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Selective Vulnerability of Aneuploid Human Cancer Cells to Inhibition of the Spindle Assembly Checkpoint

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Selective Vulnerability of Aneuploid Human Cancer Cells to Inhibition of the Spindle Assembly Checkpoint bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159038; this version posted June 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Selective vulnerability of aneuploid human cancer cells to inhibition of the spindle assembly checkpoint Yael Cohen-Sharir1, James M. McFarland2, Mai Abdusamad2, Carolyn Marquis3, Helen Tang2, Marica R. Ippolito4, Sara V. Bernhard5, Kathrin Laue1, Heidi L.H. Malaby3, Andrew Jones2, Mariya Kazachkova2, Nicholas Lyons2, Ankur Nagaraja2,6, Adam J. Bass2,6, Rameen Beroukhim2,6, Stefano Santaguida4,7, Jason Stumpff 3, Todd R. Golub2,4, Zuzana Storchova5 and Uri Ben-David1,# 1 Department of Human Molecular Genetics and Biochemistry, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA 3 Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT, USA 4 Department of Experimental Oncology at IEO, European Institute of Oncology IRCCS, Milan, Italy 5 Department of Molecular Genetics, TU Kaiserlautern, Kaiserlautern, Germany 6 Dana Farber Cancer Institute, Boston, MA, USA 7 Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy # Corresponding author: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159038; this version posted June 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Selective targeting of aneuploid cells is an attractive strategy for cancer treatment. Here, we mapped the aneuploidy landscapes of ~1,000 human cancer cell lines and classified them by their degree of aneuploidy. Next, we performed a comprehensive analysis of large-scale genetic and chemical perturbation screens, in order to compare the cellular vulnerabilities between near- diploid and highly-aneuploid cancer cells. We identified and validated an increased sensitivity of aneuploid cancer cells to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis. Surprisingly, we also found highly-aneuploid cancer cells to be less sensitive to short-term exposures to multiple inhibitors of the SAC regulator TTK. To resolve this paradox and to uncover its mechanistic basis, we established isogenic systems of near-diploid cells and their aneuploid derivatives. Using both genetic and chemical inhibition of BUB1B, MAD2 and TTK, we found that the cellular response to SAC inhibition depended on the duration of the assay, as aneuploid cancer cells became increasingly more sensitive to SAC inhibition over time. The increased ability of aneuploid cells to slip from mitotic arrest and to keep dividing in the presence of SAC inhibition was coupled to aberrant spindle geometry and dynamics. This resulted in a higher prevalence of mitotic defects, such as multipolar spindles, micronuclei formation and failed cytokinesis. Therefore, although aneuploid cancer cells can overcome SAC inhibition more readily than diploid cells, the proliferation of the resultant aberrant cells is jeopardized. At the molecular level, analysis of spindle proteins identified a specific mitotic kinesin, KIF18A, whose levels were drastically reduced in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to KIF18A depletion, and KIF18A overexpression restored the sensitivity of aneuploid cancer cells to SAC inhibition. In summary, we identified an increased vulnerability of aneuploid cancer cells to SAC inhibition and explored its cellular and molecular underpinnings. Our results reveal a novel synthetic lethal interaction between aneuploidy and the SAC, which may have direct therapeutic relevance for the clinical application of SAC inhibitors. bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159038; this version posted June 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Aneuploidy, defined as copy number changes were independent of the specific that encompass an entire chromosome-arm or chromosome15,16. As cancer cells are almost a whole chromosome, is the most prevalent invariably aneuploid3, whereas normal cells genetic alteration in human cancer. ~90% of are (almost) always euploid17, the the solid tumors and ~75% of the identification of aneuploidy-targeting drugs hematologic malignancies are aneuploid1, has been a long sought-after goal of cancer and >25% of the tumor genome is affected, research. on average, by aneuploidy2,3. The process that gives rise to aneuploidy is chromosomal In yeast, several aneuploidy-specific cellular 18–20 instability (CIN), characterized by high vulnerabilities have been described . For prevalence of chromosome mis-segregation example, aneuploid budding yeast have and other mitotic aberrations4. Aneuploidy elevated levels of proteotoxic stress, and CIN are associated with clinical features, presumably due to the perturbation of the such as tumor stage, differentiation status and required stoichiometry of protein 19 disease aggressiveness5,6. However, our complexes . Another example is the knowledge of how CIN and aneuploidy drive increased sensitivity of aneuploid yeast to 20,21 tumorigenesis is rather limited4,7. perturbation of sphingolipid metabolism . Consequently, no existing therapeutics Although some of these findings were directly exploit this hallmark of cancer for the successfully recapitulated in mammalian 21,22 treatment of cancer patients. cells , the systematic identification of aneuploidy-induced vulnerabilities remains Aneuploidy comes with a substantial fitness elusive in human cancer. A major reason for cost, yet is well-tolerated by cancer cells1,8. that is that aneuploidy is notoriously difficult The genetic or epigenetic events that lead to to study: it affects multiple genes at once, it aneuploidy formation during tumorigenesis, plays distinct roles in different contexts, and or that allow cancer cells to tolerate it, may it is hard to engineer experimentally. also generate unique vulnerabilities in Moreover, a high degree of aneuploidy is aneuploid cells (compared to their euploid closely associated with other genomic counterparts). While some aneuploidy- alterations, such as whole-genome induced dependencies would be specific to duplication (WGD) and p53 the altered chromosome4,9–12, others may be inactivation3,23,24; and with cell division the consequence of more general cellular defects, such as CIN and micronuclei burdens associated with an abnormal number formation25–27. Large-scale studies are of chromosomes, independently of specific therefore required in order to control for karyotypes. Indeed, aneuploidy causes potentially-confounding factors, and isogenic various types of cellular stress responses: in vitro systems are then required to validate proteotoxic, metabolic, replicative, mitotic differential dependencies and dissect them and hypo-osmotic4,8,13,14. Consistent with a mechanistically. general cellular response to aneuploidy, introduction of aneuploidy into mammalian cells activated transcriptional programs that bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159038; this version posted June 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Aneuploid cancer cells are selectively Data Fig. 1c). The full results of these sensitive to genetic perturbation of SAC comparisons are presented in core members Supplementary Table 2. DAVID functional enrichment analysis revealed that the list of To identify dependencies associated with a genes that are selectively essential in high degree of aneuploidy, we evaluated the aneuploid cancer cells is highly enriched for aneuploidy landscapes of 997 human cancer cell cycle-related pathways; in particular, the cell lines, using published copy number regulation of mitotic progression and the profiles from the Cancer Cell Line spindle assembly checkpoint came up as the 28 Encyclopedia . Each cell line was assigned top selectively-essential pathway (Fig. 1c, 3,11 an “aneuploidy score” (AS) based on the Extended Data Fig. 1d and Supplementary number of chromosome arms gained or lost Table 3). Two core members of the spindle in that cell line, relative to its basal ploidy assembly checkpoint (SAC; also known as (Fig. 1a, Extended Data Fig. 1a and the mitotic checkpoint), BUB1B (also known Supplementary Table 1). We then analyzed as BUBR1) and MAD2 (also known as the association of aneuploidy with gene MAD2L1), were at the top of the hit list (Fig. essentiality, using two distinct data sets of 1b,d, Extended Data Fig. 1b,e and loss-of-function shRNA screens across 689 Supplementary Table 2). Analysis of the 29–31 and 712 cell lines (Methods). Using the Achilles-CRISPR/Cas9 data set32 confirmed calculated aneuploidy scores as a novel that highly-aneuploid cell lines
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