Abstract: Somatic copy number alterations that result in loss of tumor suppressor gene function are important drivers of tumorigenesis. However, few existing therapeutic options to target oncogenic processes evoked by tumor suppressor gene inactivation exist. The discovery of synthetic lethal interactions with genetic drivers of cancer may yield new therapeutic strategies with cancer selective potential. We examined genome-scale CRISPR-SpCas9 and RNA interference screens to uncover new synthetic lethal vulnerabilities associated with the loss of common tumor suppressor genes (TSGs). The ATPases Vacuolar protein sorting 4 homolog A (VPS4A) and B (VPS4B) scored as strong synthetic lethal dependencies, with VPS4A selectively essential in cancers harboring loss of VPS4B adjacent to SMAD4 and VPS4B required in tumors with co-deletion of VPS4A and CDH1 (encoding E-cadherin). VPS4B resides 12.3 Mb away from the SMAD4 TSG on chromosome 18q and is lost in approximately 33% of all cancers, suggesting broad clinical applicability. Moreover, VPS4B is commonly lost in pancreatic cancer due to the frequent loss of SMAD4, highlighting VPS4A represents a promising target for this deadly cancer. VPS4A and VPS4B function as AAA ATPases forming a multimeric protein complex within the endosomal sorting complex required for transport (ESCRT) pathway to regulate membrane remodeling in a range of cellular processes. VPS4A suppression in cells with VPS4B/SMAD4 loss led to accumulation of ESCRT-III filaments, cytokinesis defects, nuclear deformation and micronucleation, which ultimately resulted in G2/M cell cycle arrest and apoptosis. Furthermore, upon VPS4A suppression, we observed potent in vivo tumor regression, which led to extended survival, in mouse subcutaneous xenograft models utilizing a pancreatic or rhabdomyosarcoma cancer cell line harboring VPS4B loss. CRISPR-SpCas9 screening and integrative genomic analysis revealed other ESCRT members, regulators of abscission and interferon signaling as modifiers of VPS4A dependency. Using the most comprehensive available CRISPR-SpCas9 and RNA-interference screening datasets to date, we provide a compendium of synthetic lethal vulnerabilities with TSG loss and credential VPS4A as a new and promising therapeutic target in cancers with VPS4B/SMAD4 deletion. Citation Format: Jasper E. Neggers, Brenton R. Paolella, Adhana Asfaw, Michael V. Rothberg, Thomas A. Skipper, Radha L. Kalekar, Michael J. Krill-Burger, Neekesh V. Dharia, Guillaume Kugener, Adam D. Durbin, Annan Yang, Nancy Dumont, Yvonne Y. Li, Brian M. Wolpin, Federica Piccioni, David E. Root, Jesse S. Boehm, Andrew D. Cherniack, Aviad Tsherniak, Andrew L. Hong, William C. Hahn, Kimberly Stegmaier, Todd R. Golub, Francisca Vazquez, Andrew J. Aguirre. Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in SMAD4 or CDH1-deleted cancers [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-011.

Abstract: Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Abstract: Background: Discovery of new biomarker-linked cancer therapeutic targets may enable novel drug development and ultimately lead to advances in clinical care. Somatic copy number alterations (CNAs) leading to loss of tumor suppressor gene function constitute important driver events in tumorigenesis. Unfortunately, there are few existing therapeutic options to target the oncogenic processes evoked by tumor suppressor inactivation. However, developing drugs that target tractable synthetic lethal interactions with common somatic CNAs represents a promising approach to attain cancer-selective therapeutics. Synthetic lethality refers to the observation that for certain gene pairs, inactivation of either gene is tolerated but combined loss-of-function of both genes results in decreased cell viability. Synthetic lethal relationships in cancer have been defined in several different contexts, including among paralog genes for which dependency on one paralog is conferred by loss of a second functionally redundant paralog gene. Since targeting synthetic lethal relationships in cancer may yield a wide therapeutic window of efficacy between tumor and normal cells, identification of pharmacologically tractable synthetic lethal targets remains a priority for oncology drug development programs. Results and Discussion: To systematically define synthetic lethal vulnerabilities associated with genomic loss of established tumor suppressor genes, we analyzed genome-scale CRISPR-SpCas9 and RNA interference loss-of-function screening data from over 600 cancer cell lines. We identified and prioritized 193 synthetic lethal interactions with genomic loss of one or more of 51 common tumor suppressor genes. In particular, we discovered that the paralog genes encoding vacuolar protein sorting 4 homolog A and B (VPS4A and VPS4B) are selective genetic vulnerabilities for