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Highlights • PCC and LG-134 had direct cytotoxicity in murine melanoma cell lines (IC50 values ranged from 0.19–1.8 µM). • PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. • PCC treatment induced G0-G1 cell cycle arrest of melanoma cells. • PCC treatment caused complete regression of established melanoma tumors in all mice. • 17β-hydroxywithanolides have the potential to improve melanoma therapeutic outcome., Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17β-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19–1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17β-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

Existing murine melanoma models, such as syngeneic transplantation of the B16 melanoma cell line, are aggressive, but poorly immunogenic, and as a result, studying the response to immunotherapy in these models has been challenging. Furthermore, in humans, a heterogeneous response to immune checkpoint inhibitor therapy has been observed between male and female melanoma patients, yet no immunogenic murine cancer model exists to study this sex-related dimorphism. To address these challenges, we created the YUMMER.G model, a melanoma cell line driven by human-relevant Braf activation and loss of Pten and Cdkn2a. YUMMER.G is related to the previously described YUMMER1.7 model; however important differences include: diploid state, female genotype, and Mc1re/e (pheomelanin producing) background. YUMMER.G was created by treatment with UV-light, resulting in a neoantigen rich subclone. Subcutaneous injection of 2 million YUMMER.G cells into C57BL/6J mice resulted in melanoma formation in both male and female mice. Treatment of established tumors with anti-CTLA-4 resulted in a comparable response between male and female mice, with complete, durable tumor regression in 100% of female mice and 88% of male mice. However, when established YUMMER.G tumors were treated with anti-PD-1, 63% of female mice, but only 13% of male mice exhibited complete tumor regression (p = 0.0330). These findings demonstrate the important role of host sex in the response to immunotherapy, and they parallel the sex-related differences in checkpoint inhibitor efficacy that have been observed in human patients. In summary, the YUMMER.G model is a human-relevant, immunogenic mouse melanoma model that will serve as a valuable platform for further investigating the mechanisms that mediate the sex-related dimorphism in anti-tumor immunity and response to immunotherapy. Citation Format: Julie Y. Ramseier, Alexandra Charos, Koonam Park, William Damsky, Marcus W. Bosenberg. The YUMMER.G mouse melanoma model recapitulates the heterogeneous response to immune checkpoint blockade based on patient sex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4622.

The sterol regulatory element-binding protein (SREBP) family member SREBP1 is a critical transcriptional regulator of cholesterol and fatty acid metabolism and has been implicated in insulin resistance, diabetes, and other diet-related diseases. We globally identified the promoters occupied by SREBP1 and its binding partners NFY and SP1 in a human hepatocyte cell line using chromatin immunoprecipitation combined with genome tiling arrays (ChIP-chip). We find that SREBP1 occupies the promoters of 1,141 target genes involved in diverse biological pathways, including novel targets with roles in lipid metabolism and insulin signaling. We also identify a conserved SREBP1 DNA-binding motif in SREBP1 target promoters, and we demonstrate that many SREBP1 target genes are transcriptionally activated by treatment with insulin and glucose using gene expression microarrays. Finally, we show that SREBP1 cooperates extensively with NFY and SP1 throughout the genome and that unique combinations of these factors target distinct functional pathways. Our results provide insight into the regulatory circuitry in which SREBP1 and its network partners coordinate a complex transcriptional response in the liver with cues from the diet., Author Summary Transcription factors (TFs) are DNA-binding proteins that regulate the transcription of their target genes. TFs typically bind in proximity to the transcription start sites of their target genes in a region called the promoter. SREBP1 is a TF that increases the transcription of numerous genes involved in cholesterol and fat metabolism and has been linked to diet-related diseases such as insulin resistance and type 2 diabetes. Using microarray technology, we identified all of the promoters in the human genome that are bound by SREBP1 and two associated TFs called NFY and SP1 in a human liver cell line. Our findings greatly expand the number of genes and biological pathways that may be regulated by SREBP1 and reveal that different combinations of SREBP1 and its partners preferentially target genes involved in different pathways. Thus, in contrast to traditional studies that focus on individual genes, we have used a genomics approach to provide a novel global view of the regulatory circuitry in which SREBP1 and its partners coordinate a transcriptional response in the liver with cues from the diet.