PROTAC-mediated degradation of Bcl-xL potentiates target therapy in preclinical melanoma models.

backgroundBcl-xL plays an important role in tumors from different origins, including melanoma, and for this reason it has been widely targeted with small-molecule BH3 mimetics, which unfortunately show several adverse effects. To overcome this limitation, selective Bcl-xL proteolysis-targeting chimera degraders have been developed. Among these, DT2216, a candidate in phase I/II clinical trials, has demonstrated antitumoral activity in preclinical cancer models from different origins, not including melanoma.

methodsBy using several established and patient-derived BRAF wild type and mutated melanoma cells, we performed western blot analysis and MTT assay to study DT2216 effect on Bcl-xL protein levels and cell viability, respectively. Combination studies were performed on BRAF mutated melanoma cells treated with DT2216 and Dabrafenib/Trametinib or on wild type melanoma cells treated with DT2216 and Trametinib or S63845. Combination index was calculated to study drug interactions. Apoptotic induction was studied through western blot (PARP-1 cleavage), cytofluorimetric (subG1 peak in the cell cycle) and live-cell fluorescent imaging of activated caspases 3/7 analyses. Group differences were analysed with a two-sided paired or unpaired Student's t-test. To investigate the effect of the combination treatment in vivo, A375luc melanoma cells were inoculated in xenograft mice, then treated with Dabrafenib/Trametinib or DT2216, alone or in combination, for three weeks. Differences between groups, were analysed with Mann-Whitney test.

resultsDT2216 induced the specific and long-lasting degradation of Bcl-xL protein, and reduced cell viability, in a concentration-dependent manner. Of note, a positive correlation between Bcl-xL degradation and sensitivity to DT2216 was observed, being cells with higher degradation the most sensitive to DT2216. In combination studies, DT2216 was able to enhance the activity of target therapy regardless BRAF mutational status. Moreover, the Mcl-1 specific inhibitor, S63845, potentiated the efficacy of DT2216 in melanoma cells in which DT2216 determined an increase of Mcl-1 protein. Interestingly, DT2216 also increased the activity of target therapy in melanoma cells resistant to Dabrafenib and Trametinib. Finally, experiments in a xenograft mouse melanoma model highlighted DT2216 potentiating effect of target therapy, not only inducing a significant reduction of tumor growth, but also showing a longer disease control.

conclusionOur findings provide new insights for combination therapy including Bcl-xL degradation for melanoma treatment.