KRAS is frequently mutated in lung cancer. Whereas the mitogen-activated protein kinase (MAPK) is a well-known effector pathway of KRAS, blocking this pathway with clinically-available MAPK inhibitors is relatively ineffective. Here, we report that epithelial-to-mesenchymal transition rewires the expression of receptor tyrosine kinases, leading to differential feedback activation of the MAPK pathway following MEK inhibition. In epithelial-like KRAS mutant lung cancers, this feedback was attributed to ERBB3-mediated activation of MEK and AKT. In contrast, in mesenchymal-like KRAS mutant lung cancers, FGFR1 was dominantly expressed but suppressed by the negative regulator sprouty proteins; MEK inhibition led to de-repression of SPRY4 and subsequent FGFR1-mediated re-activation of MEK and AKT. Therapeutically, the combination of MEK inhibitor and FGFR inhibitor induced cell death in vitro and tumor regressions in vivo. These data establish the rationale and a therapeutic approach to treat mesenchymal-like KRAS mutant lung cancers effectively with clinically available FGFR1 and MAPK inhibitors.

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.

SummaryFewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.

High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential. We performed high-throughput drug screening of epigenetic-targeted therapies across a large and diverse tumor cell line panel and uncovered the hypersensitivity of neuroblastoma cells to GSK-J4, a small-molecule dual inhibitor of lysine 27 of histone 3 (H3K27) demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and histone demethylase Jumonji D3 (JMJD3). Mechanistically, GSK-J4 induced neuroblastoma differentiation and endoplasmic reticulum (ER) stress, with accompanying up-regulation of p53 up-regulated modulator of apoptosis (PUMA) and induction of cell death. Retinoic acid (RA)–resistant neuroblastoma cells were sensitive to GSK-J4. In addition, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Furthermore, GSK-J4 and RA combination increased differentiation and ER stress over GSK-J4 effects and limited the growth of neuroblastomas resistant to either drug alone. In MYCN-amplified neuroblastoma, PUMA induction by GSK-J4 sensitized tumors to the B cell lymphoma 2 (BCL-2) inhibitor venetoclax, demonstrating that epigenetic-targeted therapies and BCL-2 homology domain 3 mimetics can be rationally combined to treat this high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust antineuroblastoma activity.

Tumor cells have devised several strategies to block the mitochondrial pathway of apoptosis despite endogenous or pharmacological cues to die. This process of cell death proceeds through the coordinated regulation of multiple anti-apoptotic and pro-apoptotic BCL-2 family proteins that ultimately impinge on the integrity of the outer mitochondrial membrane. Once compromised, mitochondria release pro-apoptotic factors to promote caspase activation and the apoptotic phenotype. Within the BCL-2 family exists a subclass of pro-apoptotic members termed the BH3-only proteins, which directly and/or indirectly functionally regulate the remaining anti- and pro-apoptotic BCL-2 proteins to compromise mitochondria and engage apoptosis. The focus of this review is to discuss the cellular and pharmacological regulation of the BH3-only proteins to gain a better understanding of the signaling pathways and agents that regulate this class of proteins. As the BH3-only proteins increase cellular sensitivity to pro-apoptotic agents such as chemotherapeutics, numerous small-molecule BH3 mimetics have been developed and are currently in various phases of clinical trials. Toward the end of the review, the discovery and application of the small-molecule BH3 mimetics will be discussed.