André Richters

Cancer genomes contain large numbers of somatic mutations but few of these mutations drive tumor development. Current approaches either identify driver genes on the basis of mutational recurrence or approximate the functional consequences of nonsynonymous mutations by using bioinformatic scores. Passenger mutations are enriched in characteristic nucleotide contexts, whereas driver mutations occur in functional positions, which are not necessarily surrounded by a particular nucleotide context. We observed that mutations in contexts that deviate from the characteristic contexts around passenger mutations provide a signal in favor of driver genes. We therefore developed a method that combines this feature with the signals traditionally used for driver-gene identification. We applied our method to whole-exome sequencing data from 11,873 tumor–normal pairs and identified 460 driver genes that clustered into 21 cancer-related pathways. Our study provides a resource of driver genes across 28 tumor types with additional driver genes identified according to mutations in unusual nucleotide contexts. MutPanning is a new method to detect cancer driver genes that identifies genes with an excess of mutations in unusual nucleotide contexts. Applying this to whole-exome sequencing data from 11,873 tumor–normal pairs identifies 460 driver genes.

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.

KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.

Receptor tyrosine kinases represent one of the prime targets in cancer therapy, as the dysregulation of these elementary transducers of extracellular signals, like the epidermal growth factor receptor (EGFR), contributes to the onset of cancer, such as non-small cell lung cancer (NSCLC). Strong efforts were directed to the development of irreversible inhibitors and led to compound CO-1686, which takes advantage of increased residence time at EGFR by alkylating Cys797 and thereby preventing toxic effects. Here, we present a structure-based approach, rationalized by subsequent computational analysis of conformational ligand ensembles in solution, to design novel and irreversible EGFR inhibitors based on a screening hit that was identified in a phenotype screen of 80 NSCLC cell lines against approximately 1500 compounds. Using protein X-ray crystallography, we deciphered the binding mode in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M, which constituted the basis for further rational design approaches. Chemical synthesis led to further compound collections that revealed increased biochemical potency and, in part, selectivity toward mutated (L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and kinetic studies were performed to substantiate our initial findings. Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed the alkylation of Cys797.

The 8p12 locus (containing the FGFR1 tyrosine kinase gene) is frequently amplified in squamous cell lung cancer. However, it is currently unknown which of the 8p12-amplified tumors are also sensitive to fibroblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifications, the 8p12 region included multiple centers of amplification, suggesting marked genomic heterogeneity. FGFR1-amplified tumor cells were dependent on FGFR ligands in vitro and in vivo. Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of FGFR1-amplified tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with FGFR1-amplified and MYC-overexpressing tumors may benefit from FGFR inhibitor therapy. Thus, both cell-autonomous and non-cell-autonomous mechanisms of transformation modulate FGFR dependency in FGFR1-amplified lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors.Amplification of FGFR1 is one of the most frequent candidate targets in lung cancer. Here, we show that multiple factors affect the tumorigenic potential of FGFR1, thus providing clinical hypotheses for refinement of patient selection.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. We provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors, such as AD80 or ponatinib, that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells, we identify the CCDC6-RETI788N mutation and drug-induced mitogen-activated protein kinase pathway reactivation as possible mechanisms by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.

Abstract The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by introducing variations to a highly conserved theme. The activity of each kinase domain is controlled by layers of regulatory mechanisms involving different combinations of post‐translational modifications, intramolecular contacts, and intermolecular interactions. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has encouraged extensive investigations of their biological function and three‐dimensional structures, yielding a more detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes. In the present review, we discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of specific inhibitors that lock protein kinases in inactive conformations. In addition, we highlight recent developments to monitor ligand‐induced structural changes in protein kinases and for screening and identifying inhibitors that stabilize enzymatically incompetent kinase conformations.

c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells.

Abstract Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small‐molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy‐induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4-hydroxy-2-pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone-inspired 4-hydroxy-2-pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4-hydroxy-2-pyridone is a selective ATP-competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases.

