Abstract 1971: Concurrent loss of PTEN and RB is sufficient to confer BRAF independence in melanomas harboring V600E BRAF mutations
Authors: Xing Feng , Persaud Yogindra , Pratilas Christine , Janakiraman Manickam , She Qing-Bai , Liu Cailian , Dolgalev Igor , Heguy Adriana , Wolchok Jedd , Cobrinik David , Houghton Alan , Taylor Barry , Rosen Neal , Solit David 
Published: February 2011

Abstract
BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. BRAF mutations are, however, common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. With the goal of identifying concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we performed MALDI-TOF mass spectrometry genotyping on a panel of 149 melanoma cell lines. Seventy-nine (53%) harbored BRAF mutations. Proteomic profiling of the class of V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cells lines harboring V600E BRAF mutations were found to express no PTEN protein and high pAKT by immunoblotting. The majority of these cell lines had small (1-2) base-pair insertions or deletions leading to a frame shift and early truncation. Array CGH confirmed focal homozygous deletion of the PTEN gene in three of the models. These deletions were small and in one case detected by the Agilent 1M but not the 244K platform. We have previously reported that mutations of BRAF are associated with enhanced and selective sensitivity to MEK inhibition when compared to either receptor tyrosine kinase-driven cells or cells harboring a RAS mutation. To determine the MAPK pathway dependence of V600E BRAF mutant cell lines as a function of PTEN expression, we used PD0325901, a selective allosteric inhibitor of MEK1/2. All V600E BRAF mutant, PTEN expressing cell lines were sensitive to PD0325901. All but two of the V600E BRAF mutant, PTEN null cells were also dependent upon MEK for proliferation with IC50 values ranging from 1.1 to &gt;500nM. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Further characterization of the two models which displayed complete resistance to MEK inhibition revealed that both harbored concurrent loss of PTEN and RB. These models were also resistant to the selective RAF inhibitor PLX4720. To determine whether loss of RB function was sufficient to confer RAF/MEK-independence in a BRAF mutant, PTEN-null context, we stably infected viral E7 into three MEK-dependent, BRAF mutant, PTEN null models. Infection of E7 led to resistance to MEK-inhibition in all three models. These data suggest that no single mutational event is likely associated with de novo resistance to MEK-inhibition in V600E BRAF mutant melanoma. Loss of PTEN expression and activation of AKT may however be associated with diminished responsiveness to RAF/MEK-inhibition. Further, tumors with concurrent PTEN and RB loss may represent a subset of BRAF mutant patients who derive no clinical benefit from inhibitors of the MAPK pathway such as PLX4032.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1971.