tumors harboring genomic copy loss of SMAD4 or CDH1 due to co-deletion of VPS4B or VPS4A, respectively. VPS4B is located on the long arm (q) of chromosome 18, 12.3 Mb away from SMAD4, while VPS4A is located 0.476 Mb downstream of CDH1 (encoding E-cadherin) on chromosome 16q. Thus, cancer cells with genomic loss of VPS4B selectively depend on expression of VPS4A for survival, and tumors with loss of VPS4A depend on VPS4B expression. Co-deletion of SMAD4 and VPS4B is commonly observed in approximately 33% of human cancer, with particularly high rates of loss in pancreatic cancers (68%), colorectal (71%) and renal cell carcinomas (17%) and to a lesser extent in cancers of the bile duct, lung, prostate, esophagus, uterus, cervix and ovary. Meanwhile, loss of CDH1 and VPS4A occurs frequently in cancers of the stomach, breast, skin, colon and prostate. VPS4A and B function as AAA ATPases which are critical for the regulation of endosomal sorting complex required for transport (ESCRT), a multimeric protein complex essential for inverse membrane remodeling. The ESCRT machinery is involved in a range of cellular processes, including cytokinesis, membrane repair, autophagy and endosomal processing. VPS4A/B are believed to form asymmetric hexameric complexes that are recruited to ESCRT-III filaments to drive ESCRT-mediated membrane fission and sealing. Here, we demonstrate that suppression of VPS4A in cancer cells with reduced copy number of VPS4B leads to accumulation of CHMP4B-containing ESCRT-III filaments, cytokinesis defects, nuclear membrane abnormalities and micronucleation, ultimately resulting in G2/M cell cycle arrest and apoptosis. We also observed that VPS4 suppression leads to defects in endosomal and endoplasmic reticulum structure. Furthermore, upon VPS4A suppression, we observed potent in vivo tumor regressions, which led to markedly prolonged survival in mouse xenograft models of pancreatic cancer and rhabdomyosarcoma harboring genomic loss of VPS4B. To understand regulators of VPS4A dependency, we performed a CRISPR-SpCas9 genome-scale screen in a pancreatic cancer cell line in the context of VPS4A suppression. We identified multiple genes that promote or suppress VPS4A dependency. Cancer cell sensitivity to VPS4A suppression was potently enhanced by disruption of regulators of the abscission checkpoint, including genes encoding the ULK3 kinase and the ESCRT-III proteins CHMP1A and CHMP1B. The abscission checkpoint is a genome protection mechanism that relies on Aurora B kinase (AURKB) and ESCRT-III subunits to delay abscission in response to chromosome mis-segregation to avoid DNA damage and aneuploidy. These findings suggest that inhibition of the ESCRT pathway and blockade of the abscission checkpoint could provide strategies to further enhance sensitivity of cancer cells to VPS4A suppression. Moreover, through CRISPR-SpCas9 screening and integrative transcriptomic and proteomic analysis, we also identified a strong correlation between baseline interferon response gene expression and VPS4A dependency. Indeed, when we treated VPS4B-deficient cells with interferon-β and interferon-γ to induce interferon signaling, we observed a pronounced sensitization of these cells to VPS4A depletion, thus suggesting that immune signals from the tumor microenvironment may influence VPS4 dependency. These data collectively suggest potential future therapeutic strategies for combination with VPS4A inhibition. Finally, we demonstrate through mutant rescue experiments that the ATPase domain is critical for the function of VPS4A in mediating survival of cells with partial copy loss of VPS4B. Furthermore, we provide data that elucidate the degree to which VPS4A and VPS4B cooperate and form functional complexes in human cancer cells. Although VPS4A and B demonstrate 80.5% homology, the development of small molecules that differentially target VPS4A in cells with VPS4B loss or VPS4B in cells with VPS4A loss remains a tractable possibility due to small structural differences near the ATP-binding pocket. Moreover, combined inhibition of VPS4A and VPS4B may also prove effective and clinically tolerable given a potential therapeutic window arising from gene dosage alterations and differences in total VPS4A/B levels in tumor versus normal cells. Citation Format: Jasper E. Neggers, Brenton Paolella, Adhana Asfaw, Michael V. Rothberg, Tom A. Skipper, Radha Kalekar, Michael Burger, Neekesh Dharia, Guillaume Kugener, Jeremie Kalfon, Nancy Dumont, Yvonne Li, Liam Spurr, Annan Yang, Wenbo Wu, AndrewAdam Durbin, Brian M. Wolpin, David E. Root, Jesse Boehm, Andrew D. Cherniack, Aviad Tsherniak, Andrew L. Hong, William C. Hahn, Kimberly Stegmaier, Todd Golub, Francisca Vazquez, Andrew J. Aguirre. Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q [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 NG01.

