Inhibition of oncogenic BRAF triggers immunogenic necrosis of human melanoma cells

Question: Mutant BRAF inhibitors such as vemurafenib and dabrafenib have achieved unprecedented responses in the treatment of melanoma, but the benefits are often of limited duration. On the other hand, durable tumor repression can be achieved by a variety of immunological agents such as the anti-CTLA4 antibody ipilimumab (Yervoy) and antibodies against PD1 in a small proportion of melanoma patients. Combinations of BARF inhibitors and immunological agents are emerging as promising therapeutic approaches. However, the mechanism(s) responsible for the enhanced efficacy remains to be determined. Methods: Here we show that inhibition of BRAFV600E induces programmed necrosis of high immunogenicity in melanoma cells. Results: Although exposure of sensitive BRAFV600E melanoma cells to the BRAF inhibitor PLX4720 triggered strong activation of the caspase cascade, it appeared dispensable for induction of cell death. Instead, PLX4720-induced killing was characterized by early rupture of the cell membrane and release of intracellular contents, indicative of programmed necrosis. Strikingly, killing of sensitive cells by PLX4720 was associated with exposure of calreticulin, characteristic of immunogenic cell death. In contrast, PLX4720 did not cause calreticulin exposure in resistant BRAFV600E melanoma cells, supporting that immunogenicity of melanoma cells triggered by PLX4720 is the consequence of cell death. Indeed, PLX4720 stimulated IFN ® production by autologous lymphocytes in mixed lymphocyte tumor cell culture (MLTC) with sensitive BRAFV600E melanoma cells, but not those resistant to PLX4720 induced killing. Conclusion: Collectively, these results provide a strong rationale for combinations of mutant BRAF inhibitors and immunological agents in the treatment of melanoma, and suggest that induction of immunogenic cell death may be a useful biomarker for prediction of responses of mutant BRAF melanomas to such combinations.2 page(s