Protein kinases constitute an attractive family of enzyme targets with high relevance to cell and disease biology. Small molecule inhibitors are powerful tools to dissect and elucidate the function of kinases in chemical biology research and to serve as potential starting points for drug discovery. However, the discovery and development of novel inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding fragments that are synthetically feasible and can be elaborated to small molecule libraries. Starting from commercially available compounds, core fragments were extracted, filtered for pharmacophoric properties compatible with hinge-region binding, and docked into a panel of protein kinases. Fragments with a high consensus score were subsequently short-listed for synthesis. Application of this strategy led to a number of core fragments with no previously reported activity against kinases. Small libraries around the core fragments were synthesized, and representative compounds were tested against a large panel of protein kinases and subjected to co-crystallization experiments. Each of the tested compounds was active against at least one kinase, but not all kinases in the panel were inhibited. A number of compounds showed high ligand efficiencies for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1, SGK1, TAK1, and GCK for which only few inhibitors are reported in the literature.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

The clinical success of covalent kinase inhibitors in the treatment of EGFR-dependent non-small cell lung cancer (NSCLC) has rejuvenated the appreciation of reactive small molecules. Acquired drug resistance against first-line EGFR inhibitors remains the major bottleneck in NSCLC and is currently addressed by the application of fine-tuned covalent drugs. Here we report the design, synthesis and biochemical evaluation of a novel class of EGFR inhibitors with a covalent yet reversible warhead. A series of WZ4002 analogs, derived from anilinopyrimidine and 3-substituted-2-cyanoacrylamide scaffolds, exhibit strong and selective inhibitory activity against clinically relevant EGFRL858R and EGFRL858R/T790M.

Discoidin domain-containing receptors (DDRs) exhibit a unique mechanism of action among the receptor tyrosine kinases (RTKs) because their catalytic activity is induced by extracellular collagen binding. Moreover, they are essential components in the assimilation of extracellular signals. Recently, DDRs were reported to be significantly linked to tumor progression in breast cancer by facilitating the processes of invasion, migration, and metastasis. Here, we report the successful development of a fluorescence-based, direct binding assay for the detection of type II and III DFG-out binders for DDR2. Using sequence alignments and homology modeling, we designed a DDR2 construct appropriate for fluorescent labeling. Successful assay development was validated by sensitive detection of a reference DFG-out binder. Subsequent downscaling led to convenient application to high-throughput screening formats. Screening of a representative compound library identified high-affinity DDR2 ligands validated by orthogonal activity-based assays, and a subset of identified compounds was further investigated with respect to DDR1 inhibition.

PRMT5 catalyzes the mono- and symmetric dimethylation of the arginine N-guanidine group of a wide variety of target proteins including histones, transcriptional elongation factors, kinases and tumor suppressors by utilizing the essential co-factor S-adenosylmethionine as methyl source. PRMT5 overexpression has been linked to the progression of various diseases, including cancer, and is oftentimes associated with a poor prognosis. Therefore, PRMT5 is promoted as a valuable target for drug discovery approaches and was a subject matter in recent endeavors aiming for the development of specific PRMT5 inhibitors. This review will embrace the significance of PRMT5 as therapeutic target with respect to its molecular interdependencies in disease states as well as its implication in drug development approaches.

Histone acetyltransferases (HATs) are epigenetic drivers that catalyze the acetyl transfer from acetyl-CoA to lysines of both histone and non-histone substrates and thereby induce transcription either by chromatin remodeling or direct transcription factor activation. Histone deacetylases (HDACs) conduct the reverse reaction to counter HAT activity. Physiological processes such as cell cycle progression or apoptosis require a thoroughly balanced equilibrium of the interplay between acetylation and deacetylation processes to maintain or, if required, alter the global acetylome status. Aberrant HAT activity has recently been demonstrated to play a crucial role in the progression of various diseases such as prostate, lung, and colon cancers as well as glioblastomas and neurodegenerative diseases. Recent investigations have aimed for the identification of HAT modulators to further decipher the complexity of acetyl transferase related signaling cascades and discover potential leads for drug design approaches. HDACs have been extensively characterized and targeted by small molecules, including four FDA-approved HDAC inhibitors; in contrast, HATs have not been active targets for therapeutic development. This review will summarize the status of HAT associated diseases and the arsenal of currently known and available HAT inhibitors with respect to their discovery, further improvements, and current applications.

The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast.

