Anti-apoptotic BCL-2 proteins govern cellular outcome following B-RAF(V600E) inhibition and can be targeted to reduce resistance.

In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.

Sensitization to the mitochondrial pathway of apoptosis augments melanoma tumor cell responses to conventional chemotherapeutic regimens.

Metastatic malignant melanoma is highly resistant to chemotherapy, and the average survival rate is under 1 year. The only FDA-approved conventional chemotherapy (i.e., dacarbazine) targets melanoma tumor cells by inducing a form of cell death referred to as apoptosis. However, dacarbazine exhibits a response rate of ~5%, and combination chemotherapies consisting of cisplatin, vinblastine, and dacarbazine often offer little clinical advantage over dacarbazine alone. Apoptosis is governed by the BCL-2 family of proteins, which is comprised of anti-apoptotic and pro-apoptotic members. To determine if the anti-apoptotic BCL-2 repertoire established the cell death threshold and chemoresistance in melanoma, a novel treatment strategy was designed to inhibit the anti-apoptotic BCL-2 members with ABT-737. Using various melanoma model systems, we determined the affects of ABT-737 on sensitivity to dacarbazine-based regimens. Strikingly, ABT-737 re-sensitized melanoma cell lines to common chemotherapeutics leading to marked BIM-mediated apoptosis. Cellular features of the ABT-737 combination treatments were loss of proliferation, mitochondrial fragmentation, nuclear condensation, phosphatidylserine exposure, and decreased clonogenic survival. We also observed significant anti-tumor activity in an in vivo melanoma model system. Our data indicate that ABT-737 may be a beneficial adjuvant therapy to improve melanoma response rates when conventional chemotherapy is the only option.