Synthetic Lethal Vulnerabilities Research Articles

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

Abstract B027: The MAT2A inhibitor IDE397: a novel combination backbone for urothelial cancer subjects with MTAP deficiency

Abstract Methylthioadenosine phosphorylase (MTAP) loss, due to focal homozygous deletions in chromosome 9 (9p21.3), occurs in over 25% of urothelial carcinoma and is an adverse prognostic factor for overall survival. Furthermore, MTAP-deleted tumors are associated with a “cold” tumor immune microenvironment and are resistant to immune checkpoint inhibitor therapy. Loss of MTAP confers a dependency on methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of the primary methyl donor for all cellular methyltransferase reactions, S-adenosyl methionine (SAM). IDE397, a potent small molecule inhibitor of MAT2A, was developed to selectively exploit this synthetic lethal vulnerability in MTAP −/− tumors. The accumulation of the metabolite MTA in MTAP −/− tumors combined with a reduction of SAM by MAT2A inhibition results in selective inhibition of PRMT5, a methyltransferase that governs spliceosome fidelity. This triggers pre-mRNA splicing defects that may induce RNA polymerase stalling and can cause R-loop accumulation. Furthermore, due to essential roles in 1-carbon metabolism, MAT2A inhibition together with MTAP loss leads to depletion of metabolites required for both de novo and salvage nucleotide biosynthesis, thus preventing effective management of DNA replication and DNA damage repair. Notably, R-loops must be resolved by topoisomerase activity (TOP1) to prevent replication conflict and mitotic catastrophe. These combinatorial mechanistic relationships underpin a hypothesis that dual inhibition of MAT2A and TOP1 will fully capitalize on the synthetic lethal vulnerabilities associated with MTAP loss in urothelial cancers (replication stress, genomic instability). Consistent with this, IDE397 in combination with TOP1 inhibitors demonstrated synergistic combination benefit in MTAP −/− bladder cancer cell lines in vitro and in cell-line derived xenograft (CDX) models in vivo. IDE397 exposure was associated with heavily disordered methionine metabolism and accumulation of DNA damage response intermediates. In the ongoing Phase 1 dose escalation/expansion study of IDE397 in MTAP −/− solid tumors (NCT04794699), measurable tumor shrinkage has been observed in urothelial cancers (including a CR per RECIST 1.1) along with molecular responses on serial ctDNA measurements. Supported by our preclinical data, and the clinical observation that MTAP −/− urothelial cancers may be more sensitive to sacituzumab govitecan (SG); a TROP-2-targeting TOP1 ADC (UNITE study; JCO.2023.41.16_suppl.4572), a combination of SG+IDE397 could represent a novel early-line therapy for these patients with high unmet need. We plan to evaluate the combination of SG+IDE397 in MTAP-/- urothelial cancers in the next stage of the ongoing IDE397 trial (NCT04794699). Citation Format: Claire L. Neilan, Marcus M. Fischer, Damien Garbett, Arjun A. Rao, Mili Mandal, Michael A. White, Jasgit C. Sachdev. The MAT2A inhibitor IDE397: a novel combination backbone for urothelial cancer subjects with MTAP deficiency [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B027.

