Exploiting Immunogenic Cell Death for Melanoma benefit

Human skin melanoma remains one of the most aggressive and difficult to treat human malignancies, with increasing incidence over years. Although sun exposure represents the main cause of melanomagenesis, it is now evident the association with several gene mutations. Among these, the RAS/RAF/MEK pathway seems to be the most affected, with BRAFV600E present in approximately 70% of all melanomas. The resulting constitutively active enzyme determines an uncontrolled cell proliferation, inactivation of cell death pathways, autophagy dysregulation and resistance to any treatment. We recently evidenced the molecular mechanisms by which BRAFV600E directly establishes a chronic ER stress status responsible for an enhanced basal autophagy and resistance to cell death. New effective therapies are therefore urgently needed to treat this malignancy and sustain patient’s survival. Immunogenic cell death have recently emerged as a powerful treatment in which the direct involvement of the immune response helps drugs to kill cancer cells, thus boosting the antitumor treatment. This relies on the stimulation of specific signaling named DAMPs with a pro-immunogenic response during the execution of the apoptotic pathway. Here we show that Doxorubicin (DOXO) and Mitoxantrone (MTX) kill melanoma cells by inducing apoptosis, with BRAFV600E cells resulting less sensitive to the treatment. Our data also show that in both DOXO- or MTX-treated cells specific DAMPs required to induce an effective immune response are stimulated. In particular, we observed an early i) production of type I interferons (a & b) and ii) secretion of ATP, iii) post-mortem release of HGMB1 and and iv) early ERp57-mediated translocation of calreticulin onto the plasma membrane. We also evidenced that both treatment resulted in the key phosphorylation of eIF2a, confirming this factor as a hallmark of ICD. However, our results indicate that the latter event is not the result of ER stress induction, as previously indicated, but possibly due to the specific activation of GCN2, since its down-regulation resulted in almost a complete abrogation of eIF2a phosphorylation, at lest in our model. Importantly, we observed no influence of oncogenic BRAF expression in the production/release of both DOXO- or MTX-stimulated DAMPs. All together our data indicate that ICD may represent a new valuable alternative pathway to efficiently kill both wt and oncogenic BRAF human skin melanoma cells.