Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments. BH3 mimetics against members of the BCL-2 family have gained excitement with the recent success in hematological malignancies. However, single drug BH3 mimetic therapy in melanoma has limited effectiveness due to escape by the anti-apoptotic protein MCL-1 and/or survival of melanoma-initiating cells (MICs). We tested the efficacy of the BH3 mimetic combination of A-1210477 (an MCL-1 inhibitor) and ABT-263 (a BCL-2/BCL-XL/BCL-W inhibitor) in killing melanoma, especially MICs. We also sought to better define Dynamin-Related Protein 1 (DRP-1)’s role in melanoma; DRP-1 is known to interact with members of the BCL-2 family and is a possible therapeutic target for melanoma treatment. We used multiple assays (cell viability, apoptosis, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing techniques. For clinical relevance, we employed patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found the BH3 mimetic combination kill both the MICs and non-MICs (bulk of melanoma) in all cell lines and patient samples irrespective of the mutation status or relapsed state (p < 0.05). Unexpectedly, the major pro-apoptotic proteins, NOXA and BIM, are not necessary for the combination-induced cell death. Furthermore, the combination impedes the activation of DRP-1, and inhibition of DRP-1 further enhances apoptosis (p < 0.05). DRP-1 effects in melanoma differ from those seen in other cancer cells. These results provide new insights into BCL-2 family’s regulation of the apoptotic pathway in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death even without involvement from major pro-apoptotic proteins. Importantly, our study also indicates that DRP-1 inhibition is a promising adjuvant for BH3 mimetics in melanoma treatment.

Dendritic cell (DC) activation is characterized by sustained commitment to glycolysis that is a requirement for survival in DC subsets that express inducible NO synthase (Nos2) due to NO-mediated inhibition of mitochondrial respiration. This phenomenon primarily has been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6) mice, where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced postactivation survival due to attenuated NO production in LPS-stimulated PWD DCs phenocopying human monocyte-derived DCs. To genetically map this phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. B6.PWD-Chr11.2 DCs show preserved mitochondrial function and produce lower NO levels than B6 DCs. We demonstrate that activated B6.PWD-Chr11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production to investigate the contribution of NO in regulating the interplay between DC metabolism and immune function. These findings suggest that reported differences between human and murine DCs may be an artifact of the limited genetic diversity of the mouse models used, underscoring the need for mouse genetic diversity in immunology research.

In 2018, 9,000 of those people will die from melanoma (American Cancer Society, 2018). In melanoma like many cancers, a mutation in the Ras protein has been implicated in excessive cell proliferation. A farnesyl transferase inhibitor, FTI‐277, has shown the ability to lower Ras membrane association by disrupting Ras prenylation. Additionally, Lovastatin, a cholesterol lowering drug, is associated with decreased Ras membrane association. Unprenylated Ras cannot attached to the cell membrane or initiate signals in downstream effector proteins. Previous studies have shown that these two drugs cannot kill melanoma tumors individually, but the combinatorial effects have not been examined. We hypothesize that FTI‐277 will be a more potent inhibitor of Ras prenylation/activation than Lovastatin due to Lovastatin's high cytotoxicity. A metastatic melanoma cell line (A375) was treated and Ras prenylation and membrane association examined using immunofluorescence. Flow cytometry showed that the minimum concentration of 10 mM of each drug individually and in combination did not result in more than 30% cell death. Results showed that high concentrations of both drugs induced cell toxicity and at 10 mM Lovastatin was more cytotoxic than FTI‐277 (mean= 30.67%, 10.69% respectively, p=0.017). Cell image analysis showed that the average corrected total cell fluorescence for individual treatment with each drug was significantly lower than both the vehicle control and treatment with both drugs (p<0.05). Together this shows that treatment with these drugs is efficacious for melanoma due to their abilities to induce apoptosis and alter Ras membrane association.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dendritic cells (DCs), the professional antigen presenting cells of the immune system, undergo metabolic reprogramming in response to stimulation through Toll-Like Receptors (TLRs). Our lab and others have shown that metabolic reprogramming in DCs is accompanied by increased reliance on glycolytic metabolism through use of glucose and glycogen as fuel. Both metabolites are required by DCs to perform immune functions associated with activation including secretion of cytokines, upregulation of costimulatory molecules, and initiation of the T cell-mediated immune response. While our lab has published that glycogen is required within 4–8 hours after activation, production of nitric oxide (NO) regulates many metabolic processes during the later stages of activation via NO-dependent suppression of mitochondrial respiration. Recent studies have shown that glycogen pathway intermediate uridine diphosphate glucose (UDPG) is continually elevated in inflammatory macrophages past 24 hours of activation. Additionally, UDPG can be exported extracellularly and act as an autocrine signaling molecule to increase proinflammatory gene expression. We tested the hypothesis, that, like inflammatory macrophages, DCs elevate production of UDPG in response to activation and that this metabolite can bolster NO-mediated processes during DC activation. To investigate the role of UDPG in DCs, we characterized DC mitochondrial respiration in response to activation by LPS and the addition of exogenous UDPG. Additionally, we performed functional assays to assess NO production, cytokine secretion, as well as inflammatory gene expression to begin elucidating an axis by which UDPG and NO work together to regulate DC metabolism and immune function. Supported by grants from UVM (COBRE VCIID Funding Support and Pilot Project) and NIH (NIAID 1R21AI135385-01A1)

Current melanoma treatment have limitations of relapse. BH3 mimetics against BCL-2 family members have gained excitement with recent success in hematological cancers. However, single drug BH3 therapy not effective in melanoma due to escape by the anti-apoptotic protein MCL-1 and/or survival of Melanoma Initiating Cells (MICs). Melanoma progression correlates with increase in Dynamin-related protein 1 (DRP1). DRP1 interacts with BCL-2 family members, but its potential effects on BH3 mimetic treatment is not defined in melanoma. This study targeted the above components to develop treatment options for melanoma. We tested the efficacy of the BH3 mimetics combination of A-1210477 (MCL-1 inhibitor) and ABT-263 (BCL-2/BCL-XL/BCL-W inhibitor), and examined how inhibiting DRP1 may influence this effect. We used multiple assays (cell viability, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing technique. To make the study clinically relevant, we utilized patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found that the BH3 mimetic combination de-bulks and kills MICs in all samples irrespective of the mutation status or relapsed state (p<0.05). Unexpectedly, cell death occurs independent of major pro-apoptotic proteins such as NOXA,BIM or BID. Moreover, the combination treatment impedes the activation of DRP1 and inhibition of DRP1 further enhances the combination-induced apoptosis (p<0.05). This finding is different from those seen in other cancers. We are currently studying how manipulation of DRP1 affects BCL-2 family members in melanoma. These results suggest that inhibiting the major anti-apoptotic proteins are sufficient to induce cell death in melanoma even without involvement from major pro-apoptotic proteins, and provide new insights into BCL-2 familys regulation of the apoptotic pathway. Importantly, our study also indicate that DRP1 Inhibition is a promising adjuvant for BH3 mimetics in melanoma treatment.