Abstract 1792: Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer

Abstract Reprogramming of metabolic processes by tumor cells is essential to cope with their increased proliferative activity, and with the challenges of the unique microenvironment they inhabit. These metabolic alterations are attractive targets to design novel therapeutic approaches.The most aggressive subtype of thyroid cancer, Anaplastic Thyroid Cancer (ATC), is often unresectable at presentation, highly resistant to therapy and usually lethal. We have recently discovered that ATCs overexpress several genes encoding enzymes involved in one-carbon metabolism (1C-Met), which utilizes serine and dietary folates to produce glycine and tetrahydrofolate-bound one-carbon units, required for nucleotide biosynthesis and for NADPH and glutathione (GSH) production. We have shown that i) the increased activity of the 1C-Met pathway supports the high purine demand of ATC; ii) inhibition of 1C-Met impairs tumor growth in vitro and in vivo, leading to growth arrest; iii) inhibition of 1C-Met renders thyroid cancer cells glycine-auxotroph.We now demonstrate that the profound addiction of ATC cells to 1C-Met creates a series of targetable vulnerabilities that can be harnessed to induce massive ATC cell death.Our new data demonstrate that:1- The purine depletion consequent impaired 1C-Met induces replicative stress and triggers the DNA Damage Response (DDR). Activation of DDR, coupled with the absence of functional TP53 in the vast majority of ATCs, leads to synthetic lethality between inhibition of 1C-Met and G2/M checkpoint kinases.2- Inhibition of 1C-Met in ATC cells generates an absolute dependence on extracellular glycine uptake. We have identified and validated the main glycine transporter(s) in ATC cells and show that inhibition of 1C-Met is synthetic lethal with inhibition of glycine uptake.3- Impaired 1C-Met leads to increased oxidative stress and decrease of NADPH and glutathione levels. These alterations create a synthetic lethality relationship with the glutathione-depleting activity of PRIMA-1 (APR246), a small molecule originally identified as a compound restoring mutant p53 conformation and function, but later shown to be converted within cells into the reactive electrophile methylene quinuclidinone, which acts in a p53-independent manner through a variety of mechanisms, including reduction of cellular GSH levels.Our data shed light on the molecular consequences of 1C-Met upregulation in ATC and support the efficacy of novel rationally designed therapeutic approaches with curative intent not only for ATC, but also for other aggressive, dedifferentiated solid tumors. Citation Format: Alexander Forrest, Anil Ahsan, Antonio Di Cristofano. Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer [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 1792.

Blocking LBH expression causes replication stress and sensitizes triple-negative breast cancer cells to ATR inhibitor treatment.

Triple-negative (ER-PR-HER2-) breast cancers (TNBC) are highly aggressive and difficult to treat. TNBC exhibit high genomic instability, which enables them to adapt and become resistant to chemo/radiation therapy, leading to rapid disease relapse and mortality. The pro-survival factors that safeguard genome integrity in TNBC cells are poorly understood. LBH is an essential mammary stem cell-specific transcription regulator in the WNT pathway that is aberrantly overexpressed in TNBC, correlating with poor prognosis. Herein, we demonstrate a novel role for LBH in promoting TNBC cell survival. Depletion of LBH in multiple TNBC cell models triggered apoptotic cell death both in vitro and in vivo and led to S-G2M cell cycle delays. Mechanistically, LBH loss causes replication stress due to DNA replication fork stalling, leading to ssDNA breaks, ɣH2AX and 53BP1 nuclear foci formation, and activation of the ATR/CHK1 DNA damage response. Notably, ATR inhibition in combination with LBH downmodulation had a synergistic effect, boosting TNBC cell killing and blocking in vivo tumor growth. Our findings demonstrate, for the first time, that LBH protects the genome integrity of cancer cells by preventing replicative stress. Importantly, they uncover new synthetic lethal vulnerabilities in TNBC that could be exploited for future multi-modal precision medicine.

Discovery and Mechanistic Elucidation of NQO1‐Bioactivatable Small Molecules That Overcome Resistance to Degraders

AbstractDegraders hold the promise to efficiently inactivate previously intractable disease‐relevant targets. Unlike traditional inhibitors, degraders act substoichiometrically and rely on the hijacked proteolysis machinery, which can also act as an entry point for resistance. To fully harness the potential of targeted protein degradation, it is crucial to comprehend resistance mechanisms and formulate effective strategies to overcome them. We conducted a chemical screening to identify synthetic lethal vulnerabilities of cancer cells that exhibit widespread resistance to degraders. Comparative profiling followed by tailored optimization delivered the small molecule RBS‐10, which shows preferential cytotoxicity against cells pan‐resistant to degraders. Multiomics deconvolution of the mechanism of action revealed that RBS‐10 acts as a prodrug bioactivated by the oxidoreductase enzyme NQO1, which is highly overexpressed in our resistance models. Collectively, our work informs on NQO1 as an actionable vulnerability to overcome resistance to degraders and as a biomarker to selectively exploit bioactivatable prodrugs in cancer.

