Synthetic Lethal Approaches Research Articles

ROSALINE: a phase II, neoadjuvant study targeting ROS1 in combination with endocrine therapy in invasive lobular carcinoma of the breast.

Invasive lobular carcinoma (ILC) is the most common histologic subtype of breast cancer after invasive ductal carcinoma (i.e., no special type [NST]). ILC differs from NST in clinical presentation, site-specific metastasesand response to conventional therapies. Loss of E-cadherin protein expression, due to alterations in its encoding gene CDH1, is the most frequent oncogenic event in ILC. Synthetic lethality approaches have shown promising antitumor effects of ROS1 inhibitors in models of E-cadherin-defective breast cancer in in vivo studies and provide the rationale for testing their clinical activity in patients with ILC. Entrectinib is a tyrosine kinase inhibitor targeting TRK, ROS1 and ALK tyrosine kinases. Here, the authors present ROSALINE (NCT04551495), a phase II study testing neoadjuvant entrectinib and endocrine therapy in women with estrogen receptor-positive, HER2-negative early ILC.

Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.

Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (β-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kdapp119µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase.

Current and Future Therapies for Pancreatic Ductal Adenocarcinoma.

Simple SummaryPancreatic cancer is the fourth leading cause of cancer-related mortality worldwide. The poor survival associated with this disease is due to delayed diagnosis, a lack of reliable biomarkers, and tumour resistance to treatment. Currently, surgery is the only curative treatment option, but few patients are eligible for this procedure. Developing resistance to current chemotherapies such as gemcitabine has led to a reduction in effective therapy options for patients and an urgent requirement for the development of novel therapeutic avenues. Potential success has been noted in therapeutic approaches such as synthetic lethality and immunotherapy. An array of clinical trials are currently recruiting, primarily in the area of monoclonal antibodies in combination with other therapies such as chemotherapy and immune checkpoint inhibitors. This review article aims to highlight the potential these therapies have to improve patient prognosis and survival.Pancreatic cancer is one of the leading causes of cancer-related death worldwide. This is due to delayed diagnosis and resistance to traditional chemotherapy. Delayed diagnosis is often due to the broad range of non-specific symptoms that are associated with the disease. Resistance to current chemotherapies, such as gemcitabine, develops due to genetic mutations that are either intrinsic or acquired. This has resulted in poor patient prognosis and, therefore, justifies the requirement for new targeted therapies. A synthetic lethality approach, that targets specific loss-of-function mutations in cancer cells, has shown great potential in pancreatic ductal adenocarcinoma (PDAC). Immunotherapies have also yielded promising results in the development of new treatment options, with several currently undergoing clinical trials. The utilisation of monoclonal antibodies, immune checkpoint inhibitors, adoptive cell transfer, and vaccines have shown success in several neoplasms such as breast cancer and B-cell malignancies and, therefore, could hold the same potential in PDAC treatment. These therapeutic strategies could have the potential to be at the forefront of pancreatic cancer therapy in the future. This review focuses on currently approved therapies for PDAC, the challenges associated with them, and future directions of therapy including synthetically lethal approaches, immunotherapy, and current clinical trials.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n=375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Redefining CD56 as a Biomarker and Therapeutic Target in Multiple Myeloma.

Multiple myeloma is an incurable, genetically heterogeneous disease, without available tailored therapeutic approaches. CD56 signaling promotes multiple myeloma growth and adhesion, by activating CREB1 target genes, MCL1 and BCL2. Inhibition of CREB1 alone or in combination with lenalidomide is an unexplored synthetic lethal approach in CD56-expressing patients with multiple myeloma.

BRAF Inhibitor Resistance Confers Increased Sensitivity to Mitotic Inhibitors.

