Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL, Jak2-V617F, or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

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.

Down regulation of Chk1 by p53 plays a role in synergistic induction of apoptosis by chemotherapeutics and inhibitors for Jak2 or BCR/ABL in hematopoietic cells

“…To test the possible involvement of p53 in these processes, we examined Ton.32D/pRevTRE-p53-DD cells. In accordance with our previous reports [16, 17], inhibition of Jak2 by JakI-1 prevented etoposide-induced accumulation of these cells in the G2/M phase and drastically enhanced apoptosis, as shown in Figure 2A. Very similar results were obtained by using the Jak1/Jak2 inhibitor ruxolitinib clinically in use for myeloproliferative neoplasms (Supplementary Figure S2) [15].…”

“…We have previously shown that inhibition of cytokine-induced activation of Jak2 signaling pathway induces apoptosis synergistically with chemotherapeutics in hematopoietic cells by down regulating Chk1-mediated G2/M checkpoint activation through mechanisms involving activation of GSK3 [16, 17]. To test the possible involvement of p53 in these processes, we examined Ton.32D/pRevTRE-p53-DD cells.…”

“…We have previously revealed that the aberrant tyrosine kinases, such as BCR/ABL and Jak2-V617F, as well as hematopoietic cytokines, such as IL-3 and Epo, enhance Chk1-mediated cell cycle checkpoint activation by chemotherapeutics through inhibition of GSK3 by activating the PI3K/Akt pathway, thus protecting hematopoietic cells from induction of apoptosis [16, 17]. Thus, apoptosis induced by chemotherapeutics was synergistically enhanced by inhibition of these aberrant kinases or the PI3K/Akt pathway by clinically relevant inhibitors, such as imatinib or pictilisib (GDC-0941), through down regulation of Chk1-mediated cell cycle arrest [17].…”

“…Thus, apoptosis induced by chemotherapeutics was synergistically enhanced by inhibition of these aberrant kinases or the PI3K/Akt pathway by clinically relevant inhibitors, such as imatinib or pictilisib (GDC-0941), through down regulation of Chk1-mediated cell cycle arrest [17]. We have also found that imatinib synergistically induced apoptosis of BCR/ABL-expressing cells caused with nutlin-3, which induces p53 expression by inhibiting its Mdm2-mediated degradation [18].…”

Investigational FMS-like tyrosine kinase 3 inhibitors in treatment of acute myeloid leukemia

“…Another area of investigation is the hypoxic bone marrow niche, as demonstrated by Konopleva et al 73,74 . Multiple studies have suggested that dual inhibition of both PI3K and FLT3 may serve as a means of overcoming resistance of the bone marrow stroma as a protective environment for leukemic cells, and should be further investigated as a novel therapeutic approach in patients with FLT3 -mutated AML 75,76 . Another potential mechanism of acquired resistance to FLT3 kinase inhibitors is the development of point mutations in the kinase domain, notably the FLT3 D835 point mutation 77 .…”