Discovery and Mechanistic Elucidation of NQO1-Bioactivatable Small Molecules That Overcome Resistance to Degraders.

Degraders hold the promise to efficiently inactivate previously intractable disease-relevant targets. Unlike traditional inhibitors, degraders act substoichiometrically and rely on the hijacked proteolysis machinery, which can also act as an entry point for resistance. To fully harness the potential of targeted protein degradation, it is crucial to comprehend resistance mechanisms and formulate effective strategies to overcome them. We conducted a chemical screening to identify synthetic lethal vulnerabilities of cancer cells that exhibit widespread resistance to degraders. Comparative profiling followed by tailored optimization delivered the small molecule RBS-10, which shows preferential cytotoxicity against cells pan-resistant to degraders. Multiomics deconvolution of the mechanism of action revealed that RBS-10 acts as a prodrug bioactivated by the oxidoreductase enzyme NQO1, which is highly overexpressed in our resistance models. Collectively, our work informs on NQO1 as an actionable vulnerability to overcome resistance to degraders and as a biomarker to selectively exploit bioactivatable prodrugs in cancer.

INPP5A phosphatase is a synthetic lethal target in GNAQ and GNA11-mutant melanomas.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients' tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

Network targeting combination therapy of synthetic lethal vulnerabilities in 9p21-deficient glioblastoma: A case report.

Patients with relapsed or progressive glioblastoma only rarely respond to salvage therapies. Nevertheless, comprehensive genomic profiling can provide insight that can identify promising approaches. Signaling pathway analyses have revealed synthetic lethal partnerships, which create the possibility of targeting vulnerabilities arising from the loss of tumor suppressor genes. For synthetic lethal vulnerabilities that are not present in normal tissues, lethal cytotoxicity against cancer cells can be achieved without the necessity of causing normal tissue toxicity. This case report describes a patient with progressive glioblastoma with homozygous deletion of chromosome 9p21. Vulnerabilities created by CDKN2A and MTAP loss were exploited with pemetrexed, bevacizumab, and candesartan to achieve a clinically meaningful remission by targeting multiple synthetic lethal nodes. Synthetic lethality can reveal the basis for exceptional responsiveness, thus extending the utility of molecular profiling and fulfilling the promise of precision medicine.

Abstract B020: SF3B1 hotspot mutations confer sensitivity to PARP inhibition through a defective replication stress response

Abstract SF3B1 hotspot mutations are highly prevalent in several tumor types, where they lead to a global disruption of canonical splicing and are associated with a poor clinical outcome. Whilst evidence suggests that spliceosomal inhibitors have a therapeutic benefit for specific SF3B1 mutant cancers, recent trials have failed to report any clinical responses. Here we used a myriad of in vitro and in vivo techniques to identify and explore a synthetic lethal vulnerability in SF3B1 mutant cancers. These included a high throughput drug screen with an in-house curated library of 80 small-molecule inhibitors in an isogenic cell line model with the most prevalent SF3B1 mutation, a resistance CRISPR knockout screen and a range of DNA fibre analyses to gain mechanistic insights into the observed sensitivity. We identified that SF3B1 mutant cells are selectively sensitive to multiple PARP inhibitors (PARPi), which validated in numerous cell lines and patient derived models in vitro and in vivo, whereby they prevent metastasis. SF3B1 selective PARPi sensitivity occurs via a previously undescribed mechanism, as a result of excessive dormant origin firing and altered fork dynamics, due to the loss of the canonical replication stress response (pCHK1, pRPA, 53BP1), when SF3B1 mutant cells are treated with PARPi. This was further substantiated by the lack of MUS81 recruitment to under-replicated DNA intermediates, leading to the accumulation of DNA damage and cell cycle stalling, via the induction of the G2/M checkpoint. These findings demonstrate that SF3B1 mutant cells have wild-type-like replication under normal growth conditions, but fail to safeguard replication when exposed to PARPi. High content proteomic profiling identified that the defective ATR response is a direct consequence of the downregulation of CINP in SF3B1 mutant cells, which was confirmed in multiple isogenic models and in primary tumors. The defective ATR response was rescued with CINP re-expression and phenocopied through CINP silencing. In SF3B1 mutant cells G2/M checkpoint activation under PARP inhibition can be further abrogated by the addition of ATM inhibitors, but not by the inhibition of ATR or Chk1. Our discovery defines a unique mechanism underpinning PARPi sensitivity in SF3B1 mutant cancers. This synthetic-lethal relationship is further validated in a Phase 1 clinical trial, which shows that those patients with SF3B1 mutant relapsed leukemia have the longest progression free survival when treated with single agent PARPi. This novel synthetic lethality extends the utility of PARPi beyond BRCA1/2 mutant cancers and introduces a new therapeutic approach ready for proof-of-concept clinical trials. Citation Format: Philip Bland, Harry Saville, Patty T Wai, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Holly E Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Patrycja Gazinska, Helen N Pemberton, Aditi Gulati, Naomi Guppy, Ioannis Roxanis, Guy Pratt, Ceri Oldreive, Tatjana Stankovic, Samantha Barlow, Helen Kalirai, Sarah E Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti S Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher J Lord, Rachael Natrajan. SF3B1 hotspot mutations confer sensitivity to PARP inhibition through a defective replication stress response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B020.