Single agent and combination therapy with BRAFV600E/K and MEK inhibitors have remarkable efficacy against melanoma tumors with activating BRAF mutations, but in most cases BRAF inhibitor (BRAFi) resistance eventually develops. One resistance mechanism is reactivation of the ERK pathway. However, only about half of BRAFi resistance is due to ERK reactivation. The purpose of this study is to uncover pharmacological vulnerabilities of BRAFi-resistant melanoma cells, with the goal of identifying new therapeutic options for patients whose tumors have developed resistance to BRAFi/MEKi therapy. We screened a well-annotated compound library against a panel of isogenic pairs of parental and BRAFi-resistant melanoma cell lines to identify classes of compounds that selectively target BRAFi-resistant cells over their BRAFi-sensitive counterparts. Two distinct patterns of increased sensitivity to classes of pharmacological inhibitors emerged. In two cell line pairs, BRAFi resistance conferred increased sensitivity to compounds that share the property of cell cycle arrest at M-phase, including inhibitors of aurora kinase (AURK), polo-like kinase (PLK), tubulin, and kinesin. Live cell microscopy, used to track mitosis in real time, revealed that parental but not BRAFi-resistant melanoma cells were able to exit from compound-induced mitotic arrest through mitotic slippage, thus escaping death. Consistent with the key role of Cyclin B1 levels in regulating mitosis at the spindle checkpoint in arrested cells, we found lower Cyclin B1 levels in parental compared with BRAFi-resistant melanoma cells, suggesting that inability to down-regulate Cyclin B1 expression levels may explain the increased vulnerability of resistant cells to mitotic inhibitors. Another BRAFi-resistant cell line showed increased sensitivity to Chk1/2 inhibitors, which was associated with an accumulation of DNA damage, resulting in mitotic failure. This study demonstrates that BRAFi-resistance, in at least a subset of melanoma cells, confers vulnerability to pharmacological disruption of mitosis and suggests a targeted synthetic lethal approach for overcoming resistance to BRAF/MEK-directed therapies.

A network-based approach to integrate nutrient microenvironment in the prediction of synthetic lethality in cancer metabolism.

Synthetic Lethality (SL) is currently defined as a type of genetic interaction in which the loss of function of either of two genes individually has limited effect in cell viability but inactivation of both genes simultaneously leads to cell death. Given the profound genomic aberrations acquired by tumor cells, which can be systematically identified with -omics data, SL is a promising concept in cancer research. In particular, SL has received much attention in the area of cancer metabolism, due to the fact that relevant functional alterations concentrate on key metabolic pathways that promote cellular proliferation. With the extensive prior knowledge about human metabolic networks, a number of computational methods have been developed to predict SL in cancer metabolism, including the genetic Minimal Cut Sets (gMCSs) approach. A major challenge in the application of SL approaches to cancer metabolism is to systematically integrate tumor microenvironment, given that genetic interactions and nutritional availability are interconnected to support proliferation. Here, we propose a more general definition of SL for cancer metabolism that combines genetic and environmental interactions, namely loss of gene functions and absence of nutrients in the environment. We extend our gMCSs approach to determine this new family of metabolic synthetic lethal interactions. A computational and experimental proof-of-concept is presented for predicting the lethality of dihydrofolate reductase (DHFR) inhibition in different environments. Finally, our approach is applied to identify extracellular nutrient dependences of tumor cells, elucidating cholesterol and myo-inositol depletion as potential vulnerabilities in different malignancies.

Fanconi Anemia Pathway in Colorectal Cancer: A Novel Opportunity for Diagnosis, Prognosis and Therapy.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and has the second highest mortality rate globally. Thanks to the advent of next-generation sequencing technologies, several novel candidate genes have been proposed for CRC susceptibility. Germline biallelic mutations in one or more of the 22 currently recognized Fanconi anemia (FA) genes have been associated with Fanconi anemia disease, while germline monoallelic mutations, somatic mutations, or the promoter hypermethylation of some FANC genes increases the risk of cancer development, including CRC. The FA pathway is a substantial part of the DNA damage response system that participates in the repair of DNA inter-strand crosslinks through homologous recombination (HR) and protects genome stability via replication fork stabilization, respectively. Recent studies revealed associations between FA gene/protein tumor expression levels (i.e., FANC genes) and CRC progression and drug resistance. Moreover, the FA pathway represents a potential target in the CRC treatment. In fact, FANC gene characteristics may contribute to chemosensitize tumor cells to DNA crosslinking agents such as oxaliplatin and cisplatin besides exploiting the synthetic lethal approach for selective targeting of tumor cells. Hence, this review summarizes the current knowledge on the function of the FA pathway in DNA repair and genomic integrity with a focus on the FANC genes as potential predisposition factors to CRC. We then introduce recent literature that highlights the importance of FANC genes in CRC as promising prognostic and predictive biomarkers for disease management and treatment. Finally, we represent a brief overview of the current knowledge around the FANC genes as synthetic lethal therapeutic targets for precision cancer medicine.

