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100

To investigate some of the cellular consequences of HIV-1 Tat expression in human astrocytic cells. This study is based on evidence that cellular factors play a critical role in facilitating transcriptional activation by Tat through its interaction with the trans-acting-response (TAR) RNA element and upstream HIV-1 long terminal repeat (LTR) promoter binding site. STUDY DESIGN-METHODS: Using the previously established astrocytic cell line of human origin stably transfected with Tat cDNA, we analyzed the formation of a nucleoprotein complex consisting of three cellular proteins associated with TAR RNA using ultraviolet (UV) crosslinking and glutathione-S-transferase (GST) pull-down assays.UV crosslinking experiments reveal that the molecular masses of the proteins range from 50 to 62 kd. Transient transfection studies demonstrate that the presence of these proteins correlates with the ability of Tat to transactivate the HIV-1 LTR in the absence of the trinucleotide bulge, a region within TAR that has been shown to be important for Tat-TAR interaction. A combination of GST pull-down assays and RNA binding studies demonstrates that the 50-kd protein interacts with both Tat and TAR and is likely to be NF-kappa B p50.Taken together, these data suggest that in the absence of a functional Tat binding site such as TAR (which tethers the viral protein to the RNA), cellular protein NF-kappa B p50 may be able to bring Tat into the RNA binding complex Tat has been shown to activate expression of a variety of cellular genes that may not contain a binding site for Tat but do contain binding sites for NF-kappa B family members. The results presented in this study may be relevant for Tat-mediated transactivation of cellular as well as viral genes, both of which might contribute to the central nervous system damage associated with HIV-1 infection.

Abstract β-Thalassemia is a common, frequently debilitating, inherited anemia caused by HBB gene mutations that reduce or eliminate the expression of the β-globin subunit of adult hemoglobin (HbA, α2β2). Consequently, excess free α-globin forms toxic precipitates in red blood cells (RBCs) and their precursors, leading to ineffective erythropoiesis and hemolytic anemia. Previously, we showed that free α-globin is eliminated by protein quality-control pathways, including the ubiquitin-proteasome system and autophagy (Khandros et al., Blood 2012;119:5265). In β-thalassemic mice, disruption of the Unc-51-like autophagy activating kinase gene (Ulk1) increased α-globin precipitates and worsened the pathologies of β-thalassemia. Treatment of β-thalassemic mice with rapamycin to inhibit mTOR (an ULK1 inhibitor) reduced α-globin precipitates, lessened ineffective erythropoiesis, and increased the lifespan of circulating RBCs in an Ulk1-dependent fashion. To investigate the therapeutic potential of rapamycin in human β-thalassemia, we treated erythroid precursors generated by in vitro differentiation of patient-derived CD34+ hematopoietic stem and progenitor cells. Reverse-phase high-performance liquid chromatography (HPLC) analysis of hemoglobinized erythroblasts generated from transfusion-dependent (TD, n = 5) or non-transfusion-dependent (NTD, n = 5) β-thalassemia patients revealed α-chain excesses (α-chain/β-like [β + γ + δ] chain) of approximately 40% and 15%, respectively (compared to 7 normal donors; P &lt; 0.001). Rapamycin (10µM or 20µM) or the proteasome inhibitor MG132 (2.5µM) was added to day 13 cultures, which contained mid- to late-stage erythroblasts, and α-globin accumulation was determined by HPLC 2 days later. As expected, proteasome inhibition by MG132 raised free α-globin levels in thalassemic erythroblasts (P &lt; 0.01) and induced cell death (P &lt; 0.01). In contrast, rapamycin reduced free α-globin in a dose-dependent manner by 40% and 85% in TD (P &lt; 0.0001) and NTD β-thalassemia (P &lt; 0.001), respectively, but had no effect on erythroblasts derived from normal CD34+ cells (figure). We also observed decreases in the accumulation of autophagic markers, such as SQSTM1/p62 protein, by Western blotting. We observed no negative effects of rapamycin on the survival of patient-derived erythroblasts. Also of note, under our experimental conditions, rapamycin treatment of erythroblasts did not induce fetal hemoglobin production, as has been previously reported, thereby excluding this potential mechanism for reducing globin chain imbalances. Overall, rapamycin treatment significantly reduced the accumulation of free α-globin in TD β-thalassemia and almost fully corrected the imbalance in NTD β-thalassemia cells. Our findings identify a new drug-regulatable pathway for ameliorating β-thalassemia. Rapamycin is approved and well studied, and it has a generally manageable toxicity profile. Moreover, there are additional pharmacologic approaches to activating ULK via mTOR inhibition or other pathways. These approaches may lead to effective drug therapies for β-thalassemia, particularly NTD or intermittently TD forms of the disease. Disclosures Cappellini: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

Abstract Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, mutations in the β-globin gene cause a build-up of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality-control systems mitigate β-thalassemia pathophysiology by degrading toxic free α-globin. We show that loss of the Unc 51–like autophagy-activating kinase gene Ulk1 in β-thalassemic mice reduces autophagic clearance of α-globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy gene Atg5 has minimal effects. Systemic treatment with rapamycin to inhibit the ULK1 inhibitor mTORC1 reduces α-globin precipitates and lessens pathologies in β-thalassemic mice, but not in those lacking Ulk1 . Similarly, rapamycin reduces free α-globin accumulation in erythroblasts derived from β-thalassemic patient CD34 + hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating β-thalassemia. One Sentence Summary Rapamycin alleviates β-thalassemia by stimulating ULK1-dependent autophagy of toxic free α-globin.