The curious case of IDH mutant acute myeloid leukaemia: biochemistry and therapeutic approaches.

Of the many genetic alterations that occur in cancer, relatively few have proven to be suitable for the development of targeted therapies. Mutations in isocitrate dehydrogenase (IDH) 1 and -2 increase the capacity of cancer cells to produce a normally scarce metabolite, D-2-hydroxyglutarate (2-HG), by several orders of magnitude. The discovery of the unusual biochemistry of IDH mutations spurred a flurry of activity that revealed 2-HG as an 'oncometabolite' with pleiotropic effects in malignant cells and consequences for anti-tumour immunity. Over the next decade, we learned that 2-HG dysregulates a wide array of molecular pathways, among them a large family of dioxygenases that utilise the closely related metabolite α-ketoglutarate (α-KG) as an essential co-substrate. 2-HG not only contributes to malignant transformation, but some cancer cells become addicted to it and sensitive to inhibitors that block its synthesis. Moreover, high 2-HG levels and loss of wild-type IDH1 or IDH2 activity gives rise to synthetic lethal vulnerabilities. Herein, we review the biology of IDH mutations with a particular focus on acute myeloid leukaemia (AML), an aggressive disease where selective targeting of IDH-mutant cells is showing significant promise.

Abstract 5727: Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH

Abstract Loss of Function (LOF) mutations in the NOTCH receptors are found in several different cancers most notably in small cell lung cancer (SCLC) and squamous cell carcinomas (SCC). We previously developed genetically engineered mouse models (GEMM) of SCLC using CRISPR/Cas9 engineered to be NOTCH1-Mutant, NOTCH2-Mutant, and NOTCH-WT. Synthetic lethality provides a paradigm for targeting cancers with LOF mutations in tumor suppressor genes. In applying this paradigm, one looks for specific vulnerabilities that are created upon loss of the gene of interest. Using 6 cell lines developed from these CRISPR-based SCLC GEMMs which are isogenic to NOTCH, we performed CRISPR/Cas9 LOF negative selection screens (using an sgRNA library enriched in druggable enzymes) to identify synthetic lethal interactors with LOF NOTCH mutations. Our CRISPR/Cas9 screen identified TRIM28 (Tripartite Motif Containing 28, or KAP1) as a highly significant synthetic lethal interactor with NOTCH1 or NOTCH2. We validated the synthetic lethal interaction between NOTCH1/2 and TRIM28 using both human and mouse NOTCH-isogenic cell lines. Interestingly, RNA-sequencing of NOTCH2-Mutant or NOTCH-WT SCLC cell lines after TRIM28 CRISPR inactivation showed robust increases in endogenous retroviral (ERV) expression and the MDA5/RIG-I/MAVS RNA-sensing machinery leading to hyperactivation of the TBK1/IRF3/7 innate immune signaling pathway and the pro-inflammatory cytokine CXCL10 selectively in cells with NOTCH inactivated. Notably, inactivation of TBK1 activity or JAK-STAT activity using specific small molecule inhibitors of TBK1 or JAK completely reversed the synthetic lethality between NOTCH and TRIM28 suggesting that the synthetic lethality phenotype is a consequence of hyperactivation of innate immune signaling, which is known to be cytotoxic. Together our findings uncover a novel vulnerability in SCLCs with LOF NOTCH mutations and perhaps other cancers with LOF NOTCH mutations and suggest a strategy that could also potentiate anti-tumor immunity by increasing innate immune signaling in tumor cells. Citation Format: Deli Hong, Matthew A. Booker, Sidrah Anjum, Yixiang Li, Tran C. Thai, Michelle Y. Wang, David A. Barbie, Michael Y. Tolstorukov, Jun Qi, Matthew G. Oser. Identification of synthetic lethal vulnerabilities in cancers with loss of function mutations in NOTCH. [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 5727.

Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids

Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [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 251.

DNAR-08. THE ROLE OF SMARCAL1 AS A SYNTHETIC LETHAL VULNERABILITY IN ATRX-DEFICIENT GLIOMAS

Abstract Mechanisms to maintain telomere length over successive cell divisions are a requirement for cancer cell immortalization. Although many cancers maintain telomere length through the activation of telomerase, ~10-15% of human cancers use telomerase-independent mechanisms of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). In gliomas, the ALT phenotype is associated with loss-of-function mutations in the ATRX gene in IDH-mutant astrocytomas, adult and pediatric glioblastomas (GBM), high-grade astrocytomas with piloid features, and pleomorphic xanthoastrocytomas. In adult GBM, we previously identified a relatively rare subset of ALT-positive ATRX wildtype tumors that harbored loss-of-function mutations in the SMARCAL1 gene, and we found that loss of SMARCAL1 activity plays a causative role in the onset of ALT in these tumors. SMARCAL1 is an annealing helicase that localizes to sites of DNA damage and replication stress and catalyzes the reversal of stalled replication forks. To better understand the relationship between ATRX mutations, SMARCAL1 activity, and the ALT phenotype, we investigated the localization and function of SMARCAL1 in ALT-positive glioma cells and xenografts with native ATRX deficiency. We found that SMARCAL1 localizes to ALT-associated PML bodies in astrocytoma cell lines with concurrent ATRX and IDH1 mutations (derived from grade 3 and 4 tumors). Our data show that inducible suppression of SMARCAL1 via doxycycline-induced RNAi leads to increased DNA double-strand breaks, increased abundance of extrachromosomal telomeric repeats (c-circles), and increased sensitivity to irinotecan, a topoisomerase I inhibitor. In an orthotopic mouse model, SMARCAL1 depletion exhibited a chemosensitizing effect in combination with irinotecan against a patient-derived xenograft (astrocytoma, IDH-mutant Grade 4). Based on these data, we hypothesize that SMARCAL1 activity is critical for resolving ALT-associated replication stress in ATRX-deficient malignant gliomas. We therefore propose that SMARCAL1 functions as a synthetic lethal vulnerability in ATRX-deficient ALT-positive gliomas and that SMARCAL1 inhibition is a viable therapeutic strategy in these tumors.

Dysregulated Lipid Synthesis by Oncogenic IDH1 Mutation Is a Targetable Synthetic Lethal Vulnerability.

Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247.