Five series of metabolically stable disubstituted dibenzo[b,e]oxepin-11(6H)-ones were synthesized and tested in a p38α enzyme assay for their inhibition of tumor necrosis factor-α (TNF-α) release in human whole blood. Compared to the monosubstituted dibenzo[b,e]oxepin-11(6H)-one derivatives, it has been shown that the additional introduction of hydrophilic residues at position 9 leads to a substantial improvement of the inhibitory potency and metabolic stability. Using protein X-ray crystallography, the binding mode of the disubstituted dibenzoxepinones and the induction of a glyince flip in the hinge region were confirmed. The most potent compound of this series, 32e, shows an outstanding biological activity on isolated p38α, with an IC50 value of 1.6 nM, extraordinary selectivity (by a factor >1000, Kinase WholePanelProfiler), and low ATP competitiveness. The ability to inhibit the release of TNF-α from human whole blood was optimized down to an IC50 value of 125 nM. With the promising dibenzoxepinone inhibitor 3i, a pharmacokinetic study in mice was conducted.

The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules.

Abstract Proteinkinasen repräsentieren wichtige Knotenpunkte intrazellulärer Signalwege und sind somit an vielen physiologischen und pathologischen Prozessen beteiligt. Konformations‐abhängige Eigenschaften dienen hierbei der Feinregulation ihrer enzymatischen Aktivität und Katalyse‐unabhängigen Funktionen. Die Stabilisierung definierter Konformationen ermöglicht detaillierte Analysen solcher Konformations‐abhängiger Funktionen. Hier beschreiben wir das strukturbasierte Design, die Synthese und die Charakterisierung Pleckstrin‐Homologie(PH)‐Domänen‐abhängiger Akt‐Inhibitoren, die einen neuartigen kovalent‐allosterischen Bindungsmodus aufweisen. Durch die kovalente Modifizierung bestimmter Cysteine stabilisieren diese Moleküle die inaktive Kinasekonformation irreversibel. Ihre beträchtliche Inhibitorwirkung und Selektivität bezüglich verwandter Proteinkinasen machen sie zu einem neuartigen Hilfsmittel für die Erforschung chemisch‐biologischer und medizinischer Fragestellungen.

Abstract Progressive loss and impaired restoration of neuronal activity are hallmarks of neurological diseases, and new small molecules with neurotrophic activity are in high demand. The militarinone alkaloids and structurally simplified analogues with 4‐hydroxy‐2‐pyridone core structure induce pronounced neurite outgrowth, but their protein target has not been identified. Reported herein is the synthesis of a militarinone‐inspired 4‐hydroxy‐2‐pyridone collection, its investigation for enhancement of neurite outgrowth, and the discovery of the stress pathway kinase MAP4K4 as a target of the discovered neuritogenic pyridones. The most potent 4‐hydroxy‐2‐pyridone is a selective ATP‐competitive inhibitor of MAP4K4 but not of the other stress pathway related kinases, as proven by biochemical analysis and by a crystal structure of the inhibitor in complex with MAP4K4. The findings support the notion that MAP4K4 may be a new target for the treatment of neurodegenerative diseases.

Abstract Introduction: The Cyclin-Dependent Kinases (CDKs) with their cyclin binding partners, are associated with cell cycle progression and transcriptional regulation. Targeting CDKs is a key oncology therapeutic strategy with CDK4/6 inhibitors demonstrating significant clinical benefit in HR+/HER2− metastatic breast cancer, the most prevalent subtype. However, 30% of patients develop acquired resistance in the clinic with cyclin E1 (CCNE1) amplification/overexpression and CDK2 activation implicated as a major resistance mechanism to CDK4/6i breast cancer therapy. Additionally, CCNE1/CDK2 activation has also been associated with poor prognosis in ovarian and endometrial cancers. Selective targeting of CDK2 using small molecule inhibitor-based approaches have recently advanced into the clinic. Targeted protein degradation of CDK2 provides an alternative strategy that has the potential to eliminate the activity of the CCNE1/CDK2 complex, as well as CDK2 complexed with other cyclins, including cyclin A, and provide improved clinical benefit. Results: Discovery efforts at Plexium have identified CDK2 bivalent degraders that consist of a CDK2-binding moiety, a linker and a high affinity cereblon-binding ligand. Cereblon binding potency was found to correlate with potent and deep degradation of CDK2. Degradation was blocked in the presence of a proteasome inhibitor as well as in a cereblon knock-out cell line, confirming that CDK2 degradation is mediated by the ubiquitin proteasome system and through engaging cereblon. Proteome-wide analysis demonstrated that CDK2 was selectively depleted without significantly modulating other CDKs or known cereblon neo-substrates. Selective degradation was achieved despite lack of selectivity for binding/inhibition of other CDKs and was confirmed to be dependent on the formation of a CRBN-CDK2 ternary complex. Dose dependent CDK2 degradation resulted in dose dependent inhibition of Rb phosphorylation, cell cycle arrest, senescence-associated phenotypes and antiproliferative activity in CCNE1 amplified cancer cell lines. The in vitro data was used to evaluate the PK/PD drug exposure-response relationship in vivo and deep CDK2 degradation was demonstrated both in vitro and in vivo. Conclusions: These data provide validation for CDK2 degradation as a therapeutic approach. Potent and selective CDK2 bivalent degraders were exploited as tools for studying the sensitivity of CCNE1 amplified tumor models to CDK2 degradation and dependence on E3 ligase. This proof-of concept study supports Plexium’s current approach of discovering novel CDK2 molecular glue degraders for the treatment of CDK4/6 inhibitor-naïve and -resistant HR+/HER2− breast cancer, and CCNE1 amplified ovarian and endometrial cancers. Citation Format: Adina Gerson-Gurwitz, Pengyu Yang, Sarah Fish, Gabrielle Blanco, Hongfeng Gao, Kyohei Hayashi, Mike Hocker, Aleks Jamborcic, Andre Richters, Mary E. Spalding, Julia Toth, Duc Tran, Linette Yang, Shu You, Andrew Burritt, Alex Campos, Gregory Parker, Kevin Freeman-Cook, Peggy A. Thompson, Simon Bailey. Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3309.

