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High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. IMPLICATIONS: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy., (©2019 American Association for Cancer Research.)

Germ-cell tumours (GCTs) are derived from germ cells and occur most frequently in the testes. GCTs are histologically heterogeneous and distinctly curable with chemotherapy. Gains of chromosome arm 12p and aneuploidy are nearly universal in GCTs, but specific somatic genomic features driving tumour initiation, chemosensitivity and progression are incompletely characterized. Here, using clinical whole-exome and transcriptome sequencing of precursor, primary (testicular and mediastinal) and chemoresistant metastatic human GCTs, we show that the primary somatic feature of GCTs is highly recurrent chromosome arm level amplifications and reciprocal deletions (reciprocal loss of heterozygosity), variations that are significantly enriched in GCTs compared to 19 other cancer types. These tumours also acquire KRAS mutations during the development from precursor to primary disease, and primary testicular GCTs (TGCTs) are uniformly wild type for TP53. In addition, by functional measurement of apoptotic signalling (BH3 profiling) of fresh tumour and adjacent tissue, we find that primary TGCTs have high mitochondrial priming that facilitates chemotherapy-induced apoptosis. Finally, by phylogenetic analysis of serial TGCTs that emerge with chemotherapy resistance, we show how TGCTs gain additional reciprocal loss of heterozygosity and that this is associated with loss of pluripotency markers (NANOG and POU5F1) in chemoresistant teratomas or transformed carcinomas. Our results demonstrate the distinct genomic features underlying the origins of this disease and associated with the chemosensitivity phenotype, as well as the rare progression to chemoresistance. These results identify the convergence of cancer genomics, mitochondrial priming and GCT evolution, and may provide insights into chemosensitivity and resistance in other cancers.

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.