Abstract 3027: Elucidating the role of glutamine metabolism in head & neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, resulting in over 14,600 deaths each year in the United States alone. HNSCC is associated with human papillomavirus (HPV) infection, tobacco use, and abusive alcohol intake. Without truly effective targeted therapies, surgery and radiotherapy represent the primary treatment options for most patients. Unfortunately, these treatments are associated with significant morbidity and a reduction in quality of life. Immunotherapies have recently revolutionized HNSCC treatment, but <20% of patients exhibit clinical responses, albeit often not durable (Saddawi-Konefka et al. Frontiers in Oncology 2021). This highlights the unmet need to identify novel therapeutic options and biomarkers predicting a more favorable response to maximize the efficacy of targeted cancer strategies for HNSCC treatment. Glutamine is a conditionally essential amino acid for rapidly proliferating cancer cells making glutamine pathway inhibition an attractive approach for anti-cancer therapy. We found that treatment with the broad glutamine antagonist sirpiglenastat (DRP-104), which irreversibly inhibits all known enzymes involved in glutamine metabolism, results in metabolically halted cell growth in a large panel (n=8) of HPV- and HPV+ HNSCC cell lines (IC50 of 0.2-25uM). Interestingly, HNSCC cells bearing genetic alterations in PIK3CA and PTEN were significantly more sensitive to glutamine antagonism than unaltered HNSCC cells. The dependence of glutamine in HNSCC growth and the increased sensitivity of PIK3CA/PTEN aberrant cells was also observed in orosphere assays and HNSCC tumor xenografts in mice in vivo. We next explored the mechanism of glutamine suppression in HNSCC by integrating the results from genome-wide CRISPR-Cas9 knockout library screens and broad-spectrum metabolomics analysis. Both approaches converged on the identification of a dysregulated metabolic pathway that represents a synthetic lethal vulnerability that can be exploited via targeted therapies. Our data suggest that broad glutamine antagonism using sirpiglenastat (DRP-104) has therapeutic potential in HNSCC by dismantling cancer metabolism and sensitizing cells to additional perturbations leading to specific cell death. A clinical trial of sirpiglenastat (DRP-104) is currently ongoing (NCT04471415). Citation Format: Michael Allevato, Yumi Yokoyama, Robert Wild, J. Silvio Gutkind. Elucidating the role of glutamine metabolism in head & neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3027.

Synthetic Vulnerabilities in the KRAS Pathway.

Simple SummaryDespite recent dramatic progress, developing drugs that target oncogenic KRAS or its key effectors remains a major challenge for cancer research. Improving our understanding of the underlying biology of KRAS in cancer will identify potential codependent vulnerabilities or synthetic lethal partners that are essential specifically in the context of KRAS mutations. Aberrant alterations in the KRAS oncogene not only favor cancer cell survival and proliferation, but also trigger oncogenic stress and compensatory mechanisms in cancer cells. These effectors are, thus, rational targets for defining synthetic lethal approaches to form the basis for effective therapies directed at KRAS-mutant tumors.Mutations in Kristen Rat Sarcoma viral oncogene (KRAS) are among the most frequent gain-of-function genetic alterations in human cancer. Most KRAS-driven cancers depend on its sustained expression and signaling. Despite spectacular recent success in the development of inhibitors targeting specific KRAS alleles, the discovery and utilization of effective directed therapies for KRAS-mutant cancers remains a major unmet need. One potential approach is the identification of KRAS-specific synthetic lethal vulnerabilities. For example, while KRAS-driven oncogenesis requires the activation of a number of signaling pathways, it also triggers stress response pathways in cancer cells that could potentially be targeted for therapeutic benefit. This review will discuss how the latest advances in functional genomics and the development of more refined models have demonstrated the existence of molecular pathways that can be exploited to uncover synthetic lethal interactions with a promising future as potential clinical treatments in KRAS-mutant cancers.

Discovery of AL-GDa62 as a Potential Synthetic Lethal Lead for the Treatment of Gastric Cancer.

Diffuse gastric cancer and lobular breast cancer are aggressive malignancies that are frequently associated with inactivating mutations in the tumor suppressor gene CDH1. Synthetic lethal (SL) vulnerabilities arising from CDH1 dysfunction represent attractive targets for drug development. Recently, SLEC-11 (1) emerged as a SL lead in E-cadherin-deficient cells. Here, we describe our efforts to optimize 1. Overall, 63 analogues were synthesized and tested for their SL activity toward isogenic mammary epithelial CDH1-deficient cells (MCF10A-CDH1-/-). Among the 26 compounds with greater cytotoxicity, AL-GDa62 (3) was four-times more potent and more selective than 1 with an EC50 ratio of 1.6. Furthermore, 3 preferentially induced apoptosis in CDH1-/- cells, and Cdh1-/- mammary and gastric organoids were significantly more sensitive to 3 at low micromolar concentrations. Thermal proteome profiling of treated MCF10A-CDH1-/- cell protein lysates revealed that 3 specifically inhibits TCOF1, ARPC5, and UBC9. In vitro, 3 inhibited SUMOylation at low micromolar concentrations.