Recombination Mediator Proteins: Misnomers That Are Key to Understanding the Genomic Instabilities in Cancer.

Recombination mediator proteins have come into focus as promising targets for cancer therapy, with synthetic lethal approaches now clinically validated by the efficacy of PARP inhibitors in treating BRCA2 cancers and RECQ inhibitors in treating cancers with microsatellite instabilities. Thus, understanding the cellular role of recombination mediators is critically important, both to improve current therapies and develop new ones that target these pathways. Our mechanistic understanding of BRCA2 and RECQ began in Escherichia coli. Here, we review the cellular roles of RecF and RecQ, often considered functional homologs of these proteins in bacteria. Although these proteins were originally isolated as genes that were required during replication in sexual cell cycles that produce recombinant products, we now know that their function is similarly required during replication in asexual or mitotic-like cell cycles, where recombination is detrimental and generally not observed. Cells mutated in these gene products are unable to protect and process replication forks blocked at DNA damage, resulting in high rates of cell lethality and recombination events that compromise genome integrity during replication.

ATM: Functions of ATM Kinase and Its Relevance to Hereditary Tumors.

Ataxia–telangiectasia mutated (ATM) functions as a key initiator and coordinator of DNA damage and cellular stress responses. ATM signaling pathways contain many downstream targets that regulate multiple important cellular processes, including DNA damage repair, apoptosis, cell cycle arrest, oxidative sensing, and proliferation. Over the past few decades, associations between germline ATM pathogenic variants and cancer risk have been reported, particularly for breast and pancreatic cancers. In addition, given that ATM plays a critical role in repairing double-strand breaks, inhibiting other DNA repair pathways could be a synthetic lethal approach. Based on this rationale, several DNA damage response inhibitors are currently being tested in ATM-deficient cancers. In this review, we discuss the current knowledge related to the structure of the ATM gene, function of ATM kinase, clinical significance of ATM germline pathogenic variants in patients with hereditary cancers, and ongoing efforts to target ATM for the benefit of cancer patients.

Loss of E-Cadherin Leads to Druggable Vulnerabilities in Sphingolipid Metabolism and Vesicle Trafficking.

Simple SummaryGermline loss of the CDH1 gene is the primary genetic basis for hereditary diffuse gastric cancer, a disease resulting in elevated risk of both diffuse gastric cancer and lobular breast cancer. Current preventative treatment consists of prophylactic total gastrectomy, a therapy with several associated long-term morbidities. To address the lack of targeted molecular therapies for hereditary diffuse gastric cancer, we have utilized a synthetic lethal approach to identify candidate compounds that can specifically kill CDH1-null cells. Inhibitors of sphingolipid metabolism and vesicle trafficking pathways were identified as promising candidate compounds in a cell line model of CDH1 loss, then further validated in murine-derived organoid models of hereditary diffuse gastric cancer. With further research, these findings may lead to the development of novel chemoprevention strategies for the treatment of hereditary diffuse gastric cancer.Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC.

Clinical Translation of Combined MAPK and Autophagy Inhibition in RAS Mutant Cancer.

RAS (rat sarcoma virus) mutant cancers remain difficult to treat despite the advances in targeted therapy and immunotherapy. Targeted therapies against the components of mitogen-activated protein kinase (MAPK) pathways, including RAS, RAF, MEK, and ERK, have demonstrated activity in BRAF mutant and, in limited cases, RAS mutant cancer. RAS mutant cancers have been found to activate adaptive resistance mechanisms such as autophagy during MAPK inhibition. Here, we review the recent clinically relevant advances in the development of the MAPK pathway and autophagy inhibitors and focus on their application to RAS mutant cancers. We provide analysis of the preclinical rationale for combining the MAPK pathway and autophagy and highlight the most recent clinical trials that have been launched to capitalize on this potentially synthetic lethal approach to cancer therapy.

