Abstract
Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.
Highlights
Constitutively-activated tyrosine kinase mutants play important roles in development and evolution of hematopoietic malignancies and are implicated in acquisition of therapy resistance
We previously reported that hematopoietic cytokines, such as IL-3 and Epo, enhance Chk1-mediated cell cycle checkpoint activation by etoposide through inhibition of GSK3 by activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, inhibiting etoposide-induced apoptosis [13]
We examined the effect of GSK3 inhibition and found that its specific inhibitor GSK3-I #5 prevented apoptosis induced by the combined treatment with etoposide and JakI-1 in UT7/Jak2-V617F cells and significantly restored the G2/M arrest (Fig. 1A), as expected from our previous observations [13]
Summary
Constitutively-activated tyrosine kinase mutants play important roles in development and evolution of hematopoietic malignancies and are implicated in acquisition of therapy resistance. BCR/ABL confers survival and proliferation advantages on hematopoietic cells by activating various intracellular signaling pathways, such as those involving Ras, Raf-1, MEK, Erk, phosphatidylinositol 3-kinase (PI3K), Akt, STAT5, and NFkB, which normally play roles in regulation of hematopoiesis by hematopoietic cytokine receptors that activate the Jak family tyrosine kinases, including Jak2 [1,2]. The tyrosine kinase mutation most frequently found in acute myeloid leukemia (AML) is the internal tandem duplication (ITD) mutation of FLT3, a receptor tyrosine kinase that plays a critical role in regulation of hematopoietic progenitor cells [5,6]. FLT3ITD and FLT3 with an activating amino acid substitution in the tyrosine kinase domain, such as FLT3-D835Y, constitutively activate the PI3K/Akt and MEK/Erk signaling pathways as well as STAT5 to stimulate proliferation and enhance survival of hematopoietic cells. Controversial results have been reported for FLT3-D835Y, FLT3-ITD has been associated with therapy resistance and established as a poor prognostic factor for AML [6]
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