Abstract: Somatic copy number alterations that result in loss of tumor suppressor gene function are important drivers of tumorigenesis. However, few existing therapeutic options to target oncogenic processes evoked by tumor suppressor gene inactivation exist. The discovery of synthetic lethal interactions with genetic drivers of cancer may yield new therapeutic strategies with cancer selective potential. We examined genome-scale CRISPR-SpCas9 and RNA interference screens to uncover new synthetic lethal vulnerabilities associated with the loss of common tumor suppressor genes (TSGs). Vacuolar protein sorting 4 homolog A (VPS4A) scored as a strong, selective dependency in cancer cells with genomic loss of the SMAD4 tumor suppressor due to co-deletion of VPS4A's paralog gene, VPS4B. VPS4B resides 12.3 Mb away from the SMAD4 TSG on chromosome 18q and is lost in approximately 33% of all cancers, suggesting broad clinical applicability. VPS4A and VPS4B function as AAA ATPases forming a multimeric protein complex within the endosomal sorting complex required for transport (ESCRT) pathway to regulate membrane remodeling in a range of cellular processes. VPS4A suppression in cells with VPS4B/SMAD4 loss led to accumulation of ESCRT-III filaments, cytokinesis defects, nuclear deformation and micronucleation, which ultimately resulted in G2/M cell cycle arrest and apoptosis. Furthermore, upon VPS4A suppression, we observerd potent in vivo tumor regression, which led to extended survival, in mouse subcutaneous xenograft models with human cancer cell lines harboring VPS4B loss. Finally, genome-scale CRISPR-SpCas9 loss-of-function screening revealed other ESCRT pathway members and regulators of cellular abscission as modifiers of VPS4A dependency. Using the most comprehensive available CRISPR-SpCas9 and RNA-interference screening datasets to date, we provide a compendium of synthetic lethal vulnerabilities with TSG loss and credential VPS4A as a new and promising therapeutic target in cancers with VPS4B/SMAD4 deletion. Citation Format: Jasper E. Neggers, Brenton R. Paolella, Adhana Asfaw, Michael V. Rothberg, Thomas A. Skipper, Radha L. Kalekar, John M. Krill-Burger, Andrew L. Hong, Guillaume Kugener, Jeremie Kalfon, Annan Yang, Chen Yuan, Nancy Dumont, Alfredo Gonzalez, Mai Abdusamad, Yvonne Y. Li, Liam F. Spurr, Westley W. Wu, Federica Piccioni, Brian M. Wolpin, David E. Root, Jesse S. Boehm, Andrew D. Cherniack, Aviad Tsherniak, Todd R. Golub, Francisca Vazquez, Andrew J. Aguirre. VPS4A is a synthetic lethal target in VPS4B-deficient cancers due to co-deletion with SMAD4 [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 LB-053.

Abstract: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Despite multimodality therapy and trials of molecularly targeted agents, limited improvements in overall survival have been realized for patients with high-risk disease. Thus, we aimed to determine the landscape of tumor-specific gene dependencies that underlie tumorigenesis in RMS and therefore provide a valuable group of targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches, we identified a set of RMS-specific tumor dependencies involved in tumor cell growth and survival. RMS dependencies were enriched for nucleic acid binding proteins, including transcription factors (TFs). We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential TFs—MYCN, MYOD1, TCF12, SOX8, ZEB2, ZNF217, and SIX1—are members of the transcriptional core regulatory circuitry (CRC) that maintains the malignant cell state of RMS. Both c-MYC and MYCN were associated with gene and enhancer copy number increases in cell lines and primary tumors and represented strong dependencies in the RMS cell lines screened. c-MYC and MYCN function to similarly invade and regulate the CRC in respectively dependent cells. To disable the CRC, we tested A485, an inhibitor of the histone acetyltransferase enzymes involved in the establishment of super-enhancer elements that are associated with high level expression of the CRC factors. A485 caused a reversible and rapid loss of CRC factor and c-MYC and/or MYCN expression, and prolonged treatment resulted in cell cycle arrest, differentiation, and apoptosis in vitro and in vivo. This phenotype is rescued by exogenous re-expression of either c-MYC or MYCN in a manner insensitive to A485, indicating a mechanism by which these genes subvert a myogenic CRC to produce an oncogenic fate. This study defines a common set of critical dependency genes in RMS and identifies key genomic events surrounding the c-MYC and MYCN loci that lead to elevated expression and tumorigenesis. Citation Format: Adam D. Durbin, Guillaume Kugener, Mark W. Zimmerman, Chuan Yan, Neekesh V. Dharia, Elizabeth S. Frank, Xiang Chen, Ken N. Ross, Brenton Paolella, Michael Krill-Burger, David E. Root, Jesse S. Boehm, Francisca Vazquez, Andrew L. Hong, Aviad Tsherniak, David M. Langenau, William C. Hahn, Todd R. Golub, Brian J. Abraham, Richard A. Young, A. Thomas Look, Kimberly Stegmaier. Rhabdomyosarcoma requires MYC family genomic events to pathogenically subvert core-regulatory circuitry [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B10.