No abstract

Purpose Vemurafenib, a selective inhibitor of BRAFV600 has shown significant activity in BRAFV600 melanoma, but not in the <10% of metastatic BRAFV600 colorectal cancers (CRC), suggesting that studies of the unique hypermethylated phenotype and concurrent oncogenic activation of BRAFmut CRC may provide combinatorial strategies Experimental Design We performed comparative proteomic analysis of BRAFV600E melanoma and CRC cell lines, followed by correlation of PI3K pathway activation and sensitivity to the vemurafenib-analog PLX-4720. Pharmacologic inhibitors and siRNA were used in combination with PLX4720 to inhibit PI3K and methyltrasnferase in cell lines and murine models. Results Compared to melanoma, CRC lines demonstrate higher levels of PI3K/AKT pathway activation. CRC cell lines with mutations in PTEN or PIK3CA were less sensitive to growth inhibition by PLX4720 (P=0.03), and knockdown of PTEN expression in sensitive CRC cells reduced growth inhibition by the drug. Combined treatment of PLX4720 with PI3K inhibitors caused synergistic growth inhibition in BRAF-mutant CRC cells with both primary and secondary resistance. In addition, methyltransferase inhibition was synergistic with PLX4720 and decreased AKT activation. In vivo, PLX4720 combined with either inhibitors of AKT or methyltransferase demonstrated greater tumor growth inhibition than PLX4720 alone. Clones with acquired resistance to PLX4720 in vitro demonstrated PI3K/AKT activation with EGFR or KRAS amplification. Conclusions We demonstrate that activation of the PI3K/AKT pathway is a mechanism of both innate and acquired resistance to BRAF inhibitors in BRAFV600E CRC, and suggest combinatorial approaches to improve outcomes in this poor prognosis subset of patients.

The function of the pro-apoptotic molecule BAD is regulated by phosphorylation of two sites, serine-112 (Ser-112) and . Phosphorylation at either site results in loss of the ability of BAD to heterodimerize with the survival proteins BCL-X L or BCL-2. Phosphorylated BAD binds to 14-3-3 and is sequestered in the cytoplasm. It has been shown that phosphorylation of BAD at Ser-136 is mediated by the serine/threonine protein kinase Akt-1/PKB which is downstream of phosphatidylinositol 3-kinase (PI3K). The signaling process leading to phosphorylation of BAD at Ser-112 has not been identi®ed. In this study, we show that phosphorylation of the two serine residues of BAD is di erentially regulated. While Ser-136 phosphorylation is concordant with activation of Akt, Ser-112 phosphorylation does not correlate with Akt activation. Instead, we demonstrate that activated Ras and Raf, which are upstream of mitogen-activated protein kinases (MAPK), stimulate selective phosphorylation of BAD at Ser-112. Furthermore, phosphorylation of Ser-112, but not Ser-136 requires activation of the MAPK pathway as the MEK inhibitor, PD 98059, blocks EGF-, as well as activated Ras-or Raf-mediated phosphorylation of BAD at Ser-112. Therefore, the PI3K-Akt and Ras-MAPK pathways converge at BAD by mediating phosphorylation of distinct serine residues.

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH 2 -terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosinephosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K-dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr 688 or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH 2 -terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr 688 and the NH 2 -terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr 688 in p85 leads to a novel mechanism of PI3K regulation. The PI3K1 signaling cascade has been linked to proliferation, cell survival, differentiation, apoptosis, cytoskeletal rearrangement, and vacuolar trafficking. Growth factor-responsive Class IA PI3Ks consist of heterodimers of a 110-kDa catalytic subunit associated with an 85-kDa noncatalytic regulatory subunit designated p85. The p85 adapter subunits are encoded by at least three different genes with splice variation generating multiple proteins potentially serving many different functions (1). Of the known p85 adapter subunits and splice variants, nearly all contain two Src-homology 2 (SH2) domains, which enable p85 to bind phosphotyrosine in an appropriate amino acid context. The p85 SH2 domains most frequently, but not exclusively, recognize phosphotyrosine embedded in a YXXM motif (2). Most p85 gene products also include a Src homology 3 (SH3) domain, as well as other domains involved in protein-protein interactions (3). All p85 family members contain a p110-binding motif located between the two SH2 domains. The diversity of protein interaction domains found among p85 family members likely contributes to the ability of multiple signaling proteins and pathways to activate PI3K. Under resting conditions, p85 serves to both stabilize p110 protein and inhibi...