FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4–11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.
Fms-like tyrosine kinase 3 (Flt3) 2 also known as fetal liver kinase-2 (Flk-2) is a receptor-tyrosine kinase expressed on hematopoietic progenitors and regulates early steps of hematopoietic progenitor cell proliferation, survival, and differentiation (1, 2). Wild-type Flt3 (Flt3-WT) has been shown to be highly expressed in several hematopoietic malignancies including 70 -100% cases of acute myeloid leukemia (AML). Oncogenic internal tandem duplication (ITD) mutations in the juxtamembrane domain of Flt3 (Flt3-ITD) and point mutations within the tyrosine kinase domain, such as the most predominant D835Y mutation, are the most frequent kinase mutations in AML, occurring in 25-30 and 5-10% of cases, respectively, and are associated with poor prognosis (1-3). Flt3-ITD results in ligand-independent autophosphorylation and activation of the receptor with subsequent activation of multiple downstream targets, including the signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase, and Akt pathways. Flt3-ITD has been shown to induce a myeloproliferative disorder in murine models. Flt3-ITD exists partially in an immature, underglycosylated, constitutively phosphorylated form (4). Aberrant intracellular localization and activity of Flt3-ITD generate oncogenic phosphorylation patterns and aberrantly activate signaling cascades (5). Because Flt3 or Flt3-ligand (FL) knock-out mice have only a subtle hematopoietic stem/progenitor cell deficit and show no significant disadvantage in viability, interference with deregulated Flt3 functions appears as a promising treatment option for AML (1-3). Thus, several Flt3 inhibitors are currently under evaluation for their efficacy in AML patients with Flt3-ITD.Recent studies showed that both activated Flt3 and mutant Flt3 are degraded through the proteasome (6 -11). The proteasomal degradation of targeted proteins, including protein kinases, requires polyubiquitination catalyzed by a series of enzymes containing ubiquitin activating enzymes (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3) (12, 13). E3 ubiquitin ligases confer substrate specificity and are responsible for mediating the transfer of ubiquitin from E2s to the substrates. The Cbl proteins are a highly conserved family of RING finger E3 ubiquitin ligases that regulate signaling by receptor and non-receptor-tyrosine kinases, including EGF receptor, PDGF receptor ␣, and FMS (14). There are three mammalian Cbl proteins encoded by separate genes: c-Cbl, Cbl-b, and Cbl-c. These proteins contain an N-terminal phosphotyrosine binding domain that allows direct interaction with activated receptortyrosine kinases and a RING finger domain that classifies Cbl
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.
The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.
) Subcutaneous versus intravenous administration of bortezomib in patients with relapsed multiple myeloma: a randomised, phase 3, noninferiority study. The Lancet Oncology, 12, 431-440. Reeder, C.B., Reece, D.E., Kukreti, V., Chen, C., Trudel, S., Hentz, J., Noble, B., Pirooz, N.A., Spong, J.E., Piza, J.G., Zepeda, V.H., Mikhael, J.R., Leis, J.F., Bergsagel, P.L., Fonseca, R
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.