Modulation of RNA Splicing Enhances Response to BCL2 Inhibition in Acute Myeloid Leukemia

Resistance to therapy is one of the most significant challenges in the treatment of acute myeloid leukemia (AML). While great efforts have uncovered genetic mechanisms of resistance to certain AML-directed therapies, to date, treatment resistance in AML has only partially explained by acquired genetic alterations. Here, we performed genome-wide CRISPR/Cas9 screens to identify drug-gene interactions that modulate therapeutic response to treatments commonly used in AML. Interestingly, our findings uncovered several genes that regulate pre-mRNA splicing whose loss strongly synergized with venetoclax, a BH3 mimetic that blocks the antiapoptotic protein BCL-2. To further delineate the role of RNA processing in response to AML treatments, we performed secondary CRISPR screens with a domain-focused gRNA library targeting 490 RNA processing factors in the presence of various AML drugs. Overall, these genetic screens identified a number of RNA splicing factors whose loss-of-function sensitized AML cells to BCL2 inhibition (Fig. A).Among the top gene candidates whose loss promoted venetoclax efficacy was the splicing factor RBM10 (Fig.B). Strikingly, loss of RBM10 exclusively synergized with venetoclax-based treatments across AML therapeutics, including in TP53 mutant lines (Fig.C-D). Moreover, RBM10 loss restored venetoclax sensitivity to AML cell line variants with acquired venetoclax resistance. Interestingly, while many RNA splicing factors are pan-essential, generation of an Rbm10 conditional knockout mouse revealed that Rbm10 is completely dispensable for steady-state normal hematopoiesis (Fig.E). Since RBM10 has not been studied previously in hematopoiesis, we mapped the impact of RBM10 on mRNA expression and splicing using RNA-seq and direct RNA binding partners genome-wide by eCLIP-Seq (Fig. F). RBM10 loss was strongly associated with downregulation of BCL2A1, an anti-apoptotic factor whose expression is correlated with venetoclax resistance in AML (Fig.G-H). This was dependent on RBM10's ability to bind RNA and expression of BCL2A1 cDNA fully rescued the growth-inhibitory effect of RBM10 KO-venetoclax treated AML cells. Overall, the above data support RBM10 as a synthetic lethal vulnerability in venetoclax therapy.Beyond RBM10, our genetic screens also identified several splicing factors belonging to the family of serine and arginine-rich (SR) proteins whose loss synergized with venetoclax treatment (Fig. I). SR proteins are essential for pre-mRNA splicing and are substrates for phosphorylation by conserved family of kinases, such as Cdc2-like kinases (CLKs) and (dual-specificity tyrosine-regulated kinases) DYRKs. We therefore utilized a series of selective pan-CLK/DYRK1A inhibitors, including SM09419 and SM08502, that potently suppress SR protein phosphorylation. Interestingly, BCL2 is one of the top genetic dependencies upon DYRK1A genetic suppression in prior work from the DepMap (Fig. J). Pharmacologic inhibition of CLK/DYRK1A exhibited high in vitro efficacy at nanomolar range across a diverse range of AML subtypes including cell lines with acquired venetoclax resistance (Fig.K). Consistent with this, combined SM09419 and venetoclax displayed synergistic anti-leukemic effects and venetoclax-sensitive AML cell lines (Fig.L). Taken together, these data support the notion of targeting CLK/DYRK1A in the context of BCL2 inhibition.In this study, we systematically defined gene interactions that mediate the response to a wide range of AML drugs. Recent studies have begun to show that dysfunctional RNA processing promotes AML development. However, the role of RNA processing in modulating drug responsiveness in AML is not well understood. Here, we have uncovered that synthetic lethal targeting of splicing factors, such as RBM10, increases sensitivity of AML cells to BCL2 inhibition. Therapeutically, pharmacologic inhibition of SR protein function via inhibiting CLK/DYRK1A-mediated phosphorylation of splicing factors is an effective strategy used in combination with venetoclax or to overcome venetoclax resistance. Overall, our findings underscore the central importance of RNA splicing in drug response and provides a therapeutic rationale for modulating RNA splicing to enhance current AML therapies. [Display omitted] DisclosuresMcMillan: Prizer: Ended employment in the past 24 months. Bossard: Biosplice Therapeutics: Current Employment. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Synthetic Lethal Screening Identifies CHK1 Inhibition As an Exploitable Vulnerability in EZH2 Deficient T-ALL

