Mutationally activated BRAF^V600E (BRAF^VE) is detected in ∼6% of human malignancies and promotes sustained MEK1/2–ERK1/2 pathway activation. We have designed BRaf^CA mice to express normal BRaf prior to Cre-mediated recombination after which BRaf^VE is expressed at physiological levels. BRaf^CA mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to adenocarcinoma. Moreover, BRaf^VE-induced lung tumors were prevented by pharmacological inhibition of MEK1/2. BRaf^VE expression initially induced proliferation that was followed by growth arrest bearing certain hallmarks of senescence. Consistent with Ink4a/Arf and TP53 tumor suppressor function, BRaf^VE expression combined with mutation of either locus led to cancer progression.

Purpose: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

Adenocarcinoma of the lung, a leading cause of cancer death, frequently displays mutational activation of the KRAS proto-oncogene but, unlike lung cancers expressing mutated EGFR, ROS1, or ALK, there is no pathway-targeted therapy for patients with KRAS-mutated lung cancer. In preclinical models, expression of oncogenic KRAS^G12D in the lung epithelium of adult mice initiates development of lung adenocarcinoma through activation of downstream signaling pathways. In contrast, mutationally activated BRAF^V600E, a KRAS effector, fails to initiate lung carcinogenesis despite highly efficient induction of benign lung tumorigenesis. To test if phosphoinositide 3-kinase (PI3K)-α (PIK3CA), another KRAS effector, might cooperate with oncogenic BRAF^V600E to promote lung cancer progression, we used mice carrying a conditional allele of Pik3ca that allows conversion of the wild-type catalytic subunit of PIK3CA to mutationally activated PIK3CA^H1047R. Although expression of PIK3CA^H1047R in the lung epithelium, either alone or in combination with PTEN silencing, was without phenotype, concomitant expression of BRAF^V600E and PIK3CA^H1047R led to dramatically decreased tumor latency and increased tumor burden compared with BRAF^V600E alone. Most notably, coexpression of BRAF^V600E and PIK3CA^H1047R elicited lung adenocarcinomas in a manner reminiscent of the effects of KRAS^G12D. These data emphasize a role for PI3K signaling, not in lung tumor initiation per se, but in both the rate of tumor growth and the propensity of benign lung tumors to progress to a malignant phenotype. Finally, biologic and biochemical analysis of BRAF^V600E/PIK3CA^H1047R-expressing mouse lung cancer cells revealed mechanistic clues about cooperative regulation of the cell-division cycle and apoptosis by these oncogenes. Cancer Res; 73(21); 6448–61. ©2013 AACR.

Pharmacological inhibition of KRAS>RAF>MEK1/2>ERK1/2 signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDAC). Interestingly, combined inhibition of MEK1/2 (with trametinib [T]) plus autophagy (with chloroquine [CQ] or hydroxychloroquine [HCQ]) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDAC cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC–mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ-resistant PDAC tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of P/HCQ.