The MYC-NFATC2 axis maintains the cell cycle and mitochondrial function in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage, with generally poor patient outcomes owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor nuclear factor of activated T cells 2 (NFATC2) is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Furthermore, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), NFATc2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a newly identified co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease.

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

Tyrosine Kinase Inhibitor Independent Gene Expression Signature in CML Offers New Targets for LSPC Eradication Therapy.

Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can re-initiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes that were differentially expressed in CML LSC compared to the healthy controls but were not affected by TKI in vitro. Two of them, CD33 and PPIF, are targeted by gemtuzumab-ozogamicin and cyclosporin A, respectively. We treated CML and the control CD34+ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A, in combination with imatinib, reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab-ozogamicin showed an EC50 of 146 ng/mL, below the plasma peak concentration of 630 ng/mL observed in the AML patients and below the EC50 of 3247 ng/mL observed in the non-CML cells. Interestingly, gemtuzumab-ozogamicin seems to promote cell cycle progression in CML CD34+ cells and demonstrated activation of the RUNX1 pathway in an RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML.

Synergistic cytotoxicity of dual PI3K/mTOR and FLT3 inhibition in FLT3-ITD AML cells.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy, characterized by a heterogeneous genetic landscape and complex clonal evolution, with poor outcomes. Mutation at the internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic alterations in AML, associated with high relapse rates and poor survival due to the constitutive activation of the FLT3 receptor tyrosine kinase and its downstream effectors, such as PI3K signaling. Thus, aberrantly activated FLT3-kinase is regarded as an attractive target for therapy for this AML subtype, and a number of small molecule inhibitors of this kinase have been identified, some of which are approved for clinical practice. Nevertheless, acquired resistance to these molecules is often observed, leading to severe clinical outcomes. Therapeutic strategies to tackle resistance include combining FLT3 inhibitors with other antileukemic agents. Here, we report on the preclinical activity of the combination of the FLT3 inhibitor quizartinib with the dual PI3K/mTOR inhibitor PF-04691502 in FLT3-ITD cells. Briefly, we show that the association of these two molecules displays synergistic cytotoxicity in vitro in FLT3-ITD AML cells, triggering 90% cell death at nanomolar concentrations after 48 h.

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival.

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence.

Acute myeloid leukemia (AML) is a highly heterogeneous hematopoietic malignancy characterized by excessive proliferation and accumulation of immature myeloid blasts in the bone marrow. AML has a very poor 5-year survival rate of just 16% in the UK; hence, more efficacious, tolerable, and targeted therapy is required. Persistent leukemia stem cell (LSC) populations underlie patient relapse and development of resistance to therapy. Identification of critical oncogenic signaling pathways in AML LSC may provide new avenues for novel therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway, is often hyperactivated in AML, required to sustain the oncogenic potential of LSCs. Growing evidence suggests that targeting key components of this pathway may represent an effective treatment to kill AML LSCs. Despite this, accruing significant body of scientific knowledge, PI3K/Akt/mTOR inhibitors have not translated into clinical practice. In this article, we review the laboratory-based evidence of the critical role of PI3K/Akt/mTOR pathway in AML, and outcomes from current clinical studies using PI3K/Akt/mTOR inhibitors. Based on these results, we discuss the putative mechanisms of resistance to PI3K/Akt/mTOR inhibition, offering rationale for potential candidate combination therapies incorporating PI3K/Akt/mTOR inhibitors for precision medicine in AML.

The Emerging Role of H3K9me3 as a Potential Therapeutic Target in Acute Myeloid Leukemia.

Growing evidence has demonstrated that epigenetic dysregulation is a common pathological feature in human cancer cells. Global alterations in the epigenetic landscape are prevalent in malignant cells across different solid tumors including, prostate cancer, non-small-cell lung cancer, renal cell carcinoma, and in haemopoietic malignancy. In particular, DNA hypomethylation and histone hypoacetylation have been observed in acute myeloid leukemia (AML) patient blasts, with histone methylation being an emerging area of study. Histone 3 lysine 9 trimethylation (H3K9me3) is a post-translational modification known to be involved in the regulation of a broad range of biological processes, including the formation of transcriptionally silent heterochromatin. Following the observation of its aberrant methylation status in hematological malignancy and several other cancer phenotypes, recent studies have associated H3K9me3 levels with patient outcome and highlighted key molecular mechanisms linking H3K9me3 profile with AML etiology in a number of large-scale meta-analysis. Consequently, the development and application of small molecule inhibitors which target the histone methyltransferases or demethylase enzymes known to participate in the oncogenic regulation of H3K9me3 in AML represents an advancing area of ongoing study. Here, we provide a comprehensive review on how this particular epigenetic mark is regulated within cells and its emerging role as a potential therapeutic target in AML, along with an update on the current research into advancing the generation of more potent and selective inhibitors against known H3K9 methyltransferases and demethylases.

Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal.

