Epigenetic Regulation of Melanoma-Associated Fibroblast Plasticity - PROJECT SUMMARY Histone variant dysfunction has profound consequences for tumor cells; however, their role in the tumor microenvironment (TME) remains unclear. Using an autochthonous, immunocompetent BRAFV600E/PTEN- deficient primary melanoma model, I showed that mice devoid of macroH2A (dKO) develop larger tumors, accompanied by accumulation of immunosuppressive monocytes and depletion of functional cytotoxic T cells. This compromised anti-tumor response stems from intrinsic upregulation of inflammatory genes, including Ccl2, Cxcl1 and Il6, in cancer-associated fibroblasts (CAFs), whose frequency and activation increase in the absence of macroH2A. dKO CAFs also downregulate myofibroblast-associated genes, raising the possibility that macroH2A impacts fibroblast polarization during tumor progression, skewing the balance between inflammatory (iCAFs) and myofibroblast (myCAF) subtypes. Mechanistically, this novel function for macrohistones in the melanoma TME involves 3D chromatin organization, as we found that macroH2A loss leads to increased looping between inflammatory genes and active enhancers. We hypothesize that epigenetic modulation of CAFs could direct their phenotype, in turn affecting immune cell activity in the melanoma TME. I aim to define how CAF hyper-activation in the absence of macroH2A shapes tumor initiation and the ensuing immune response. To this aim, I will leverage primary CAF cultures derived from human melanoma (PDMCAFs). I will first characterize how transcription of macroH2A genes is regulated in PDMCAFs to give rise to variable macroH2A levels. Next, I will establish to what extent PDMCAF cultures represent the functional CAF subtypes present in vivo in melanoma. Third, I will determine if macroH2A epigenetically regulates inflammatory gene loci in human cells by coordinating chromatin looping. Finally, I will test if macroH2A deficiency skews fibroblast polarization towards an inflammatory vs. myofibroblastic phenotype in CAFs during tumor progression. Therapeutic approaches that leverage the TME, such as immunotherapy, cure a subset of otherwise deadly melanomas. However, CAF plasticity hampers their efficacy and can have both tumor-promoting and restraining effects. The published study I led was the first to demonstrate a role for a histone variant in modulating the inflammatory properties of the melanoma stroma. Addressing whether similar mechanisms affect CAF plasticity in human melanoma is particularly timely, as epigenetic deregulation may promote a cold immune microenvironment. Therefore, this proposed project will make an important contribution to understanding how epigenetic deregulation within the TME could potentially impact melanoma initiation, response to therapy, progression, recurrence, and/or dormancy.