Mammalian ULK1 (unc-51 like kinase 1) and ULK2, <i>Caenorhabditis elegans</i> UNC-51, and <i>Drosophila melanogaster</i> Atg1 are serine/threonine kinases that regulate flux through the autophagy pathway in response to various types of cellular stress. <i>C. elegans</i> UNC-51 and <i>D. melanogaster</i> Atg1 also promote axonal growth and defasciculation; disruption of these genes results in defective axon guidance in invertebrates. Although disrupting ULK1/2 function impairs normal neurite outgrowth in vitro, the role of ULK1 and ULK2 in the developing brain remains poorly characterized. Here, we show that ULK1 and ULK2 are required for proper projection of axons in the forebrain. Mice lacking <i>Ulk1</i> and <i>Ulk2</i> in their central nervous systems showed defects in axonal pathfinding and defasciculation affecting the corpus callosum, anterior commissure, corticothalamic axons and thalamocortical axons. These defects impaired the midline crossing of callosal axons and caused hypoplasia of the anterior commissure and disorganization of the somatosensory cortex. The axon guidance defects observed in <i>ulk1/2</i> double-knockout mice and central nervous system-specific (<i>Nes-Cre</i>) <i>Ulk1/2</i>-conditional double-knockout mice were not recapitulated in mice lacking other autophagy genes (i.e., <i>Atg7</i> or <i>Rb1cc1</i> [RB1-inducible coiled-coil 1]). The brains of <i>Ulk1/2</i>-deficient mice did not show stem cell defects previously attributed to defective autophagy in <i>ambra1</i> (autophagy/Beclin 1 regulator 1)- and <i>Rb1cc1</i>-deficient mice or accumulation of SQSTM1 (sequestosome 1)⁺ or ubiquitin⁺ deposits. Together, these data demonstrate that ULK1 and ULK2 regulate axon guidance during mammalian brain development via a noncanonical (i.e., autophagy-independent) pathway.

Abstract Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the CBF β/RUNX1 complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1lacZ/lacZ embryonic stem (ES) cells that have homozygous disruption of the RUNX1 gene, as well as Runx1+/lacZ mice with N-ethyl-N-nitrosurea (ENU). The heterozygous Runx1+/lacZ mice did not show increased incidence of any malignancy. On the other hand, we observed an increased incidence of precursor T-lymphoblastic lymphoma in Runx1lacZ/lacZ compared to wild-type chimeras, and confirmed that the tumors were of ES cell origin. It was determined by PCR that Runx1lacZ/lacZ ES cells contributed to the T cell progenitor population in the chimeras prior to leukemia development, which may explain the tissue-specificity of the malignancy we observed. Our results suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.

Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific <i>rb1cc1-</i> and <i>atg14-</i>conditional knockout (cKO) mice by using <i>Ckm/Ckmm2-Cre</i> and compared their phenotypes to those of <i>ulk1 ulk2</i>-conditional double-knockout (cDKO) mice. <i>atg14</i>-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both <i>atg14</i>-cKO and <i>rb1cc1</i>-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin⁺ SQSTM1⁺ deposits, but only those of <i>rb1cc1</i>-cKO mice showed TARDBP/TDP-43⁺ pathology and other features of the inclusion body myopathy–like disease we previously described in <i>ulk1 ulk2</i>-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP⁺ pathology. <b>ABBREVIATIONS</b>:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&amp;E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100

Defects in macroautophagy/autophagy are implicated in the pathogenesis of neuromuscular and heart diseases. To precisely define the roles of autophagy-related genes in skeletal and cardiac muscles, we generated muscle-specific <i>rb1cc1-</i> and <i>atg14-</i>conditional knockout (cKO) mice by using <i>Ckm/Ckmm2-Cre</i> and compared their phenotypes to those of <i>ulk1 ulk2</i>-conditional double-knockout (cDKO) mice. <i>atg14</i>-cKO mice developed hypertrophic cardiomyopathy, which was associated with abnormal accumulation of autophagic cargoes in the heart and early mortality. Skeletal muscles of both <i>atg14</i>-cKO and <i>rb1cc1</i>-cKO mice showed features of autophagic vacuolar myopathy with ubiquitin⁺ SQSTM1⁺ deposits, but only those of <i>rb1cc1</i>-cKO mice showed TARDBP/TDP-43⁺ pathology and other features of the inclusion body myopathy–like disease we previously described in <i>ulk1 ulk2</i>-cDKO mice. Herein, we highlight tissue-specific differences between skeletal and cardiac muscles in their reliance on core autophagy proteins and unique roles for ULK1-ULK2 and RB1CC1 among these proteins in the development of TARDBP⁺ pathology. <b>ABBREVIATIONS</b>:AVM: autophagic vacuolar myopathy; cDKO: conditional double knockout; cKO: conditional knockout; H&amp;E: hematoxylin and eosin; IBM: inclusion body myopathy; mtDNA: mitochondrial DNA; PFA: paraformaldehyde; RNP: ribonucleoprotein; TBST: Tris-buffered saline with 0.2% Triton X-100