CSIG-02. NRF2-GUIDED GLUTATHIONE SYNTHESIS IS AN ESSENTIAL METABOLIC PATHWAY IN IN IDH1-MUTATED GLIOMA

Abstract BACKGROUND Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in WHO Grade II/III glioma, which result in the reprogramming of cellular metabolism and redox homeostasis. Many lines of evidence showed that IDH mutations are critical for glioma formation, whereas the therapeutic options for IDH-mutated cancers remain limited. METHODS In the present study, we used the patient-derived glioma cell lines to investigate the role of nuclear factor erythroid 2-related factor 2 (NRF2) governed glutathione de novo synthesis. Further, we evaluated the therapeutic value of NRF2 inhibitors in IDH1-mutated cells and preclinical orthotopic models. RESULTS The neomorphic activity of mutant IDH reprogrammed the metabolic pathways involving enzyme cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The depletion of NAD(P) in IDH1-mutated cells resulted in elevated oxidative stress and constitutive activation of NRF2-governed cytoprotective pathways through the decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1 (Keap1). Activation of NRF2 enhanced glutathione synthesis by enhancing the gene transcription of GCLC, GCLM, and SLC7A11, which are the critical for glutathione de novo synthesis. Further, evidence from both in vitro assays and patient cohort indicated that NRF2 governed glutathione synthesis is important for maintaining the redox homeostasis and cell survival, especially in IDH1-mutated glioma. Finally, Blockade of the NRF2/glutathione metabolic pathway exhibited synergistic cytotoxicity with the metabolic stress in IDH1-mutated cells, which results in overwhelming oxidative damage, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION In this study, we highlighted that NRF2 plays critical roles in the disease progression of IDH1-mutated glioma by prompting glutathione synthesis. Targeting NRF2 governed glutathione metabolism could serve as a valuable synthetic lethality approach for IDH1-mutated malignancies.

