Analysis of B-RafV600E Regulated MicroRNAs and Proteins in Melanoma

Genome sequencing studies have identified oncogenic B-Raf mutations in over 70% of all sporadic melanomas. The B-RafV600E mutation leads to elevated kinase activity and constitutive activation of the mitogen-activated protein (MAP) kinase signaling pathway, which supports a variety of oncogenic functions critical to melanoma formation and progression. Metastatic melanomas are highly resistant to conventional chemotherapeutic treatments, and pharmacological inhibitors of B-RafV600E have been incompletely effective in pre-clinical studies. Understanding the global molecular responses to constitutive MAP kinase signaling in melanomas would allow the development of more effective therapeutics. Manipulation of microRNA (miRNA) expression is emerging as an important molecular mechanism by which oncogenes can broadly support tumorigenesis. In this study, strategic microarray profiling of melanoma cells identified a network of over 25 miRNAs that are controlled by B-RafV600E. Functional screening of all regulated microRNAs revealed that many were capable of altering cell growth and/or invasion, and I was able to identify important melanoma genes that were targeted by one or more B-RafV600E miRNA(s). Interestingly, several genes suppressed by multiple miRNAs were direct targets for both induced and repressed miRNAs, suggesting a highly complex interaction of co-regulated miRNAs may contribute to overall gene regulation. The strong overlap in targets and functions suggested that BRafV600E miRNAs might work cooperatively, and I showed that miRNAs work combinatorially to further augment cellular invasion responses. Previously, a similar DNA microarray profiling analysis discovered a set of axon guidance genes repressed by activated MAP kinase signaling in melanoma cells. I validated one of these genes, semaphorin 3C (sema3C), as suppressed by B-RafV600E through a transcriptional mechanism involving the co-regulation of TFAP2A, ATF3, and JUN transcription factors. Functional analyses in both primary and metastatic melanoma cell lines using strategies for sema3C inhibition or over-expression identified a novel role for this semaphorin in melanoma cell migration and invasion, as well as tumor growth and metastasis. By using microarray profiling studies to identify global molecular responses to activated MAP kinase signaling in melanoma cells, I established multiple microRNAs and an axon guidance gene, sema3C, as novel molecular targets controlled by B-RafV600E and functionally relevant in melanoma progression

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials

BRAF Modulates Lipid Use and Accumulation

There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers

Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity

Abstract Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity

Melanoma and microbiota:Current understanding and future directions

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of “omics” technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.</p

Melanoma and microbiota:Current understanding and future directions

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of “omics” technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.</p