Chronic Myelogenous Leukemia Progenitors Research Articles

Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells

AbstractTyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1–mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

Metabolic Adaptation to Tyrosine Kinase Inhibition in Chronic Myelogenous Leukemia Stem Cells

Objectives: BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myelogenous leukemia (CML) but fail to eliminate leukemia stem cells, which persist as a source of disease recurrence. Previous studies have suggested that primitive CML cells rely on upregulated oxidative metabolism for survival. However, there is limited knowledge about the effects of TKI treatment on CML stem cell metabolism and the role of metabolic adaptations in stem cell persistence after TKI treatment. Here we evaluated the metabolic response to TKI treatment in CML stem and progenitor cells using a representative SCL-tTA/BCR-ABL mouse model of CML. Methods: CML mice were treated with the TKI Nilotinib or vehicle for 2 days, to evaluate initial effects of treatment, and for 2 weeks, to evaluate response to continued treatment. Bioenergetics, metabolite profiling, and stable isotope metabolic labeling was performed on BM c-Kit+ progenitor cells obtained from vehicle and TKI treated mice. The effects of TKI treatment on metabolism-related gene expression in CML hematopoietic stem cells (HSC) was studied using single cell RNA-Seq (scRNA) analysis of BM Lin- c-Kit+ Sca-1+ (LSK) cells from CML mice treated with vehicle or TKI for 2 days or 2 weeks. Results: Extracellular flux analysis revealed that TKI treatment initially inhibited oxygen consumption rate (OCR), representing mitochondrial respiration, and extracellular acidification rate (ECAR), representing glycolysis, in CML progenitor cells, but that these were restored with continued TKI treatment. Metabolite profiling using high-resolution mass spectrometry (HRMS) identified significant initial reduction in glycolytic, glutaminolytic and tricarboxylic acid cycle (TCA) intermediates after 2 days of TKI treatment. However, metabolite levels were restored to levels similar to controls with continued TKI treatment. Steady-state metabolic labeling with [U-13C6]-glucose or [U-13C5]-Glutamine showed reduced glycolytic flux, glutaminolysis and TCA intermediates after treatment with TKI for 2 days, but restoration of glycolytic activity and increased glucose-mediated lactate generation, and increased glutaminolytic and TCA cycle activity with continued treatment. The above studies indicate initial inhibition of energy metabolism in CML progenitors with TKI treatment, but metabolic adaptation to continued TKI exposure. scRNA analysis of CML HSC populations showed that TKI exposure led to selective enrichment of primitive HSC subsets characterized by reduced OXPHOS, glycolysis, nucleotide metabolism, and MYC gene signatures. Treatment with TKI for 2 days further reduced metabolic gene signatures, but continued treatment led to restoration of MYC and OXPHOS gene signature expression coupled with enrichment of HSC and quiescence signatures in persistent HSC. TKI-treated stem cells showed increased levels of Hif1a protein and enhanced activity of HIF-1, a key transcriptional regulator of energy metabolism. Treatment of CML mice with a HIF-1 inhibitor, Echinomycin, in combination with TKI treatment, resulted in significant depletion of CML stem cells compared to TKI alone. The combination of HIF-1 inhibitor with TKI treatment also depleted human CML stem cells in xenografted mice. Conclusions: Our results demonstrate metabolic adaptation in CML and progenitor stem cells following TKI treatment, and identify HIF-1 activation as a targetable mechanism to enhance elimination of CML stem cell that persist after TKI treatment.

The Bone Marrow Niche Uses Mir-300 As a Biological Rheostat to Selectively Control Stem Cell-Driven Malignant Hematopoiesis and Innate Anti-Cancer Immunity

Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance, disease evolution and innate anti-cancer immunity. In CML, persistence of ABL tyrosine kinase inhibitor (TKI)-resistant quiescent LSCs, disease development and inhibition of natural killer (NK) cell growth and anti-cancer activity depend on cell-autonomous and bone marrow (BM)-generated signals.Here we show that expression profiling of CD34+ BM progenitors from healthy individuals and CML patients in chronic (CP) and blastic (BC) phase identified a discrete number of microRNA (miRs) altered in CML and/or during blastic transformation. Among the miRs downregulated in CML, we focused on miR-300 because it directly inhibited expression of multiple components of the PP2A inhibitory pathway (i.e. Jak2, hnRNPA1, SET) and of other factors essential for LSC maintenance and disease progression (e.g. CCND1/2, b-catenin, Myc, Twist-1), when ectopically expressed in CML but not in normal CD34+ cells.MiR-300 acted as a potent tumor suppressor in leukemic CD34+ progenitors by inducing cell cycle exit and promoting spontaneous and (TKI)-induced apoptosis, whereas no effects were exerted in normal miR-300-transduced CD34+ cells. In CD34+ CML-BC progenitors miR-300 downregulation required the BCR-ABL1 kinase-dependent inhibition of C/EBPb, which was capable of binding and transcriptionally activating miR-300 promoter.Conversely, low O2 levels and mesenchymal stromal cell (MSC)-derived exosomes abnormally increased miR-300 expression in a TKI-insensitive manner to induce and/or preserve LSC quiescence and suppress NK cell growth. Indeed, lentiviral-mediated miR-300 inhibition in MSCs significantly prevented the MSC growth inhibitory activity on CD34+ CML-BC and NK cells. Accordingly, miR-300 levels were found higher in CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals.Induction/maintenance of high miR-300 levels in LSCs (CD34+ CFSEMAX and CD34+CD38- cells) required the hypoxia-induced inhibition of BCR-ABL1 activity and induction of C/EBPb expression/activity. However, MSC-derived exosomal miR-300 also contributed to increase miR-300 expression in LSCs.LSCs escaped from the miR-300 pro-apoptotic activity through an autocrine/paracrine mechanism that required the release by CD34+ CML stem/progenitors of TGFb1 that, in turn, induced the expression of TUG1, a lncRNA acting as a miR-300 sponge. In fact, shRNA-mediated TUG1 suppression or Ab-dependent TGFb1 inhibition decreased the quiescent (CFSEMAX) CD34+ LSC number. By contrast, miR-300 inhibition did not alter LSC survival and self-renewal, further supporting a role for TUG1 as a miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low ectopic miR-300 expression induced growth arrest (decreased LTC-ICs and CD34+CD38- CFC/replating activities) without affecting the number of CFSEMAX LSCs.By contrast, high doses of miR-300 (CPG-miR-300; 500nM) but not scramble oligonucleotides impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced a marked killing of quiescent LSCs (decreased CD34+ CFSEMAX cells) and dividing CD34+ CML progenitors, and impaired CML-BC engraftment in NRG-SGM3 mice. Notably, the association of high CPG-miR-300 with TUG1 silencing (CPG-anti-TUG1) further reduced CD34+ CFSEMAX LSC number. Notably, TUG1 was highly expressed in normal CD34+ stem/progenitor cells; in fact, neither low nor high miR-300 levels altered their survival and/or self-renewal.Altogether our results indicate that while loss of miR-300 expression is essential for survival/proliferation of leukemic progenitors and, therefore, for development of a full-blown leukemia, increased miR-300 expression is necessary for reduced NK cell number/activity and maintenance of the LSC reservoir. In this scenario, induction of TUG1 may occur to preserve LSC survival after migration into the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through marked induction of intracellular (endogenous and exosomal) miR-300 levels. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias and restoration of innate immunity.This work is supported in part by NHI-NCI R01CA163800-01 DisclosuresGambacorti-Passerini:Pfizer: Consultancy, Honoraria, Research Funding; BMS: Consultancy. Deininger:ARIAD: Consultancy; Incyte: Consultancy; BMS: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding. Milojkovic:ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau. Apperley:Pfizer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Therakos: Honoraria; Incyte: Honoraria; Sun Pharma: Honoraria; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses , Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding.

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

<h3></h3> Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that <i>MIR300</i> has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. <i>MIR300</i> is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates <i>MIR300</i> in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating <i>TUG1</i> long noncoding RNA that uncouples and limits <i>MIR300</i> function to cytostasis. Genetic and pharmacologic <i>MIR300</i> modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis <i>in vitro</i> and in patient-derived xenografts; hence, the importance of <i>MIR300</i> and PP2A activity for CML development and therapy. <h3>Significance:</h3> Tumor-naïve microenvironment–induced <i>MIR300</i> is the only tumor suppressor miRNA that induces CML LSC quiescence while inhibiting NK cell antitumor immune response, and CML LSC/progenitor cell apoptosis through its anti-proliferative and PP2A-activating functions, respectively. Thus, the importance of <i>MIR300</i> and PP2A-activating drugs for formation/survival and eradication of drug-resistant CML LSCs, respectively. <i>See related commentary by Broxmeyer, p. 13</i>. <i>This article is highlighted in the In This Issue feature, p. 5</i>

Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions.

Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.

A 14q32.31 Genomic-Imprinted DLK1-DIO3 microrna promotes Leukemogenesis By Inducing Stem Cell Quiescence and Inhibiting NK Cell Anti-Cancer Immunity

