MEK1/2 inhibitors sensitize Bcr/Abl+ human leukemia cells to the dual Abl/Src inhibitor BMS-354/825

Abstract

IntroductionChronic myelogenous leukemia (CML) is a stem-cell disease characterized in 95% of cases by the reciprocal translocation of the long arms of chromosomes 9 and 22, resulting in a chimeric fusion protein with constitutively active tyrosine kinase activity (Bcr/ Abl). 1,2 Bcr/Abl signals downstream to multiple survival pathways, including STAT5, Bcl-x L , ERK1/2 (extracellular signal regulated kinase 1/2), and NF-B, among others, which collectively confer a survival advantage on CML cells compared with their normal counterparts. 2,3 The therapy of CML has changed dramatically with the introduction of imatinib mesylate (Gleevec), a tyrosine kinase inhibitor that inhibits Bcr/Abl as well as other kinases including c-Kit. 4,5 Despite the success of imatinib mesylate in CML patients, it is less effective in patients with more advanced disease (eg, accelerated or blast phase). [6][7][8] In addition, patients who initially respond eventually become refractory to imatinib due to the development of increased expression of Bcr/Abl, or more commonly, the appearance of mutations in the kinase domain that prevent drug binding and inhibitory activity. [9][10][11] For these reasons, attempts to circumvent or overcome imatinib mesylate resistance represent the focus of intense interest.One approach to this problem involves combining imatinib mesylate with other signaling inhibitors, and combination studies involving agents such as flavopiridol, 12 farnesyltransferase inhibitors, 13,14 histone deacetylase inhibitors, 15,16 and Akt inhibitors 17 have been described. Another strategy involves the design of second-generation Bcr/Abl kinase inhibitors that are more active than imatinib mesylate and/or able to kill Bcr/Abl ϩ cells that have become resistant to imatinib mesylate. An example of such agents is BMS-354825 (dasatinib), a dual Bcr/Abl and Src kinase inhibitor that is active against Bcr/Abl ϩ cells when administered at nanomolar concentrations. 18,19 Notably, dasatinib is active against cells exhibiting certain Bcr/Abl mutations (eg, E255K, M351T), but is relatively ineffective against cells with T315I mutation, which occupies a "gatekeeper" position in the Bcr/Abl kinase region. 18,20 The relative contribution of Bcr/Abl and Src kinase inhibition in the lethality of dasatinib remains to be fully elucidated. Recent preclinical studies suggest potential benefit for combining imatinib mesylate with Bcr/Abl kinase inhibitors such as dasatinib. 21 The Raf1/MEK1/2/ERK1/2 pathway is an important survival signaling cascade involved in cell proliferation, differentiation, and transformation. [22][23][24] It has also been implicated in the antiapoptotic actions of Bcr/Abl. 2 While MEK activity appears restricted to only one class of substrates, ERK activates more than 70 substrates including nuclear transcription factors. [22][23][24][25] For this reason, several pharmacologic MEK1/2 inhibitors have recently entered the clinic, and have been shown to inhibit their targets (ie, ERK1/2 phosphorylation) when administered at we...

Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik

“…In our hands, the two mechanisms of BimEL inhibition by p-Erk1/2 were observed. In COLO205, NCI-H460, HCT116 and v-src-transformed 3T3 cells, the p-Erk1/2-dependent phosphorylation of BimEL led to a band-shift already described 30 that correlated with its inactivation. 29 In LS174T, the p-Erk1/2-dependent phosphorylation of BimEL led to its degradation.…”

“…29 In NCI-H460 cells, in which Src drove Erk1/2 activation, both dasatinib and PD184352 restored high Bik levels and led to a band-shift described as unphosphorylated BimEL. 30 However, BimEL levels were low and apoptosis restored by Src or Mek1/2 inhibitors depended mainly on Bik, as in 3T3 cells. In COLO205 cells, in which BRAF drove Erk1/2 activation, Bik accumulation and Bim dephosphorylation were observed on Mek1/2 or BRAF (PLX-4720) inhibitions but not Src inhibition.…”

Tumour cell survival signalling by the ERK1/2 pathway

“…The combination of the dual Src/Abl inhibitor dasatinib and PD184352 gave a marked enhancement of apoptosis compared with either drug given alone, including in an imatinib-resistant clone of K562 cells; death under these conditions appears to be due to expression of BIM and the loss of MCL-1. 85 These results highlight the importance of coordinated changes in expression and activity of BCL-2 proteins in the cytotoxic response to TKIs and the potential of augmenting this by co-administration of inhibitors of the ERK1/2 pathway or BH3 mimetics.…”

“…82 In addition, both imatinib and MEK inhibitors induce a similar loss of MCL-1 in CML cells. 80 Despite this, as with EGFR in NSCLC, inhibition of ERK1/2 signalling alone with PD184352 caused little cell death in BCR/ABL-positive leukaemic cells 85 indicating that BCR/ABL uses multiple pathways to ensure cell survival. The combination of the dual Src/Abl inhibitor dasatinib and PD184352 gave a marked enhancement of apoptosis compared with either drug given alone, including in an imatinib-resistant clone of K562 cells; death under these conditions appears to be due to expression of BIM and the loss of MCL-1.…”

BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism

“…Targeting MEK has been reported to synergize with dasatinib and imatinib. 9,15 In cells with mutated BCR-ABL, blocking JAK2 alone induces apoptosis. 14 In DA1-3b/M1, DA1-3b/M2 and DA1-3b/M3, UO126 had limited effect, alone, or associated with imatinib, or with dasatinib ( Figure 5b).…”