Project 3 will focus on efforts to understand and overcome acute resistance to BRAF(V600E) suppression in melanoma by understanding how BRAF(V600E) modulates death signals. Multiple studies show that BRAF(V600E) suppression strongly induces BIM, a pro-apoptotic factor. However very little apoptosis occurs. Instead either MCL1 or BCL2A1 bind &sequester BIM. We report BCL2A1 as a newly recognized genomically amplified melanoma oncogene in about 1/3 of patients. It is also transcriptionally regulated by MITF, a lineage specific master transcription factor which is also amplified in ~20% of melanomas. BCL2A1 and MITF are thus lineage-restricted anti-apoptotic regulators. We found that BRAF-MEK-MAPK phosphorylates MITF, triggering ubiquitin-dependent degradation. Conversely BRAF(V600E) suppression blocks MITF degradation, thereby upregulating MITF. In turn MITF's targets are potently induced?some of which are anti-apoptotic while others serve as immune antigens. The immune system may contribute to efficacy of BRAF(V600E) inhibitors because 1) treatment induces T cell tumor infiltrates within 10 days, 2) melanocytic antigen expression is strongly induced via MITF, and 3) BRAF( /600E) targeting is significantly weaker for melanomas in an immunodeficient background. To pursue these questions Aim1 will examine MITF's and BCL2A1's roles as apoptosis antagonists to BRAF inhibitors. We will utilize clinically annotated patient specimens to determine whether genomic amplifications in MITF or BCL2A1 are prognostic for response to BRAF(V600E) targeting. Further, the use of gene targeting (knockdown) suggests that MITF and BCL2A1 are lineage specific antagonists to inhibition of BRAF sensitivity. Therefore several small molecules which antagonize MITF or BCL2A1, and which are clinically available, will be tested alone or with BRAF suppression in vitro, in mice, and (for MITF antagonism) in a recently opened clinical trial.
Aim 2 will dissect interactions between BRAF(V600E), MITF, and melanoma immune responses in immune-intact mice. Inhibitors to BRAF, MEK, or ERK will be combined with immune checkpoint blocking antibodies (anti-CTLA4 anti-PDLI, or anti-PDI) and tested in mice, and in correlative studies for a human trial for vemurafenib+ipilimumab. Distinct vulnerabilities in "resistant" lines will be analyzed against copy number and deep sequencing data (Project 1, 2 &Cores A, B) and shRNA functional screens (Projects 1 &2) to identify drugable combination pathways for preclinical and clinical development.

Public Health Relevance

The melanoma field has seen significant therapeutic progress, but little durable benefit (cure) from the new, targeted drugs. This Project seeks to improve melanoma therapy by identifying drug combinations and incorporation of immune modulation, which we believe may profoundly enhance the ability to kill melanoma cells upon suppression of BRAF(V600E). Successful implementation of this work will lead to molecular predictors of treatment efficacy and new lead-combinations for clinical development in man.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Massachusetts General Hospital
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Roider, Elisabeth M; Fisher, David E (2014) The impact of MITF on melanoma development: news from bench and bedside. J Invest Dermatol 134:16-7
Konieczkowski, David J; Johannessen, Cory M; Abudayyeh, Omar et al. (2014) A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov 4:816-27
Van Allen, Eliezer M; Wagle, Nikhil; Sucker, Antje et al. (2014) The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma. Cancer Discov 4:94-109
Lo, Jennifer A; Fisher, David E (2014) The melanoma revolution: from UV carcinogenesis to a new era in therapeutics. Science 346:945-9
Wagle, Nikhil; Van Allen, Eliezer M; Treacy, Daniel J et al. (2014) MAP kinase pathway alterations in BRAF-mutant melanoma patients with acquired resistance to combined RAF/MEK inhibition. Cancer Discov 4:61-8
Sullivan, Ryan J; Fisher, David E (2014) Understanding the biology of melanoma and therapeutic implications. Hematol Oncol Clin North Am 28:437-53
Pop, Marius S; Stransky, Nicolas; Garvie, Colin W et al. (2014) A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein. Mol Cancer Ther 13:1492-502
Chen, Hongxiang; Weng, Qing Y; Fisher, David E (2014) UV signaling pathways within the skin. J Invest Dermatol 134:2080-5
Fell, Gillian L; Robinson, Kathleen C; Mao, Jianren et al. (2014) Skin ?-endorphin mediates addiction to UV light. Cell 157:1527-34
Hsiao, Jennifer J; Fisher, David E (2014) The roles of microphthalmia-associated transcription factor and pigmentation in melanoma. Arch Biochem Biophys 563:28-34

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