The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr 253 and Tyr 257 ) in the ATP binding loop (P-loop) of BCR-ABL when Thr 315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr 315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.chronic myelogenous leukemia ͉ kinase inhibitor resistance ͉ phosphoproteomics ͉ imatinib ͉ dasatinib P oint mutations in the kinase domain of BCR-ABL are primarily responsible for resistance to the ABL inhibitor imatinib (Gleevec) in chronic myelogenous leukemia (CML) patients. The majority of imatinib-resistant BCR-ABL point mutations (of Ͼ50 distinct examples now reported clinically) impair drug binding by restricting flexibility of the enzyme, precluding adoption of the inactive conformation required for imatinib binding (1-4). The second generation Abl inhibitor dasatinib is effective against imatinib-resistant CML because it binds the BCR-ABL kinase domain regardless of activation loop conformation (5-7). As a result, the number of BCR-ABL mutations capable of conferring resistance to dasatinib is small and is limited almost exclusively to direct contact sites (8, 9). One mutation, T315I, confers resistance to imatinib, dasatinib, and the imatinib-related compound nilotinib (AMN-107) (10, 11).Although discovered in the context of drug resistance, there is growing evidence that these mutations may confer other fitness advantages to BCR-ABL. First, the T315I and E255K mutants were each detected by our group in imatinib-naïve CML blast crisis patients by direct sequencing of total BCR-ABL cDNA and are therefore estimated to account for at least 20% of the CML tumor burden in these patients (1). In addition, these and other kinase domain mutations have been identified before treatment in CML patients using mutation-specific quantitative PCR (12-18). Furthermore, the analogous mutation to T315I in the...

Objective-Previous studies have shown that oxidized products of PAPC (Ox-PAPC) regulate cell transcription of interleukin-8, LDL receptor, and tissue factor. This upregulation takes place in part through the activation of sterol regulatory element-binding protein (SREBP) and Erk 1/2. The present studies identify vascular endothelial growth factor receptor 2 (VEGFR2) as a major regulator in the activation of SREBP and Erk 1/2 in endothelial cells activated by Ox-PAPC. Methods and Results-Ox-PAPC induced the phosphorylation of VEGFR2 at Tyr 1175 in human aortic endothelial cells. Inhibitors and siRNA for VEGFR2 decreased the transcription of interleukin-8, LDL receptor, and tissue factor in response to Ox-PAPC and the activation of SREBP and Erk 1/2, which mediate this transcription. We provide evidence that the activation of VEGFR2 is rapid, sustained, and c-Src-dependent. Conclusions-These data point to a major role of VEGFR2 in endothelial regulation by oxidized phospholipids which accumulate in atherosclerotic lesions and apoptotic cells.

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