A Novel Naphthoquinone-Coumarin Hybrid That Inhibits BCR-ABL1-STAT5 Oncogenic Pathway and Reduces Survival in Imatinib-Resistant Chronic Myelogenous Leukemia Cells.
ABSTRACT: BCR-ABL1-STAT5 is an oncogenic signaling pathway in human chronic myelogenous leukemia (CML) and it represents a valid target for anti-CML drug design. Resistance to direct BCR-ABL1 inhibitors is a common clinical issue, so STAT5 inhibition has become an interesting alternative target. In this study, the effects of NPQ-C6, a novel naphtoquinone-coumarin conjugate, were evaluated on human CML-derived K562 cells. Live-Cell Imaging analysis revealed that NPQ-C6 inhibited 2D (IC50AUC = 1.4 ± 0.6 ?M) growth of CML cells. NPQ-C6 increased sub-G1 and reduced G0/G1 cell cycle phases in a dose- and time-dependent manner. This effect on cell cycle was related to increased levels of apoptotic nuclei, cleavage of caspase-3, -9, and PARP and annexin V-positive cells. NPQ-C6 increased ?H2AX, a double-strand DNA break marker. NPQ-C6 showed a wide range of modulatory effects on cell signaling through an early increased phosphorylation of JNK, P38-MAPK and AKT, and decreased phosphorylation of ERK1/2, BCR-ABL1, and STAT5. NPQ-C6 inhibited expression of c-MYC and PYM-1, two target gene products of BCR-ABL1/STAT5 signaling pathway. Cytokine-induced activation of STAT5/STAT3-dependent transcriptional and DNA binding activities were also inhibited by NPQ-C6. Notably, NPQ-C6 maintained its activity on BCR-ABL1/STAT5/c-MYC/PIM-1 oncogenic pathway in imatinib-resistant cells. Molecular modeling suggested BCR-ABL1 and JAK2 proteins as NPQ-C6 targets. In summary, our data show a novel multikinase modulator that might be therapeutically effective in BCR-ABL1-STAT5-related malignancies.
Project description:We recently reported that chronic myeloid leukaemia (CML) patients harbour high levels of STAT5 when they progress to advanced phases of disease. Advanced disease is characterized by an increased incidence of BCR-ABL1 mutations. We now describe a highly significant correlation between STAT5 expression and the incidence of BCR-ABL1 mutations in primary CML. Forced expression of STAT5 in murine BCR-ABL1 transformed cells sufficed to enhance the production of reactive oxygen species (ROS) and to trigger DNA damage. STAT5-mediated ROS production is independent of JAK2 but requires concomitant BCR-ABL1 signalling as forced STAT5 expression in untransformed BCR-ABL1 negative cells has no impact on ROS levels. Only within the context of a BCR-ABL1 positive cell does STAT5 transcriptionally regulate a target gene or set of genes that causes the enhanced ROS production. Our study suggests the existence of a feed-forward loop accelerating disease progression, in which BCR-ABL1 enhances its own mutation rate in a STAT5-ROS dependent manner. This model explains the increased occurrence of inhibitor-resistant BCR-ABL1 mutations in advanced disease stages driven and characterized by high STAT5 expression.
Project description:Although the use of ATP-competitive tyrosine kinase inhibitors of oncoprotein BCR-ABL1 has enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. To test whether the degradation of BCR-ABL1 kinase could offer improved response, we developed a series of proteolysis-targeting chimera (PROTAC) that allosterically target BCR-ABL1 protein and recruit the E3 ligase Von Hippel-Lindau, resulting in ubiquitination and subsequent degradation of the oncogenic fusion protein. In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared with diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34+ cells, with no effect on healthy CD34+ cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells. Together, these findings suggest that combined BCR-ABL1 kinase inhibition and protein degradation may represent a strategy to address BCR-ABL1-dependent drug resistance, and warrant further investigation into the eradication of persistent leukemic stem cells, which rely on neither the presence nor the activity of the BCR-ABL1 protein for survival. SIGNIFICANCE: Small-molecule-induced degradation of BCR-ABL1 in CML provides an advantage over inhibition and provides insights into CML stem cell biology. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/18/4744/F1.large.jpg.
Project description:Chronic Myeloid Leukaemia (CML) is caused by the BCR/ABL1 fusion gene. Both the presence and the levels of BCR/ABL1 expression seem to be critical for CML progression from chronic phase (CP) to blast crisis (BC). After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown.A region of 1443bp of the functional BCR promoter was studied for transcription factor binding sites through in-silico analysis and Chromatin Immunoprecipitation experiments. BCR and BCR/ABL1 expression levels were analysed in CML cell lines after over-expression or silencing of MYC transcription factor. A luciferase reporter assay was used to confirm its activity on BCR promoter.In the present study we demonstrate that MYC and its partner MAX bind to the BCR promoter, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, which consequently leads to decreased proliferation and induction of cell death.Here we describe a regulatory pathway modulating BCR and BCR/ABL1 expression, showing that the BCR promoter is under the transcriptional control of the MYC/MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL1 up-regulation and blast aggressiveness acquired during CML evolution.