Protein kinases are key enzymes in the complex regulation of cellular processes in almost all living organisms. For this reason, protein kinases represent attractive targets to stop the growth of eukaryotic pathogens such as protozoa and fungi. However, using kinase inhibitors to fight against these organisms bears several challenges since most of them are unselective and will also affect crucial host kinases. Here we present the X-ray structure of glycogen synthase kinase 3 from the fungal plant pathogen Ustilago maydis (UmGSK3) and its inhibition by type-II kinase inhibitors. Despite the high sequence homology between the human and the fungal variant of this vital kinase, we found substantial differences in the conformational plasticity of their active sites. Compounds that induced such conformational changes could be used to selectively inhibit the fungal kinase. This study serves as an example of how species-specific selectivity of inhibitors can be achieved by identifying and addressing the inactive state of a protein kinase. In addition to this, our study gives interesting insights into the molecular plasticity of UmGSK3 by revealing a previously unknown inactive conformation of this important kinase family.

Mutations in the catalytic domain at the gatekeeper position represent the most prominent drug-resistant variants of kinases and significantly impair the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular and atomic levels will aid in the design and development of inhibitors that have the potential to overcome these resistance mutations. Herein, by introducing adaptive elements into the inhibitor core structure, we undertake the structure-based development of type II hybrid inhibitors to overcome gatekeeper drug-resistant mutations in cSrc-T338M, as well as clinically relevant tyrosine kinase KIT-T670I and Abl-T315I variants, as essential targets in gastrointestinal stromal tumors (GISTs) and chronic myelogenous leukemia (CML). Using protein X-ray crystallography, we confirm the anticipated binding mode in cSrc, which proved to be essential for overcoming the respective resistances. More importantly, the novel compounds effectively inhibit clinically relevant gatekeeper mutants of KIT and Abl in biochemical and cellular studies.

Abstract Autophagy is a critical regulator of cellular homeostasis and metabolism. Interference with this process is considered a new approach for the treatment of disease, in particular cancer and neurological disorders. Therefore, novel small‐molecule autophagy modulators are in high demand. We describe the discovery of autophinib, a potent autophagy inhibitor with a novel chemotype. Autophinib was identified by means of a phenotypic assay monitoring the formation of autophagy‐induced puncta, indicating accumulation of the lipidated cytosolic protein LC3 on the autophagosomal membrane. Target identification and validation revealed that autophinib inhibits autophagy induced by starvation or rapamycin by targeting the lipid kinase VPS34.

