Abstract 3562: Loss of the PTEN and RB1 tumor suppressors attenuates RAF-dependence in melanomas

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Oncogenic BRAF mutations are found in ∼8% of human tumors, with the highest frequency observed in melanoma (40-70%). BRAF mutations are common in nevi suggesting that BRAF mutation alone is insufficient for tumorigenesis. To identify concurrent mutations that condition BRAF/MEK dependence in V600E BRAF melanoma, we genotyped a panel of 149 melanoma cell lines. Seventy-eight cell lines (52%) harbored BRAF mutations with 72 of which are V600E mutants. Proteomic profiling of 41 out of the 72 V600E BRAF mutant cell lines demonstrated significant variability in the expression of PTEN and pAKT. Nine cell lines harboring V600E BRAF mutations express no PTEN protein and high pAKT by immunoblotting. All of the nine cell lines harbored small (1-2) base-pair insertions or deletions, missense mutations or focal deletions encompassing the PTEN locus. 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. These data suggest that PTEN loss is insufficient to confer resistance to MAPK-pathway inhibition. Examination of the two models which displayed complete resistance to MEK inhibition (IC50 > 3,000 nM) revealed the presence of concurrent loss of PTEN and RB1. Inactivation of Rb functions by expressing human papillomavirus E7 protein in three BRAF mutant, PTEN-null models led to failure of the cells to arrest in G1 and complete resistance to MEK-inhibition. Moreover, RB1 alterations were mutually exclusive with loss of p16INK4A, suggesting that whereas p16INK4A and RB1 may have overlapping roles in preventing tumor formation, tumors with functional loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. V600EBRAF/RB-null/PTEN-null models were also resistant to the selective RAF inhibitor PLX4720. Consistent with these in vitro results, V600EBRAF/RB-null/PTEN-null xenografts were resistant to RAF and MEK inhibition whereas V600EBRAF/WTRB/PTEN-null cells exhibited an intermediate phenotype. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the MAP kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2011-3562