Novel Synthetic Lethal Targets for Myeloid Neoplasms with Loss of Chromosome 7

Loss of chr7 (-7) and partial deletions of its long arm (del7q) are observed in 10% of de novo myeloid neoplasms (MNs), 50% of therapy related MDS, up to 60% of post aplastic anemia MNs and occur frequently as evolution of congenital bone marrow failure syndromes (e.g., GATA2 and SAMD9L deficiency, FA). LOH of one or more chr7 genes has been considered the culprit in the pathogenesis of -7/del7q MNs. In addition to the loss of protective alleles, deletion resulting in haploinsufficiency (HI) of tumor suppressor genes (TSGs) in CDRs might be also a cause of leukemogenic drive behind -7/del7q. To date, albeit many candidate genes have been associated with -7/del7q, the search for genes responsible for clinical phenotype has failed to identify causative -7/del7q TSGs and their putative loss would be difficult to target. Irrespective of the important goal of clarification of leukemogenic effects of -7/del7q, loss of genes in CDRs may create a vulnerability phenotype, which could be exploited with synthetic lethal approaches. Such strategies would rely on the higher resistance of diploid vs -7/del7q cells, thus allowing for a therapeutic window.Here, we studied the molecular profile of 8160 MN patients (del7q: 1.7%; -7: 6%). EZH2 mutations were enriched in -7/del7q compared to chr7 diploid cases (3.8 vs 1.2%, P<.0001) also by absolute numbers heterozygous mutations were more numerous. We also detected somatic CUX1 mutations (1.7 vs 0.9%), SAMD9/SAMD9L (0.3 vs 0.1%), and LUC7L2 (0.3 vs 0.1%) in -7/del7q vs diploid. In -7/del7 cases somatic alterations were detected in BRAF (n=7), POT1 (n=3), PCLO (n=5) and PSMC2 (n=1) while no mutations in CUL1 and KMT2C were found. We then investigated the presence of driver mutations located on other chromosomes in -7/del7q. Del7q/-7 cases showed a lower frequency of TET2 and SF3B1 mutations vs diploid cases. In isolated/+1 del7q/-7 cases, TP53 mutations were significantly less frequent, but were increased in -7/del7q with complex karyotype (P<.0001). Higher frequencies of RAS genes, RUNX1 and ETV6 hits were also found. Del7q and TP53 mutations were founder lesions (dominant) in 38% and 54% of -7/del7q, while -7 was dominant in 63% of -7 cases. TP53 was the only mutation significantly associated with further worsening the already poor prognosis of -7/del7q cases (HR=1.629 P< .01). Germline alterations were more common in -7/del7q as compared to diploid cases (13 vs 5% P< .0001) of which most were FA or DNA repair gene variants also in other genes including (e.g., SAMD9L, 7%, DDX41, 3.7%).Having defined the genotype of -7/del7q, we set to identify genes which could be possible targets for the therapy chiefly synthetic lethality. Criteria for selection included: consistent HI in most of the patients, genes not affected by hemizygous LOF mutations and embryonic lethality in knockout (KO) configuration. Expression data of -7/del7q (n=86), diploid cases (n=1066) and healthy controls (n=84; MLL and BEAT AML to increase precision) were analyzed. Our algorithm included selection of genes with mRNA expression inversely correlating with copy number (deletion copy number). Out of 694 genes on chr7, 147 genes were deleted in all patients and 101 genes had more inconsistent HI levels. In total 35 genes showed significant negative correlation with -7/del7q ploidyincluding ACTR3B,AGK,ATP06V0E2,CUL1,FASTK,GALNT11,GSTK1, IMPDH1, PLXNA4, SLC37A3, ZNF277, KMT2C, NUP205, TMEM209, ZC3HC1 and GIMAP1/2/4/6, a cluster ofnucleotide binding proteins. Following adjustment to ploidy, HI was found for EZH2 (76% cases), CUX1 (76%), KMT2C (70%), LUC7L2 (60%), and SAMD9/9L (32%/50%) but also even more consistently in SSBP1 (88%), PSMC2 (86%), CUL1, ZNF398, and RHEB (all 84%) and TNPO3 (82%). Among those genes homozygous KO of Ezh2 and Cul1 lead to embryonic lethality, Gimap family deletion reduces normal hematopoiesis, Samd9l +/-and Samd9l-/- mice develop MDS and die after 1.5yrs and Cux1 knockdown causes an MDS like phenotype. Existing inhibitors are available for CUL1 (MLN4924), CUX1 (BER modulating agents) and EZH2 (EPZ6438, GSK343), but the presence of homozygous mutations (UPD7q) argues that EZH2 inhibition is unlikely to be successful.In conclusion, we showed a comprehensive molecular topography of -7/del7q and identified novel HI genes which could be targeted by novel or repurposed drugs. Ongoing drug screens for identified targets performed in cells with -7/del7q will be presented at the meeting. DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.

Playing on the Dark Side: SMYD3 Acts as a Cancer Genome Keeper in Gastrointestinal Malignancies.

Simple SummaryThe activity of SMYD3 in promoting carcinogenesis is currently under debate. Growing evidence seems to confirm that SMYD3 overexpression correlates with poor prognosis, cancer growth and invasion, especially in gastrointestinal tumors. In this review, we dissect the emerging role played by SMYD3 in the regulation of cell cycle and DNA damage response by promoting homologous recombination (HR) repair and hence cancer cell genomic stability. Considering the crucial role of PARP1 in other DNA repair mechanisms, we also discuss a recently evaluated synthetic lethality approach based on the combined use of SMYD3 and PARP inhibitors. Interestingly, a significant proportion of HR-proficient gastrointestinal tumors expressing high levels of SMYD3 from the PanCanAtlas dataset seem to be eligible for this innovative strategy. This promising approach could be taken advantage of for therapeutic applications of SMYD3 inhibitors in cancer treatment.The SMYD3 methyltransferase has been found overexpressed in several types of cancers of the gastrointestinal (GI) tract. While high levels of SMYD3 have been positively correlated with cancer progression in cellular and advanced mice models, suggesting it as a potential risk and prognosis factor, its activity seems dispensable for autonomous in vitro cancer cell proliferation. Here, we present an in-depth analysis of SMYD3 functional role in the regulation of GI cancer progression. We first describe the oncogenic activity of SMYD3 as a transcriptional activator of genes involved in tumorigenesis, cancer development and transformation and as a co-regulator of key cancer-related pathways. Then, we dissect its role in orchestrating cell cycle regulation and DNA damage response (DDR) to genotoxic stress by promoting homologous recombination (HR) repair, thereby sustaining cancer cell genomic stability and tumor progression. Based on this evidence and on the involvement of PARP1 in other DDR mechanisms, we also outline a synthetic lethality approach consisting of the combined use of SMYD3 and PARP inhibitors, which recently showed promising therapeutic potential in HR-proficient GI tumors expressing high levels of SMYD3. Overall, these findings identify SMYD3 as a promising target for drug discovery.

