SUMMARY Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RASG12V mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RASG12V-induced mitochondrial dysfunction, and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1S616 phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1S616 phosphorylation status dichotomizes BRAFWt from BRAFV600E positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with unexpected leverage in chemotherapeutic success.

SUMMARY Pro-apoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP, and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define pro-apoptotic BCL-2 protein composition after stress, and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for pro-apoptotic BCL-2 family function. Cells with hyper-fragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies an unexpected mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

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In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis, and eliminate cancer cells. In practice, drugs (e.g., PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, due 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. While 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.