Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q
Authors: Neggers Jasper E. , Paolella Brenton , Asfaw Adhana , Rothberg Michael V. , Skipper Tom A. , Kalekar Radha , Burger Michael , Dharia Neekesh , Kugener Guillaume , Kalfon Jeremie , Dumont Nancy , Li Yvonne , Spurr Liam , Yang Annan , Wu Wenbo , Durbin AndrewAdam , Wolpin Brian M. , Root David E. , Boehm Jesse , Cherniack Andrew D. , Tsherniak Aviad , Hong Andrew L. , Hahn William C. , Stegmaier Kimberly , Golub Todd , Vazquez Francisca , Aguirre Andrew J. 
Published: July 2021

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.