<p>PDF file 136K,Methods: Additional information on experimental techniques and data analysis. Legends: describing supplementary figures S1-S14</p>

<p>PDF file 34156K, Table of genes from 16 recurrent amplified genomic regions (Figure S1), amplicon focality on TCGA copy number aberrations (Figure S2), hierarchical clustering of CLCGP copy number data (Figure S3), centrality frequencies of common cancer genes (Figure S4), genotypic cell line annotation (Figure S5), receptor signaling activation of H520 and HCC15 lines (Figure S6), experimental draft for ligand dependency (Figure S7), whole transcriptome analysis FGFR1 splicing (Figure S8), validation of gene expression by qPCR (Figure S9), tumor formation of FGFR1α cells in nude mice (Figure S10), quantification of MYC stains in murine tumor samples (Figure S11), apoptosis is accompanied by cytochrome c release and breakdown of mitochondrial potential (Figure S12), IHC scores of FGFR phosphorylation and MYC expression (Figure S13), High MYC expression identifies a second responder to FGFR inhibitory therapy (Figure S14)</p>

Abstract Cyclin-dependent kinase 9 (CDK9) has long been considered an attractive therapeutic target for modulating transcription in cancers of high unmet clinical need. CDK9 coordinates signaling events that regulate RNA polymerase II (Pol II) pause-release state and is considered an important co-factor for oncogenic transcription factors that drive transcription in an addictive manner. CDK9 modulation offers an approach for attenuating transcriptional dysregulation driven by amplified or overexpressed transcription factors, such as c-MYC. However, targeting CDK9 in the clinic has proven very challenging. This stems from the often highly intolerable cytotoxic effects likely due to off-target effects. Targeted protein degradation offers a novel approach for engineering enhanced selectivity and mitigating other liabilities of the conventional inhibition-driven approach that have been applied to date. Here, we present the design and characterization of a focused library of highly selective CDK9 degrader molecules with rapid kinetics and potency (half-degrading concentration of <1nM). We carry out transcriptional and phosphoproteomics profiling to comprehensively characterize the downstream effects and cellular adaptations resulting from CDK9 degradation. We gain new insights into adaptive responses to CDK9 modulation and the biological contexts most responsive for therapeutic application. Citation Format: Mohammed A. Toure, Keisuke Motoyama, André Richters, Angela N. Koehler. Design and characterization of highly potent and selective CDK9 heterobifunctional degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3409.

<p>PDF file 34156K, Table of genes from 16 recurrent amplified genomic regions (Figure S1), amplicon focality on TCGA copy number aberrations (Figure S2), hierarchical clustering of CLCGP copy number data (Figure S3), centrality frequencies of common cancer genes (Figure S4), genotypic cell line annotation (Figure S5), receptor signaling activation of H520 and HCC15 lines (Figure S6), experimental draft for ligand dependency (Figure S7), whole transcriptome analysis FGFR1 splicing (Figure S8), validation of gene expression by qPCR (Figure S9), tumor formation of FGFR1α cells in nude mice (Figure S10), quantification of MYC stains in murine tumor samples (Figure S11), apoptosis is accompanied by cytochrome c release and breakdown of mitochondrial potential (Figure S12), IHC scores of FGFR phosphorylation and MYC expression (Figure S13), High MYC expression identifies a second responder to FGFR inhibitory therapy (Figure S14)</p>

<p>PDF file 136K,Methods: Additional information on experimental techniques and data analysis. Legends: describing supplementary figures S1-S14</p>

<div>Abstract<p>The 8p12 locus (containing the <i>FGFR1</i> tyrosine kinase gene) is frequently amplified in squamous cell lung cancer. However, it is currently unknown which of the 8p12-amplified tumors are also sensitive to fibroblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifications, the 8p12 region included multiple centers of amplification, suggesting marked genomic heterogeneity. <i>FGFR1</i>-amplified tumor cells were dependent on FGFR ligands <i>in vitro</i> and <i>in vivo</i>. Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of <i>FGFR1</i>-amplified tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with <i>FGFR1-</i>amplified and MYC-overexpressing tumors may benefit from FGFR inhibitor therapy. Thus, both cell-autonomous and non–cell-autonomous mechanisms of transformation modulate FGFR dependency in <i>FGFR1</i>-amplified lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors.</p><p><b>Significance:</b> Amplification of <i>FGFR1</i> is one of the most frequent candidate targets in lung cancer. Here, we show that multiple factors affect the tumorigenic potential of <i>FGFR1</i>, thus providing clinical hypotheses for refinement of patient selection. <i>Cancer Discov; 4(2); 246–57. ©2013 AACR</i>.</p><p>See related commentary by Lockwood and Politi, p. 152</p><p>This article is highlighted in the In This Issue feature, p. 131</p></div>

