Phosphorylation of tyrosine 393 in the kinase domain of Bcr-Abl influences the sensitivity towards imatinib in vivo

Abstract

The Bcr-Abl fusion protein arising through the t(9;22)(q34;q11) reciprocal translocation is the causative agent in chronic myeloid leukemia and a subset of acute lymphocytic leukemia. Imatinib mesylate is a specific inhibitor of the Bcr-Abl kinase and has shown promising results in clinical studies. The structural relation between the Bcr-Abl oncogene and the tyrosine kinase inhibitor imatinib has recently been elucidated by an elegant crystal structure analysis, emphasizing the importance of dephosphorylated tyrosine 393 (Tyr393) in BcrAbl for access of the inhibitor to the kinase domain. By mutating this tyrosine to phenylalanine and thereby mimicking a constitutively dephosphorylated state, we now show that Ba/ F3 cells transformed by this mutant demonstrate an increased sensitivity towards imatinib in vivo. This effect is not due to an impaired kinase activity of Bcr-Abl Y393F, since a synthetic substrate is phosphorylated with similar kinetics. Treatment of Ba/F3 cells transfected with Bcr-Abl wild type with a phosphatase inhibitor diminished the effect of imatinib, but did not influence the growth of Ba/F3 cells transfected with BcrAblY393F. The results support the findings of the crystal structure and indicate that Tyr393 indeed plays a significant role for the sensitivity of Bcr-Abl towards imatinib in vivo. These data implicate the regulation of Tyr393 phosphorylation as a potential mechanism of imatinib resistance. Leukemia (2003) IntroductionThe tyrosine kinase inhibitor imatinib has recently emerged as an important new treatment option in patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph + ) acute lymphocytic leukemias (ALL) carrying the t(9;22) translocation. 1,2 Unfortunately, many patients with advanced CML and nearly all patients with ALL quickly develop resistance to imatinib treatment. [3][4][5] Imatinib has been shown to bind specifically to the nucleotide-binding pocket in the catalytic domain of Bcr-Abl. 6 Tyrosine 393 (Tyr393) is located in the activation loop of the Bcr-Abl kinase domain and has been proposed to stabilize the activation loop in the open formation when phosphorylated, thereby restricting the access of imatinib to the catalytic region and compromising its inhibitory function. Thus, phosphorylation of Tyr393 may function as a switch, regulating accessibility of imatinib to its binding site in the Bcr-Abl protein. Therefore, other molecules that are able to 'flick the switch' may influence imatinib sensitivity in a Bcr-Abl tranformed cell, and overexpression of kinases or downregulation of phosphatases targeting Tyr393 may induce imatinib resistance. As the importance of this amino acid for imatinib binding has been shown in vitro, we aimed at examining the relevance of phosphorylation of Tyr393 in vivo. We demonstrate here that a mutant Bcr-Abl imitating a constitutively dephosphorylated Tyr393 renders Ba/F3 cells more sensitive towards imatinib inhibition. Materials and methods DNA constructs, cells and transfectionsThe mutation of Tyr3...

JAK of all trades: JAK2-STAT5 as novel therapeutic targets in BCR-ABL1+ chronic myeloid leukemia

“…52,53 The phosphorylation of BCR-ABL1 and JAK2 is reciprocal. Besides v-Src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) kinase family members like v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN), [54][55][56][57][58] JAK2 is able to phosphorylate BCR-ABL1 on tyrosine residue 177. 52 This particular residue is critical for BCR-ABL1-induced disease maintenance as it allows binding of the SH2/SH3 domaincontaining growth factor receptor-bound protein 2 (GRB2) protein and the rat sarcoma (RAS)-activating nucleotide exchange factor son-of-sevenless (SOS), critical components of the pathway by which tyrosine kinases induce RAS activation.…”

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib

“…For the methylcellulose assays, colonies were picked after 14 days and analyzed by flow cytometry. On average, 20 colonies were analyzed from each plate (range [15][16][17][18][19][20][21][22][23][24][25]. Generally, the colony number decreased with higher imatinib concentrations (data not shown) and revealed a strong selection of EGFP þ /Bcr-Abl mutant expressing cells upon addition of imatinib, whereas without imatinib there was no significant difference in the percentage of EGFP þ /Bcr-Abl mutant cells compared to the initially plated ratio (Figure 3c and d).…”

“…It has previously been shown that the bicistronic MIGRI vector which contains the MSCV retroviral LTR promoter followed by an IRES-EGFP cassette allows a slightly higher expression than the MSCV vector carrying a phosphoglycerate promoter driving a neomycin resistance gene. 22 The small difference in activity of the two MSCV-based vectors is thought to be caused by promoter competition in the MSCV-pgk-neo vector. For this reason, we expressed the Bcr-Abl mutants in the MIG-constructs to ensure expression of the Bcr-Abl mutants is at least as high as expression of the Bcr-Abl wt construct.…”

“…22 Briefly, 293 cells were transfected with p210 wt p210 T315I or p210 Y253H constructs in the MIG-vector and empty MIG-vector as control. After 48 h, the cells were lysed and the Bcr-Abl protein was precipitated with an anti-Abl antibody (K-12, Santa Cruz, Heidelberg, Germany) from the lysates of p210 wt and p210 mut transfected cells.…”

“…22 For BcrAbl wt and mutant expression analysis, NIH3T3 murine fibroblast cells or Ba/F3 murine pre-B lymphocyte cells were infected with the different retroviral constructs. The infected cells were lysed, run on a polyacrylamide gel, blotted onto nylon membrane and probed with anti-Abl (8E9), antiphosphotyrosine (4G10) or antib-Actin antibodies (BD Pharmingen, Heidelberg, Germany and Biomol, Hamburg, Germany).…”