Overview of Ferroptosis and Synthetic Lethality Strategies.

Ferroptosis, a term first proposed in 2012, is iron-dependent, non-apoptotic regulatory cell death induced by erastin. Ferroptosis was originally discovered during synthetic lethal screening for drugs sensitive to RAS mutant cells, and is closely related to synthetic lethality. Ferroptosis sensitizes cancer stem cells and tumors that undergo epithelial−mesenchymal transition and are resistant to anticancer drugs or targeted therapy. Therefore, ferroptosis-inducing molecules are attractive new research targets. In contrast, synthetic lethal strategies approach mechanisms and genetic abnormalities that cannot be directly targeted by conventional therapeutic strategies, such as RAS mutations, hypoxia, and abnormalities in the metabolic environment. They also target the environment and conditions specific to malignant cells, have a low toxicity to normal cells, and can be used in combination with known drugs to produce new ones. However, the concept of synthetic lethality has not been widely adopted with ferroptosis. In this review, we surveyed the literature on ferroptosis-related factors and synthetic lethality to examine the potential therapeutic targets in ferroptosis-related molecules, focusing on factors related to synthetic lethality, discovery methods, clinical application stages, and issues in drug discovery.

Suppression of DNA Polymerase β Activity Is Synthetically Lethal in BRCA1-Deficient Cells.

People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase β (Pol β) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol β or the treatment with a Pol β pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol β as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.

Abstract 975: Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer

Abstract Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype and c-Myc amplification has been shown to be frequent in esophageal cancer. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. We therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a CDK inhibitor flavopiridol. We found frequent overexpression of c-Myc in esophageal cancer cell lines and tissues. c-Myc overexpression correlated with accelerated esophageal cancer subcutaneous xenograft tumor growth without association with improved overall survival in esophageal cancer patients. c-Myc expression was enhanced by Wnt signaling activator (6-bromoindirubin-3′ -oxime (BIO) or pcDNA3-cMyc and c-Myc expression was depleted by c-Myc siRNAs. Interestingly, esophageal cancer cells with elevated c-Myc expression were preferentially more sensitive to induction of apoptosis by CDK inhibition flavopiridol compared to esophageal cancer cells with lower c-Myc expression. In addition, we observed that flavopiridol alone or in combinations with chemotherapeutic agent nanoparticle albumin-bound paclitaxel (NPT) or in combinations with targeted agent BMS-754807 significantly inhibited esophageal cancer cell proliferation and subcutaneous xenograft tumor growth with significantly enhancing overall mice survival. These results indicate that aggressive esophageal cancer cells with elevated c-Myc expression are sensitive to CDK inhibitor flavopiridol and flavopiridol alone or in combination with cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing esophageal cancer. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Jun Li, Margaret A. Schwarz, Urs von Holzen. Antitumor effect of cyclin- dependent kinase inhibitor flavopiridol in c-Myc overexpressing esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 975.