<div>Abstract<p>The 8p12 locus (containing the <i>FGFR1</i> tyrosine kinase gene) is frequently amplified in squamous cell lung cancer. However, it is currently unknown which of the 8p12-amplified tumors are also sensitive to fibroblast growth factor receptor (FGFR) inhibition. We found that, in contrast with other recurrent amplifications, the 8p12 region included multiple centers of amplification, suggesting marked genomic heterogeneity. <i>FGFR1</i>-amplified tumor cells were dependent on FGFR ligands <i>in vitro</i> and <i>in vivo</i>. Furthermore, ectopic expression of FGFR1 was oncogenic, which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of <i>FGFR1</i>-amplified tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting that patients with <i>FGFR1-</i>amplified and MYC-overexpressing tumors may benefit from FGFR inhibitor therapy. Thus, both cell-autonomous and non–cell-autonomous mechanisms of transformation modulate FGFR dependency in <i>FGFR1</i>-amplified lung cancer, which may have implications for patient selection for treatment with FGFR inhibitors.</p><p><b>Significance:</b> Amplification of <i>FGFR1</i> is one of the most frequent candidate targets in lung cancer. Here, we show that multiple factors affect the tumorigenic potential of <i>FGFR1</i>, thus providing clinical hypotheses for refinement of patient selection. <i>Cancer Discov; 4(2); 246–57. ©2013 AACR</i>.</p><p>See related commentary by Lockwood and Politi, p. 152</p><p>This article is highlighted in the In This Issue feature, p. 131</p></div>

The development of adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) has been studied in rat liver, in the cytosol compartment and in the mitochondria, from 8 days before birth to the adult age. The activity of both compartments increased about 10-fold over the period investigated. It could be demonstrated that, despite the lower activity of adenylate kinase in the fetal liver, the enzyme establishes near equilibrium under anaerobic conditions.

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

(Cell 162, 146–159; July 2, 2015) Our paper presented a new algorithm, named PreCISE, designed to identify synergistic drug interactions that are effective at killing cancer cells harboring specific driver mutations. Using this platform and a cell-line-based screen, we identified a synergistic drug interaction between Chk1- and MK2 inhibitors in KRAS- or BRAF-driven cells, and that combination of therapy focused on these two kinase inhibitors is effective at inducing cell death of KRAS- and BRAF mutant tumors in vivo. While reviewing the paper after publication, we noticed that we had included two erroneous duplications of western blot loading control bands in the final version of Figure 2E. This figure shows that simultaneous inhibition of Chk1 and MK2 induces genotoxic stress and apoptosis in several KRAS-driven cancer cell lines and respective controls. The incorrect loading controls are presented for the HSP27 blot for the H1703 cell line, as well as for the CDC25B blot for the H1437 cell line. The errors occurred when we were copying each blot to construct the final figure. We recovered the original autoradiographs of these experiments and now provide a new version of the figure containing the correct loading controls. The correct data supports the original interpretation of the experiment, and the conclusions of the paper remain unchanged. In addition, we observed a typo in Figure S3A, showing the distribution of all cell lines used in the initial screen of our paper. In the pie chart, the slice of the pie in purple representing “lung squamous” cell lines was incorrectly labeled with n = 18. The correct value is n = 3, as depicted in the figure legend and in the main text. Both figures are now corrected online. We regret not being able to identify these errors before and sincerely apologize for any inconvenience they may have caused. A Synergistic Interaction between Chk1- and MK2 Inhibitors in KRAS-Mutant CancerDietlein et al.CellJuly 02, 2015In BriefPreCISE, a new platform that reliably captures synergic drug interactions from large-scale cell-line-based screens, shows that simultaneous inhibition of the cell-cycle checkpoint kinases Chk1 and MK2 effectively eradicates KRAS-mutant cancer cells directly isolated from patients and in distinct Kras-driven murine tumor models. Full-Text PDF Open Archive

Many promising therapeutic protein targets were previously considered “undruggable” due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging. In this chapter we present a protocol for leveraging in vitro translation strategies to enable array-based screening of short-lived proteins against large small-molecule libraries for ligand discovery.

