Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML).
ABSTRACT: Bcr-Abl is the predominant therapeutic target in chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) that inhibit Bcr-Abl have been successful in treating CML. With progression of CML disease especially in blast crisis stage, cells from CML patients become resistant to imatinib mesylate (IM) and other TKIs, resulting in relapse. Because Bcr-Abl is known to drive multiple signaling pathways, the study of the regulation of stability of Bcr-Abl in IM-resistant CML cells is a critical issue as a possible therapeutic strategy. Here, we report that a new dual-kinase chemical inhibitor, ON044580, induced apoptosis of Bcr-Abl+ IM-sensitive, IM-resistant cells, including the gatekeeper Bcr-Abl mutant, T315I, and also cells from blast crisis patients. In addition, IM-resistant K562-R cells, cells from blast crisis CML patients, and all IM-resistant cell lines tested had reduced ability to form colonies in soft agar in the presence of 0.5 µM ON044580. In in vitro kinase assays, ON044580 inhibited the recombinant Jak2 and Abl kinase activities when the respective Jak2 and Abl peptides were used as substrates. Incubation of the Bcr-Abl+ cells with ON044580 rapidly reduced the levels of the Bcr-Abl protein and also reduced the expression of HSP90 and its client protein levels. Lysates of Bcr-Abl+ cell lines were found to contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90, and its client proteins as detected by a gel filtration column chromatography, which was rapidly disrupted by ON044580. Therefore, targeting Jak2 and Bcr-Abl kinases is an effective way to destabilize Bcr-Abl and its network complex, which leads to the onset of apoptosis in IM-sensitive and IM-resistant Bcr-Abl+ cells. This inhibitory strategy has potential to manage all types of drug-resistant CML cells, especially at the terminal blast crisis stage of CML, where TKIs are not clinically useful.
Project description:Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) become drug resistant by mutations within the kinase domain of Bcr-Abl, and by other changes that cause progression to advanced stage (blast crisis) and increased expression of the Lyn tyrosine kinase, the regulation of which is not understood yet. In Bcr-Abl+ cells inhibition of Jak2, a downstream target of Bcr-Abl, by either Jak2 inhibitors or Jak2-specific short interfering RNA (siRNA) reduced the level of the SET protein, and increased PP2A Ser/Thr phosphatase and Shp1 tyrosine phosphatase activities, which led to decreased levels of activated Lyn. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn kinase compared with Jak2 inhibition alone. In contrast, inhibition of either PP2A or Shp1 combined with Jak2 inhibition interfered with the loss of Lyn kinase activation more so than Jak2 inhibition alone, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase caused by Jak2 inhibition. Inhibition of Jak2 induced apoptosis and reduced colony formation in IM-sensitive and -resistant Bcr-Abl mutant cell lines. Jak2 inhibition also induced apoptosis in CML cells from blast crisis patients but not in normal hematopoietic cells. These results indicate that Lyn is downstream of Jak2, and Jak2 maintains activated Lyn kinase in CML through the SET-PP2A-Shp1 pathway.
Project description:Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.
Project description:Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr-Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr-Abl+ cells reduced levels of the Bcr-Abl protein and also the phosphorylation of Tyr177 of Bcr-Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr-Abl+ cells with Jak2 inhibitors for 4-6?h but not with IM also reduced Bcr-Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr-Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr-Abl in immune complexes but did not reduce levels of Bcr-Abl, suggesting that the reduction of Bcr-Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr-Abl stability and oncogenic signaling in CML cells are under the control of Jak2.
Project description:BACKGROUND:Imatinib mesylate (IM) induces clinical remission of chronic myeloid leukemia (CML). The Abelson helper integration site 1 (AHI-1) oncoprotein interacts with BCR-ABL and Janus kinase 2 (JAK2) to mediate IM response of primitive CML cells, but the effect of the interaction complex on the response to ABL and JAK2 inhibitors is unknown. METHODS:The AHI-1-BCR-ABL-JAK2 interaction complex was analyzed by mutational analysis and coimmunoprecipitation. Roles of the complex in regulation of response or resistance to ABL and JAK2 inhibitors were investigated in BCR-ABL (+) cells and primary CML stem/progenitor cells and in immunodeficient NSG mice. All statistical tests were two-sided. RESULTS:The WD40-repeat domain of AHI-1 interacts with BCR-ABL, whereas the N-terminal region interacts with JAK2; loss of these interactions statistically significantly increased the IM sensitivity of CML cells. Disrupting this complex with a combination of IM and an orally bioavailable selective JAK2 inhibitor (TG101209 [TG]) statistically significantly induced death of AHI-1-overexpressing and IM-resistant cells in vitro and enhanced survival of leukemic mice, compared with single agents (combination vs TG alone: 63 vs 53 days, ratio = 0.84, 95% confidence interval [CI] = 0.6 to 1.1, P = .004; vs IM: 57 days, ratio = 0.9, 95% CI = 0.61 to 1.2, P = .003). Combination treatment also statistically significantly enhanced apoptosis of CD34(+) leukemic stem/progenitor cells and eliminated their long-term leukemia-initiating activity in NSG mice. Importantly, this approach was effective against treatment-naive CML stem cells from patients who subsequently proved to be resistant to IM therapy. CONCLUSIONS:Simultaneously targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may improve outcomes in patients destined to develop IM resistance.