Leukaemic stem cell (LSC) persistence remains a major obstacle to curing chronic myeloid leukaemia (CML). The bone morphogenic protein (BMP) pathway is deregulated in CML, with altered expression and response to the BMP ligands shown to impact on LSC expansion and behaviour. In this study, we determined whether alterations in the BMP pathway gene signature had any predictive value for therapeutic response by profiling 60 CML samples at diagnosis from the UK SPIRIT2 trial and correlating the data to treatment response using the 18-month follow-up data. There was significant deregulation of several genes involved in the BMP pathway with ACV1C, INHBA, SMAD7, SNAIL1 and SMURF2 showing differential expression in relation to response. Therapeutic targeting of CML cells using BMP receptor inhibitors, in combination with tyrosine kinase inhibitor (TKI), indicate a synergistic mode of action. Furthermore, dual treatment resulted in altered cell cycle gene transcription and irreversible cell cycle arrest, along with increased apoptosis compared to single agents. Targeting CML CD34+ cells with BMP receptor inhibitors resulted in fewer cell divisions, reduced numbers of CD34+ cells and colony formation when compared to normal donor CD34+ cells, both in the presence and absence of BMP4. In an induced pluripotent stem cell (iPSC) model generated from CD34+ hematopoietic cells, we demonstrate altered cell cycle profiles and dynamics of ALK expression in CML-iPSCs in the presence and absence of BMP4 stimulation, when compared to normal iPSC. Moreover, dual targeting with TKI and BMP inhibitor prevented the self-renewal of CML-iPSC and increased meso-endodermal differentiation. These findings indicate that transformed stem cells may be more reliant on BMP signalling than normal stem cells. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to target LSC self-renewal and improve long-term outcome for patients.

Nano-curcumin safely prevents streptozotocin-induced inflammation and apoptosis in pancreatic beta cells for effective management of Type 1 diabetes mellitus.

BACKGROUND AND PURPOSE: Approaches to prevent selective and progressive loss of insulin-producing beta cells in Type 1 diabetes mellitus (T1DM) will help to manage this prevalent and devastating disease. Curcumin (CUR), a natural anti-inflammatory substance, suppresses diabetes-associated inflammation and cell death. However, very high doses need to be used because of poor oral bioavailability, making it difficult to translate the anti-inflammatory actions to clinical situations. EXPERIMENTAL APPROACH: We have prepared biodegradable nanosystems encapsulating curcumin (nCUR), resulting in at least nine-fold improvement in oral bioavailability. Here, we tested the ability of nCUR to prevent streptozotocin (STZ)-induced inflammation and apoptosis in pancreatic islets and beta cells, in rats. KEY RESULTS: Non-fasted rats pretreated with 10 or 50 mg·kg-1 nCUR 6 h prior to STZ challenge had up to 37% reduction in the glucose levels, while plain CUR (50 mg·kg-1 ) results in 12% reduction. This treatment with nCUR was accompanied by decreased islet or beta cell death, as shown by TUNEL assay and H&E staining. Both CUR and nCUR significantly decreased levels of inflammatory cytokines in pancreatic tissue homogenates that correlated well with minimal histiocytic infiltration. Pre-treatment with nCUR, but not CUR, decreased 8-oxo-2'-deoxyguanosine, a sensitive biomarker of ROS-induced DNA damage, in pancreas. In normal rodents, daily dosing for 28 days, with nCUR (25-100 mg·kg-1 ) did not cause any deleterious health issues by the carrier. CONCLUSIONS AND IMPLICATIONS: Together, these data indicate a potentially translatable dose of nCUR that is safe and efficacious in improving beta cell function, which could prevent T1DM.

Axl Blockade by BGB324 Inhibits BCR-ABL Tyrosine Kinase Inhibitor-Sensitive and -Resistant Chronic Myeloid Leukemia.

Purpose: BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1-independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML.Experimental Design: We used primary cells from patients with CML and TKI-sensitive and -resistant BCR-ABL1+ CML cell lines and a novel ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small-molecule Axl inhibitor BGB324 in vitro and in vivo In BCR-ABL1-unmutated cells, we also investigated BGB324 in combination with imatinib.Results: We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of patients with CML compared with healthy individuals and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34+ cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status.Conclusions: Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as -resistant CML and support the need for clinical trials. Clin Cancer Res; 23(9); 2289-300. ©2016 AACR.

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression.

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34+ cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34+ CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4+CD69+ T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells.

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition.

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE: In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197.

Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor-treated CML stem cells.

Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-κB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML.

Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia.

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation.

JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of CML CD34+ cells in vitro and in vivo.

Chronic myeloid leukemia (CML) stem cell survival is not dependent on BCR-ABL protein kinase and treatment with ABL tyrosine kinase inhibitors cures only a minority of CML patients, thus highlighting the need for novel therapeutic targets. The Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)5 pathway has recently been explored for providing putative survival signals to CML stem/progenitor cells (SPCs) with contradictory results. We investigated the role of this pathway using the JAK2 inhibitor, ruxolitinib (RUX). We demonstrated that the combination of RUX, at clinically achievable concentrations, with the specific and potent tyrosine kinase inhibitor nilotinib, reduced the activity of the JAK2/STAT5 pathway in vitro relative to either single agent alone. These effects correlated with increased apoptosis of CML SPCs in vitro and a reduction in primitive quiescent CML stem cells, including NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice repopulating cells, induced by combination treatment. A degree of toxicity toward normal SPCs was observed with the combination treatment, although this related to mature B-cell engraftment in NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice with minimal effects on primitive CD34(+) cells. These results support the JAK2/STAT5 pathway as a relevant therapeutic target in CML SPCs and endorse the current use of nilotinib in combination with RUX in clinical trials to eradicate persistent disease in CML patients.

Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.

Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common ß-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.

Targeting primitive chronic myeloid leukemia cells by effective inhibition of a new AHI-1-BCR-ABL-JAK2 complex.

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.

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia.

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34(+) selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34(+) cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 -ve CD34(+) cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.