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Acquired resistance to chemotherapy is perhaps the major barrier to cure of advanced cancers. Testicular germ cell tumors (TGCTs) are the most common cancers of young males. TGCTs are one of the few solid tumors that are cured at a high rate with standard cisplatin-based chemotherapy. However, TGCTs become resistance to cisplatin resulting in poor outcomes. While the mechanisms accounting for cisplatin hypersensitivity in TGCT remain elusive, it has been suggested that DNA hypermethylation may play a role. However, there is little direct experimental evidence to support this claim. In the present study, we have leveraged unique isogenic cell models to provide strong evidence for net DNA hypermethylation as a key mechanism leading to acquired cisplatin resistance in TGCT cells. Genome-wide DNA methylation analysis revealed that hypomethylated CpG loci were enriched for SUZ12 and EZH2 binding and H3K27me3 sites. In contrast hypermethylated CpG loci were significantly enriched for CTCF and RAD21 domains as well as repressive DNA segments indicating the global remodeling of nuclear chromatin structure in resistant cells. Integrative computational analysis of RNA-seq and EPIC DNA methylation array data revealed a strong inverse correlation between gene expression and CpG gene promoter methylation. This analysis also revealed a strong bidirectional shift between gene promoter and gene body DNA methylation that correlated closely with upregulated expression of PRC2 target genes and down regulation of tumor suppression genes in resistant cells. Hence the data further suggests a functional interplay between DNA methylation and the PRC2 pathway that modulates cisplatin sensitivity of TGCTs. Further studies including H3K27me3 ChIP-seq are ongoing to further elucidate this relationship. In conclusion, our finding supports the hypothesis that genome wide remodeling of DNA methylation is associated with cisplatin hypersensitivity and resistance of TGCTs and further supports the use of hypomethylation therapy for relapsed TGCT patients. Citation Format: Zeeshan Fazal, Ratnakar Singh, Megan Tomlin, Doha Shokry, Aleyah Hattab, Sarah Spinella, Michael Spinella. Genome wide remodeling of DNA methylation and CpG hypermethylation is tightly linked to acquired resistance in testicular 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 2096.

Testicular germ cell tumors (TGCTs) are the most frequent solid cancers of males between the ages of 16 to 39 and have the highest mortality in this age group. Testicular cancer is highly curable with platinum-based chemotherapeutics. However, 15-20% of patients are resistant and succumb to their disease. The mechanisms for cisplatin sensitivity/resistance in testicular cancer is unclear. Previously we showed that cisplatin refractory testicular cancer is highly sensitive to the DNA methyltransferase inhibitors (DNMTi), 5-Aza-2'-deoxycytidine (5-AZA-DC) and guadecitabine in vivo and in vitro. High expression of DNMT3B correlates with sensitivity to DNMTi in TGCT cells. Based on these preclinical findings we recently completed a phase 1 clinical trial which demonstrated the combination of guadecitabine and cisplatin was tolerable and effective in treating patients with platinum refractory germ cell cancer. To better understand the mechanism of chemotherapy sensitivity/resistance in testicular cancer we generated a series of cisplatin resistant cell models. Genome wide methylome array and de novo transcriptional profiling of cisplatin resistant TGCT cells revealed decreased global H3K27 methylation due to suppression of the polycomb repressive complex 2 (PRC2 ). To better understand the relationship between cisplatin and 5-AZA-DC sensitivity, 5-AZA-DC resistant testicular cancer cells were also generated. Remarkably, there was a reciprocal relationship between cisplatin and 5-AZA-DC resistance, with cisplatin resistance associated with increased sensitivity to 5-AZA-DC and 5-AZA-DC resistance associated with increased sensitivity to cisplatin. Transcriptomics profiling and other studies in 5-AZA-DC resistant cells revealed increased H3K27me3, repression of polycomb target genes and a dramatic decreased in DNMT3B expression, the exact opposite of the situation in cisplatin resistant cells. Further knockdown of DNMT3B resulted in induction of H3K27me3, decreased sensitivity to 5-AZA-DC and increased sensitivity to cisplatin. These data suggest that DNA methylation and polycomb signaling are interconnected drivers of chemotherapy and epigenetic therapy responses in TGCTs. Ongoing H3K27 me3 ChIP-Seq, global methylation analysis, and validation studies will be described to further elucidate these mechanisms with the goal of devising novel epigenetic-based therapies for testicular and potentially other cancers. Citation Format: Ratnakar Singh, Emmanuel Bikorimana, Zeeshan Fazal, Cliff Yerby, Hannah Baldwin, Aleyah Destiny Hattab, Megan Tomlin, Andrea K. Corbet, Raya Iman Boyd, Khadeeja Shahid, Doha Naguib Ahmed Mohamed Shokry, Sarah Joy Spinella, Michael J. Spinella. Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents [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 2127.