Project description:Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on the treatment of chronic myeloid leukemia (CML). However, TKI monotherapy is not curative, with relapse and persistence of leukemic stem cells (LSCs) remaining a challenge. We have recently identified an AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance in CML stem/progenitor cells. JAK2 thus emerges as an attractive target for improved therapies, but off-target effects of newly developed JAK2 inhibitors on normal hematopoietic cells remain a concern. We have examined the biological effects of a highly selective, orally bioavailable JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34+ treatment-naïve IM-nonresponder cells. Combination therapy reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits proliferation and colony growth, and eliminates CML LSCs in vitro. Importantly, BMS-911543 selectively targets CML stem/progenitor cells while sparing healthy stem/progenitor cells. Oral BMS-911543 combined with the potent TKI dasatinib more effectively eliminates infiltrated leukemic cells in hematopoietic tissues than TKI monotherapy and enhances survival of leukemic mice. Dual targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may consequently lead to more effective disease eradication, especially in patients at high risk of TKI resistance and disease progression.
Project description:Chronic myeloid leukemia (CML) is caused by the fusion of the BCR activator of RhoGEF and GTPase activating protein (BCR) and ABL proto-oncogene, the nonreceptor tyrosine kinase (ABL) genes. Although the tyrosine kinase inhibitors (TKIs) imatinib (IM) and nilotinib (NI) have remarkable efficacy in managing CML, the malignancies in some patients become TKI-resistant. Here, we isolated bone marrow (BM)-derived mesenchymal stem cells (MSCs) from several CML patients by Ficoll-Hypaque density-gradient centrifugation for coculture with K562 and BV173 cells with or without TKIs. We used real-time quantitative PCR to assess the level of interleukin 7 (IL-7) expression in the MSCs and employed immunoblotting to monitor protein expression in the BCR/ABL, phosphatidylinositol 3-kinase (PI3K)/AKT, and JAK/STAT signaling pathways. We also used a xenograft tumor model to examine the in vivo effect of different MSCs on CML cells. MSCs from patients with IM-resistant CML protected K562 and BV173 cells against IM- or NI-induced cell death, and this protection was due to increased IL-7 secretion from the MSCs. Moreover, IL-7 levels in the BM of patients with IM-resistant CML were significantly higher than in healthy donors or IM-sensitive CML patients. IL-7 elicited IM and NI resistance via BCR/ABL-independent activation of JAK1/STAT5 signaling, but not of JAK3/STAT5 or PI3K/AKT signaling. IL-7 or JAK1 gene knockdown abrogated IL-7-mediated STAT5 phosphorylation and IM resistance in vitro and in vivo Because high IL-7 levels in the BM mediate TKI resistance via BCR/ABL-independent activation of JAK1/STAT5 signaling, combining TKIs with IL-7/JAK1/STAT5 inhibition may have significant utility for managing CML.
Project description:Chronic myeloid leukemia (CML) is a clonal malignant disease caused by the expression of BCR/ABL. MDM2 (human homolog of the murine double minute-2) inhibitors such as Nutlin-3 have been shown to induce apoptosis in a p53-dependent manner in CML cells and sensitize cells to Imatinib. Here, we demonstrate that JNJ-26854165, an inhibitor of MDM2, inhibits proliferation and triggers cell death in a p53-independent manner in various BCR/ABL-expressing cells, which include primary leukemic cells from patients with CML blast crisis and cells expressing the Imatinib-resistant T315I BCR/ABL mutant. The response to JNJ-26854165 is associated with the downregulation of BCR/ABL dependently of proteosome activation. Moreover, in all tested CML cells, with the exception of T315I mutation cells, combining JNJ-26854165 and tyrosine kinase inhibitor (TKI) Imatinib or PD180970 leads to a synergistic effect. In conclusion, our results suggest that JNJ-26854165, used either alone or in combination with TKIs, represents a promising novel targeted approach to overcome TKI resistance and improve patient outcome in CML.
Project description:Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).
Project description:Interactions between the dual BCR/ABL and Src inhibitor bosutinib and the Chk1 inhibitor PF-00477736 were examined in BCR/ABL(+) leukemia cells, particularly imatinib-resistant cells, including those with the T315I mutation. Bosutinib blocked PF-00477736-induced ERK1/2 activation and sharply increased apoptosis in association with Mcl-1 inhibition, p34(cdc2) dephosphorylation, BimEL up-regulation, and DNA damage in imatinib-resistant CML or Ph(+) ALL cell lines. Inhibition of Src or MEK1 by shRNA significantly enhanced PF-0047736 lethality. Bosutinib/PF-00477736 co-treatment also potentiated cell death in CD34(+) CML patient samples, including dasatinib-resistant blast crisis cells exhibiting both T315I and E355G mutations, but was minimally toxic to normal CD34(+) cells. Finally, combined in vivo treatment significantly suppressed BaF3/T315I tumor growth and prolonged survival in an allogeneic mouse model. Together, these findings suggest that this targeted combination strategy warrants attention in IM-resistant CML or Ph(+) ALL.
Project description:Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.