Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Hirundo is the most species-rich genus of the passerine swallow family (Hirundinidae) and has a cosmopolitan distribution. Here we report the complete, annotated mitochondrial genomes for 25 individuals from 10 of the 14 extant Hirundo species; these include representatives from four subspecies of the barn swallow, H. rustica. Mitogenomes were conserved in size, ranging from 18,500 to 18,700 base pairs. They all contained 13 protein-coding regions, 22 tRNAs, a control region, and large and small ribosomal subunits. Phylogenetic analysis resolved most of the relationships between the studied species and subspecies which were largely consistent with previously published trees. Several new relationships were observed within the phylogeny that could have only been discovered with the increased amount of genetic material. This study represents the largest Hirundo mitochondrial phylogeny to date, and could serve as a vital tool for other studies focusing on the evolution of the Hirundo genus.

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Treatment options for melanoma have progressed in recent years, with the rise of targeted therapy and immunotherapy. Options are still limited for patients that are BRAF wildtype, treatment refractory, and patients with non-sun exposed melanoma subtypes that typically do not respond to standard therapies. Therefore, it is necessary to explore other biological processes that may provide new therapeutic approaches. One of most appealing is the BCL-2 family of apoptotic/anti-apoptotic system that is effective in leukemias, with BH3 mimetics venetoclax alone or in combination with the hypomethylating agent 5-azacitidine. We examined similar combinations on the group of eleven patient-derived and commercially available melanoma cell lines. The BH3 mimetics we tested include MCL1 inhibitors (S63845, AMG 176, AZD5991), an inhibitor of BCL2, BCLXL, and BCLW (navitoclax/ABT-263), a BCL2 inhibitor (venotoclax/ABT-199), and a BCLXL inhibitor (A1331852). Our data showed that treatments with MCL1 inhibitors plus 5-azacitidine are effective in reducing melanoma cell viability. S63845 was the most potent compound when combined with 5-azacitidine, with nine out of eleven (~80%) melanoma cell lines achieving less than 50% viability at the highest dose. There was an overall weaker response to treatment with 5-azacitidine in combination with ABT-263 or A1331852, with only 54% and 45% of cell lines responding at the highest dose, respectively. Response to ABT-199 with 5-Azacitidine was highly limited, 18% of cell lines responded at the highest concentration. Our data demonstrate that treatment with S63845 in combination with 5-azacitidine is highly effective in cell lines from a variety of melanoma subtypes, including the rare uveal and mucosal subtypes. In vivo and mechanistic experiments are planned. The novel combination of the MCL1 inhibitor S63845 and 5-azacitidine may be a promising alternative therapeutic target in melanoma. Citation Format: Chiara Dart, Nabanita Mukherjee, Carol Amato, Robert J. Van Gulick, Morgan MacBeth, Kasey Couts, Jacqueline Turner, Stacey Bagby, Mayumi Fujita, William Robinson, Yiqun Shellman. The therapeutic potential of 5-Azacitidine plus various BH3 mimetics in melanoma [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 1805.