Abstract SY17-03: Targeting BRAF and PI3′-kinase signaling for therapy of melanoma.
Authors: McMahon Martin , Das Thakur Meghna , Marsh Victoria , Silva Jillian , Landman Allison , Deuker Marian , Salangsang Fernando , Pryer Nancy , Phillips Wayne , Levesque Mitchell , Dummer Reinhard , Bosenberg Marcus , Sellers William R. , Stuart Darrin 
Published: August 2014

Abstract
Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with greater than 50% of tumors expressing the BRAF(V600E) oncoprotein. BRAF(V600E) is a constitutively active protein kinase that promotes sustained activation of BRAF→MEK→ERK signaling in the melanoma cell. Importantly, the marked tumor regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF(V600E) in melanoma maintenance. However, since most patients relapse with lethal vemurafenib resistant disease, understanding and preventing mechanism(s) of drug resistance will be critical to extending the durability of patients' responses.
Although mutational activation of BRAF is often the initiating event, progression of BRAF mutated melanomas is frequently associated with silencing of the PTEN tumor suppressor, a negative regulator of PI3′-kinase signaling. Whilst PTEN silencing is common in human melanoma, mutational activation of PIK3CA (encoding the alpha catalytic subunit of PI3′-kinase) is extremely rare, even though PIK3CA mutations are common in other human cancers. Since PTEN is reported to have tumor suppressor functions independent of its PI3′-lipid phosphatase activity, PTEN silencing may have more profound tumor promoting activity than mutational activation of PIK3CA. To test this, we generated mice carrying a conditional knock-in allele of Pik3ca that permits Cre-induced expression of PIK3CA(H1047R), a mutationally activated form of PIK3CA detected in many cancer types. Expression of PIK3CA(H0147R) in melanocytes elicited no obvious phenotype, consistent with the absence of phenotype in PTEN(Null) melanocytes. As observed previously, melanocyte-specific expression of BRAF(V600E) combined with PTEN silencing led to metastatic melanoma. Interestingly, metastatic melanoma also arose from melanocytes expressing both BRAF(V600E) and PIK3CA(H1047R), although these tumors grew more slowly than their BRAF(V600E)/PTEN(Null) counterparts. Both BRAF(V600E)/PTEN(Null) and BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the tumor prevention effects of pharmacological blockade of class 1 PI3′-kinases. However, the clinically relevant subset of BRAF(V600E)/PTEN(Null) melanoma was resistant to pharmacological blockade of AKT. Using genetically engineered mouse models and melanoma-derived cell lines, we demonstrate cooperative anti-tumor effects of combining RAF and PI3′-kinase inhibitors, suggesting that combined pathway-targeted chemotherapy may maximize melanoma patients' therapeutic response while delaying the onset of lethal drug resistant disease.
Using patient derived xenografts propagated in immunocompromised mice we explored the cause and consequences of drug resistance, which was selected by continuous vemurafenib administration. As observed previously, resistant tumors displayed continued dependence on BRAF(V600E)→MEK→ERK signaling due to elevated BRAF(V600E) expression. However, remarkably, we observed that vemurafenib-resistant melanomas displayed a striking drug dependency for their continued proliferation, such that cessation of drug administration led to regression of established drug-resistant tumors. We further demonstrated that a discontinuous dosing strategy, which exploits the fitness deficit displayed by drug-resistant cells in the absence of the vemurafenib, forestalls the onset of lethal drug resistant disease. These data highlight the concept that drug-resistant cells may also show dependency, such that altered drug dosing may prevent the emergence of lethal drug resistance.
Citation Format: Martin McMahon, Meghna Das Thakur, Victoria Marsh, Jillian Silva, Allison Landman, Marian Deuker, Fernando Salangsang, Nancy Pryer, Wayne Phillips, Mitchell Levesque, Reinhard Dummer, Marcus Bosenberg, William R. Sellers, Darrin Stuart. Targeting BRAF and PI3′-kinase signaling for therapy of melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY17-03. doi:10.1158/1538-7445.AM2013-SY17-03