Project description:The fusion oncoprotein BCR-ABL1 exhibits aberrant tyrosine kinase activity and it has been proposed that it deregulates signaling networks involving both transcription factors and non-coding microRNAs that result in chronic myeloid leukemia (CML). Previously, microRNA expression profiling showed deregulated expression of miR-150 and miR-155 in CML. In this study, we placed these findings into the broader context of the MYC/miR-150/MYB/miR-155/PU.1 oncogenic network. We propose that up-regulated MYC and miR-155 in CD34+ leukemic stem and progenitor cells, in concert with BCR-ABL1, impair the molecular mechanisms of myeloid differentiation associated with low miR-150 and PU.1 levels. We revealed that MYC directly occupied the -11.7 kb and -0.35 kb regulatory regions in the MIR150 gene. MYC occupancy was markedly increased through BCR-ABL1 activity, causing inhibition of MIR150 gene expression in CML CD34+ and CD34- cells. Furthermore, we found an association between reduced miR-150 levels in CML blast cells and their resistance to tyrosine kinase inhibitors (TKIs). Although TKIs successfully disrupted BCR-ABL1 kinase activity in proliferating CML cells, this treatment did not efficiently target quiescent leukemic stem cells. The study presents new evidence regarding the MYC/miR-150/MYB/miR-155/PU.1 leukemic network established by aberrant BCR-ABL1 activity. The key connecting nodes of this network may serve as potential druggable targets to overcome resistance of CML stem and progenitor cells.
Project description:The product of the Ph chromosome, the BCR-ABL1 tyrosine kinase activates diverse signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph(+) B-cell acute lymphoblastic leukemia (B-ALL). Previous studies showed that nuclear factor ?B (NF-?B) is activated in BCR-ABL1-expressing cells, but the mechanism of activation and importance of NF-?B to the pathogenesis of BCR-ABL1-positive myeloid and lymphoid leukemias are unknown. Coexpression of BCR-ABL1 and a superrepressor mutant of inhibitory NF-?B ? (I?B?SR) blocked nuclear p65/RelA expression and inhibited the proliferation of Ba/F3 cells and primary BCR-ABL1-transformed B lymphoblasts without affecting cell survival. In retroviral mouse models of CML and B-ALL, coexpression of I?B?SR attenuated leukemogenesis, prolonged survival, and reduced myeloid leukemic stem cells. Coexpression of dominant-negative mutants of I?B kinase ? (IKK?)/IKK1 or IKK?/IKK2 also inhibited lymphoid and myeloid leukemogenesis by BCR-ABL1. Blockade of NF-?B decreased expression of the NF-?B targets c-MYC and BCL-X and increased the sensitivity of BCR-ABL1-transformed lymphoblasts to ABL1 kinase inhibitors. These results demonstrate that NF-?B is activated through the canonical IKK pathway and plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL1, validating NF-?B and IKKs as targets for therapy of Ph(+) leukemias.
Project description:The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A.In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis, and clonogenic assays. Efficacy of target inhibition by OP449 was evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model.We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34(+) CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells.We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML.
Project description:BACKGROUND: The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, de novo or secondary TKI-resistance is a significant problem in CML. We screened a panel of BCR-ABL1 positive ALL and CML cell lines to find models for imatinib-resistance. RESULTS: Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3K? E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR. CONCLUSION: We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.
Project description:BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, are essential for BCR-ABL1+, but not WT, pre-B-cell proliferation. The mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells.
Project description:BCR gene fused ABL kinase is the critical driving force for the Philadelphia Chromosome positive (Ph+) Chronic Myeloid Leukemia (CML) and has been extensively explored as a drug target. With a structure-based drug design approach we have discovered a novel inhibitor CHMFL-074, that potently inhibits both the native and a variety of clinically emerged mutants of BCR-ABL kinase. The X-ray crystal structure of CHMFL-074 in complex with ABL1 kinase (PDB ID: 5HU9) revealed a typical type II binding mode (DFG-out) but relatively rare hinge binding. Kinome wide selectivity profiling demonstrated that CHMFL-074 bore a high selectivity (S score(1) = 0.03) and potently inhibited ABL1 kinase (IC50: 24 nM) and PDGFR α/β (IC50: 71 nM and 88 nM). CHMFL-074 displayed strong anti-proliferative efficacy against BCR-ABL-driven CML cell lines such as K562 (GI50: 56 nM), MEG-01 (GI50: 18 nM) and KU812 (GI50: 57 nM). CHMFL-074 arrested cell cycle into the G0/G1 phase and induced apoptosis in the Ph+ CML cell lines. In addition, it potently inhibited the CML patient primary cell's proliferation but did not affect the normal bone marrow cells. In the CML cell K562 inoculated xenograft mouse model, oral administration of 100 mg/kg/d of CHMFL-074 achieved a tumor growth inhibition (TGI) of 65% without exhibiting apparent toxicity. As a potential drug candidate for fighting CML, CHMFL-074 is under extensive preclinical safety evaluation now.
Project description:Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 ?M) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker ?H2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.