A Three-Genes Signature of Anti-Apoptotic Genes Showed Different Mechanisms in Preventing Apoptosis in Acute Myeloid Leukemia Cells and Could Provide an Useful Model to Establish the Proper BH3-Mimetich Drug or Combination

BACKGROUNDSeveral biological mechanisms sustain leukemic development, but the inhibition of apoptotic death, which should be induced by the genetic instability of the disease, is surely one the key neoplastic alterations. Apoptotic regulation involves complex pathways of positive and negative regulators which may and do represent a target for personalized therapy. Furthermore, the overexpression of specific anti-apoptotic genes likely represent a useful prognostic instrument. Nowadays, BCL-2 inhibitors have successfully passed preliminary phases of clinical experimentation, however the involvement of other essential genes, such as MCL-1 and BCL2L1 (coding for bcl-xL), has been proposed as resistance mechanism to BCL-2 inhibitors and as novel therapeutic target in Acute Myeloid Leukemia (AML).Our study aims to investigate the expression of anti-apoptotic genes in AML patients to cluster them in different categories and study their impact on prognosis, in order to reveal possible mechanism of resistance to BCL-2 inhibitors and stratify patients that are more likely to take advantage of administration or co-administration of one between MCL-1 and BCL-2/BCL2L2 inhibitors. METHODSWe performed Human Transcriptome Array 2.0 (Affymetrix) in a cohort of 59 new diagnosed AML. Gene Expression Profiling (GEP) analysis has been conducted with Transcriptome Array V3.0 Software (Affymetrix). K-means clustering has been used to categorize different cluster of patients basing on BCL-2, MCL-1 and BCL2L1 gene expression. A pool of 7 healthy donor was used to normalize expression data. Survival analysis was conducted with Kaplan-Meyer method and differences in survival were assessed with Log-Rank test. RESULTSAnalyzing BCL-2, MCL-1 and BCL2L1 gene expression values in 59 AML patients at diagnosis we established 5 categories where patients are almost uniformly assigned (Figure 1a): 11/59 patients in Cluster 1, characterized by the only one overexpression of MCL1; 27/59 patients in Cluster 2, which presents low expression of all analyzed genes; 2/59 patients in Cluster 3, where BCL2L1 is strongly overexpressed; 6/59 patients in Cluster 4, distinguished by BCL-2 overexpression and 13/59 patients in Cluster 5, which high values of expression of all 3 genes. Thus, Clusters 2 and 5, where no gene is overexpressed in comparison with the others, include the majority of patients (40/59, approximately 68%), suggesting that apoptotic death control is realized thanks to an intricate debate between these 3 considered proteins and BCL-2 should not be considered the unique oncogenic anti-apoptotic mechanism. Furthermore, the 3-D Scatter Plot (Figure 1A) allows us to observe how the patients out of the central cloud (Clusters 2-5) always present one of the three anti-apoptotic genes overexpressed, and when BCL-2 level is progressively low (Cluster 1-2-3), MCL-1 and BCL2L1 values are high, respectively. Therefore, this documented behavior may indicate the presence of many ways used by leukemic cell to prevent apoptosis, with one of them almost always activated.In term of Overall Survival (OS), unsurprisingly no statistically significant difference arised between Cluster 1-3-4 where one different anti-apoptotic gene is overexpressed (Figure 1b), underling the probable equal effect of each mechanism in inhibiting apoptotic death and in sustaining leukemic progression. Finally, analyzing the impact only of BCL-2 expression on OS in a cohort of 36 young patients treated with chemotherapy it is possible to observe a trend close to statistically significance (p=0.07)(Figure 1c), unreached probably because of the relative limitation of the sample, confirming the BCL-2 induced chemoresistance. CONCLUSIONSApoptotic death regulation is precisely controlled equally by BCL-2, MCL-1 and BCL2L2, their expression is quite balanced in AML patients evaluated. One out of these three genes is almost always overexpressed, reaffirming the key role of anti-apoptotic oncogenes in leukemogenesis. Even though BCL-2 expression influence patients prognosis, MCL-1 and BCL2L2 overexpression has been documented when BCL-2 is normally or underexpressed. In conclusion, BCL-2 inhibitors resistance may be caused by MCL-1 and BCL2L2 overexpression detected in patients BCL-2 independents. Moreover, a synthetic lethality approach should be realized to switch-off leukemic anti-apoptotic multiple mechanisms. [Display omitted] DisclosuresMartinelli:Celgene: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy; Pfizer: Consultancy; Roche: Consultancy.