Many cancer genomes contain large numbers of somatic mutations, but few of these mutations drive tumor development. Current approaches to identify cancer driver genes are largely based on mutational recurrence, i.e. they search for genes with an increased number of nonsynonymous mutations relative to the local background mutation rate. Multiple studies have noted that the sensitivity of recurrence-based methods is limited in tumors with high background mutation rates, because passenger mutations dilute their statistical power. Here, we observe that passenger mutations tend to occur in characteristic nucleotide sequence contexts, while driver mutations follow a different distribution pattern determined by the location of functionally relevant genomic positions along the protein-coding sequence. To discover new cancer genes, we searched for genes with an excess of mutations in unusual nucleotide contexts that deviate from the characteristic context around passenger mutations. By applying this statistical framework to whole-exome sequencing data from 12,004 tumors, we discovered a long tail of novel candidate cancer genes with mutation frequencies as low as 1% and functional supporting evidence. Our results show that considering both the number and the nucleotide context around mutations helps identify novel cancer driver genes, particularly in tumors with high background mutation rates.

Substituted anthranilic acid and piperazines were used as building blocks to prepare two libraries of compounds, with the aim being that they would exhibit biochemical activity as small molecule kinase inhibitors. The synthesized anthranilamide- piperazine compounds were subsequently tested against a panel of kinases including EGFR, Abl, Akt and Aurora B.

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. Here, we provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors such as AD80 or ponatinib that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells we identify the CCDC6-RETI788N mutation and drug-induced MAPK pathway reactivation as possible mechanisms, by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors.

Abstract Castration resistant prostate cancers (CRPCs) lose sensitivity to hormone therapy, but remain dependent on oncogenic transcription programs driven by the androgen receptor (AR) and other oncogenic transcription factors such as MYC. Using small molecule microarrays (SMMs), we screened HEK293 cellular lysates for compounds binding to exogenously expressed ARv7, a mutant splice form of AR that drives castration resistance. Although transcription factors like ARv7 and MYC are considered classically undruggable, SMMs are able to identify small molecule interactors of druggable co-factors and other proteins in complex with the target protein – in this case ARv7. SMM hits were triaged for the ability to selectively inhibit an AR dependent transcriptional reporter, and also for their ability to reduce proliferation in AR dependent tumor cells. From this screen, we identified KI-ARv3, a potent and selective inhibitor of CDK9. CDK9 is a cyclin-dependent kinase (CDK) that functions primarily as a general co-factor in RNA Polymerase II (RNA Pol II) transcription elongation. CDK9 is a well-characterized and important cofactor for AR, MYC, and other oncogenic transcription factors. In prostate cancer, CDK9 has been shown to modulate and be required for AR-specific gene expression. More broadly, transcriptional CDK inhibitors including those selective for CDK9 have shown strong potential as therapeutic agents owing to their ability to selectively downregulate oncogenic transcription programs and target tumors addicted to transcription factors such as AR or MYC. However, as CDK9 also plays a global role in transcription, it is unclear whether there exists a sufficient therapeutic index for clinical benefit. Prior clinical investigation of transcriptional CDK inhibitors has also been confounded by off-target interactions with other kinases and especially other CDKs that also play important roles in transcription and the cell cycle. We found that KI-ARv3 demonstrated excellent selectivity for CDK9 versus other CDKs and kinases, and further optimization of KI-ARv3 resulted in KB-00130742, an oral bioavailable CDK9 inhibitor with a biochemical IC50 of 15nM against CDK9 and greater than 50-fold selectivity for all profiled CDKs and greater than 100-fold selectivity against cell cycle CDKs. Both KI-ARv3 and KB-00130742 exhibited potent anti-tumor activity in CRPC models, as well as other models known to be dependent on MYC-driven transcription. In 22Rv1 CRPC cells, KB-00130742 rapidly downregulated nascent transcription, and preferentially depleted short half-life transcripts and AR driven oncogenic programs. In vivo, oral administration of KB-00130742 was well-tolerated and significantly reduced tumor growth in models of CRPC and leukemia. Overall these data support CDK9 inhibition using KB-00130742 as a therapeutic strategy to target AR dependence in CRPC and oncogenic transcription in other tumor types. Citation Format: André Richters, David Freeman, Christina Lee, Florian Kabinger, Shelby Doyle, Becky Leifer, Peter Mikochik, Sajjeev Jagannathan, Jost Vrabic Koren, Kristen Karlin, Calla M. Olson, Christopher Wilfong, Charles Y. Lin, Doug Saffran, Joseph Vacca, Norbert Bischofberger, Marius Pop, Angela N. Koehler. Targeting oncogenic transcription in prostate cancer with a novel, oral bioavailable, and ultra-selective CDK9 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1771.