Loss of function mutations of Enhancer of Zeste (Drosophila) homolog 2 (EZH2), the enzymatic component of Polycomb Repressive Complex 2 (PRC2), are particularly frequent in the early T cell precursor (ETP) subtype of T cell Acute Lymphoblastic Leukaemia (T-ALL), and have been associated with chemotherapy-resistance and a poor prognosis. There is thus a need to identify novel treatment strategies for this subgroup of patients.We hypothesized that inactivation of EZH2 in T-ALL cells induces specific synthetic lethal vulnerabilities that can be exploited using drugs targeting other dependency pathways, potentially resulting in a clinically meaningful therapeutic strategy. Using isogenic Jurkat T-ALL cells, with and without EZH2 -inactivating mutations generated by double-nicking CRISPR Cas9, we performed cell-based drug screening using 220 well-characterized compounds, enriched for drugs targeting epigenetic, cell cycle and DNA repair machinery. Strikingly, we observed that EZH2 -knock out (KO) cells are hypersensitive to checkpoint kinase 1 (CHK1) inhibition by the clinical-grade small molecule MK8776, showing an IC50 reduction of over 2-fold. This effect was similarly observed when testing other CHK1 inhibitors (LY2603618 and CHIR-124), but was absent in the presence of inhibitors of other DNA damage-response kinases such as ATM, ATR and CHK2, demonstrating the specificity of this synthetic lethal interaction. Furthermore, pharmacological inhibition of EZH2 methyltransferase activity by GSK126 in human T-ALL cell lines resulted in sensitization to CHK1 inhibition. Notably, MK8776 resulted in apoptosis of EZH2-deficient cells as determined by Annexin V-PI staining.CHK1 inhibition has previously been shown to result in enhanced DNA damage and accumulation of cells with incompletely replicated DNA. In line with this, EZH2 -KO cells treated with MK8776 exhibited markedly elevated levels of the DNA damage marker yH2AX. Interestingly, the highest proportion of apoptotic cells were found at the G1 phase of the cell cycle when analyzed by TUNEL-PI, suggesting that MK8776 treated EZH2 -KO cells with accumulated DNA damage override the G2 checkpoint, exit mitosis and undergo mitotic catastrophe-related cell death during G1, a feature characteristic of mitotic slippage.To investigate the mechanism further, gene expression of two EZH2 -KO Jurkat clones was compared to their isogenic parental control by RNA-seq. EZH2 -KO cells showed a striking upregulation of genes associated with human ETP-ALL including LYL1, MYCN, KIT and HHEX, a finding that could be recapitulated with GSK126 treatment. Gene set enrichment analysis (GSEA) showed significant changes of expression in MYC target genes. Importantly, MYCN protein was markedly upregulated in both EZH2 -KO and GSK126 treated cells. We speculate that increased MYCN expression induces high levels of replication stress and increased dependency on CHK1 for cell survival. Our findings suggest that specific inhibition of the CHK1 pathway can preferentially target PRC2-deficient leukemic cells, providing a potentially less toxic and more effective treatment for this high-risk subgroup of patients. DisclosuresNo relevant conflicts of interest to declare.