Transcriptome of DNA Repair Genes in CMML: Rationale for Targeting ERCC1 and CDKN1A As a Synthetic Lethality Approach

Background: Chronic myelomonocytic leukemia (CMML) is characterized by features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Despite the main role of DNA repair pathways in cancer, in myelodysplastic disorders, somatic mutations are not found in these pathways. The novel discovery of non-classical leukaemogenesis mediated by splicing defects, profound influence of epigenetic anomalies, deeper knowledge of the DNA repair network, and development of unbiased high-throughput sequencing approaches, prompted us to revisit whether these processes might be disrupted by non-mutational mechanisms in CMML and related myeloid disorders. Study aims: i) to screen the transcriptome of DNA repair genes in a CMML discovery cohort; ii) to validate their misregulation in an independent large CMML cohort; and iii) determining how those candidates behave through the spectrum of myeloid malignancies. Methods: RNA-seq was performed in the discovery cohort: 27 CMML bone marrow samples (BM) at diagnosis and 9 healthy BM samples, and in 7 patients treated with Azacitidine in BM collected before and after 4 cycles. RT-qPCR was used to validate 9 DEGs through the myeloid spectrum and in a CMML validation cohort: 74 additional CMML, 70 MDS, 66 AML , and 25 CML patients. Candidates were chosen based on clinical considerations: i) druggable oncogenes highly overexpressed ii) oncogenes infra-expressed with inhibitory molecules already being tested in myeloid neoplasms. Global pattern of DNA repair gene expression was compared with MDS and AML extracted from MILE study data. The DEGs methylation status was studied using available public data and we confirmed by pyrosequencing the methylation status of our main candidates. The CMML validation cohort was mutationally characterized by targeted sequencing of 18 CMML recurrently mutated genes. Alternative splice sites, skipped exons, mutually exclusive exons and retained introns were quantified using rMATS.Results: Of 27 CMML patients and 9 healthy donors, the expression of 30 genes was significantly different between the two groups (8 genes up-regulated and 22 down-regulated). Defects on genes predominantly unique to a single strand breaks repair pathway included: NEIL1 and OGG1 in base excision repair, XPA and MSS19 in nucleotide excision repair and RPA4 in mismatch repair. XRCC4 and MSH4 overexpression and PRKDC down-regulation were found as significant changes among the genes associated exclusively to double strand breaks repair. We next extracted the DNA repair transcriptional components from MILE study: 206 MDS, 47 ckAML and 73 healthy donors. MDS misregulation was characterized by upregulated genes while ckAML showed a global disruption, mostly downregulation of Homologous Recombination genes. Some genes showed opposite sense of misregulation depending on the myeloid entity: TDP1 was upregulated in CMML cases and downregulated in ckAML, and viceversa for BAP1 . CDK1 and EXO1 were upregulated in MDS cases but downregulated in ckAML cases. Statistically significant and direction-concordant dysregulation of CDKN1A, ERCC1, XRCC4, NEIL1, MSH4, PARP1 and BAP1 were confirmed in our independent CMML validation cohort. The methylation study showed a global demethylated status of our misregulated DNA repair genes in CMML. Either responder s (n=3) or non-responders (n=2) to azacitidine failed to achieve a substantial expression change in their DNA Repair DEGs. PARP1 infraexpresion was associated with TET2 mut cases. Lower CDKN1A and BAP1 expression was related to chromatin regulator mutations. SRSF2 mutations induced widespread aberrant splicing events in DNA repair genes in CMMLConclusions: We have identified by means of an unbiased high-throughput approach, and validated in an independent cohort, a subset of DNA repair genes consistently misregulated in CMML. The genes identified warrant further study as potential novel therapeutic targets, both directly and through modulation of associated compensatory pathways. CDKN1A and ERCC1 in particular emerge as realistic candidates for a synthetic lethality approach. Our findings suggest that the promise of PARP1 inhibition appears less pertinent to CMML than for other myeloid diseases. These differences might also partially explain the different genomic and phenotypic manifestations of different myeloid neoplasms, and shed insights into their distinctive biology. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.