Leukemia emergence, maintenance, relapse and/or progression are causally linked to the presence of drug-resistant leukemia-initiating cells and impaired natural killer (NK) cell anti-tumor immune-response. Bone marrow microenvironment (BMM)- and/or leukemia-derived signals induce aberrant non-coding RNA expression and inhibit protein phosphatase 2A (PP2A) tumor suppressor activity. PP2A loss-of-function is essential for NK cell activity and leukemic but not normal stem and progenitor cell proliferation and survival. The human MIR300 gene is an intergenic miRNA that belongs to the 14q32.31 DLK1-DIO3 genomic-imprinted tumor suppressor miRNA cluster B. MIR300 was found involved in loss-of heterozygosity, inhibited in several tumor types with high mitotic index and during epithelial-to-mesenchymal transition (EMT), and associated with a cancer stem cell phenotype. By using primary cells from Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) in chronic (CP) and blastic (BC) phase, and complex karyotype (CK) acute myeloid leukemia (AML) patients, as paradigmatic examples of stem cell-derived neoplasms characterized by constitutive expression of oncogenic kinases, PP2A loss-of-function, altered microRNA expression and impaired NK cell proliferation and cytotoxicity, we found that MIR300 is a cell context-independent tumor suppressor with anti-proliferative and PP2A-dependent pro-apoptotic activities which are sequentially activated in a MIR300 dose-dependent manner through inhibition of CCND2/CDK6 and SET (PP2A inhibitor), respectively. To prevent PP2A-induced apoptosis, MIR300 is inhibited by oncogenic signals in CD34+CML (CP and BC) and CK-AML progenitors. Conversely, tumor-naïve BMM-induced C/EBPbeta-mediated signals (hypoxia and MSC exosomes) markedly upregulate MIR300 expression in primary CML and AML CD34+CFSEmax leukemic stem (LSC) and CD56+CD3-NK cells to induce/maintain quiescence (increased CD34+leukemic blasts in G0) and impair immune-response (suppression of NK cell proliferation and cytotoxic activity toward CD34+ leukemic blasts and CFSEmaxCD34+ CML-BC quiescent LSCs), respectively. Inhibition of MIR300 expression/activity rescues NK cell proliferation and anti-tumor cytotoxicity and prevented MSC- and hypoxia-induced growth-suppression of CD34+leukemic blasts by inhibiting degradation of MIR300 targets (e.g. SET, CCND2). We found that CML and AML LSCs escape MIR300-induced PP2A-mediated apoptosis through the hypoxia- and tumor-dependent TGFb1-FoxM1-mediated upregulation of TUG1 lncRNA. TUG1 is an oncogenic lncRNA described as a MIR300sponge and found upregulated in solid tumors, in which it has strong diagnostic, prognostic and therapeutic relevance and is associated with cancer stem cell maintenance and EMT. In quiescent CML and AML LSCs, TUG1 uncouples and limits MIR300 tumor suppressor functions to cytostasis by maintaining unbound MIR300 at levels sufficient to inhibit CCND2 and CDK6 but not SET expression. Exposure to clinically-relevant CpG-modified oligonucleotides modulating MIR300levels and/or inhibiting TUG1 MIR300-sponging activity, restores NK cell proliferation and cytotoxic activity, and suppresses human leukemic but not normal hematopoiesis by eradicating nearly all (&gt; 95% reduction) CFSEmaxCD34+ and CD45+CD34+CD38-CD90+ LSCs and CD34+leukemic CML (CP and BC) and CK-AML blasts in vitro (CFCs, LTC-IC, and CFSEmaxCD34+cell tracking) and/or in NRG-SGM3 PDX mouse models of acute and chronic myeloid leukemias. Altogether, this work highlights the therapeutic importance of altering MIR300 expression in anti-LSC and NK cell-based approaches for myeloid leukemias, and indicates that tumor-naïve BMM-induced MIR300 tumor suppressor anti-proliferative and PP2A-activating functions may support leukemogenesis by promoting the formation and initial expansion of the quiescent LSC pool through the induction of LSC dormancy and inhibition of quiescent LSC killing by cytokine-activated NK cells, respectively. (G.S. and R.T. equally contributed to this work) Disclosures Stagno: BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Deininger:TRM: Consultancy; Sangoma: Consultancy; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Ascentage Pharma: Consultancy, Honoraria; Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Fusion Pharma: Consultancy; Adelphi: Consultancy. Milojkovic:BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baer:Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.

Abstract 1134: The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance

Abstract Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance and disease development. Persistence of drug-resistant quiescent LSCs depends on cell-autonomous and bone marrow (BM) signals. Herein, we identified miR-300 as an miRNA inhibited in CD34+ CML progenitors and during blastic transformation (BC) through the BCR-ABL1-dependent inhibition of C/EBPβ-induced miR-300 transcription. In CML progenitors, ectopic mir-300 expression directly targets the PP2A inhibitory pathway (i.e., Jak2, hnRNPA1, SET) and other factors essential for LSC maintenance and disease progression (e.g., CCND1/2, b-catenin, Myc, Twist-1). In leukemic but not normal CD34+ cells, miR-300 acts as a potent tumor suppressor by inducing cell cycle exit and promoting apoptosis. Conversely, hypoxia-induced BCR-ABL1 inhibition and induction of C/EBPβ were found essential for increased miR-300 levels in quiescent LSCs, although mesenchymal stromal cells (MSC)-derived exosomal miR-300 also contributed to it. Moreover, low O2 levels and MSC-derived exosomes induced quiescence of CD34+ CML cells. Notably, expression of an anti-miR-300 in MSCs prevented exosome-induced CD34+ CML growth arrest. LSCs escaped miR-300-induced apoptosis through the autocrine/paracrine TGFβ1-induced expression of TUG1, a lncRNA acting as an miR-300 sponge. In fact, TUG1 or TGFβ1 inhibition decreased quiescent (CFSEMAX) LSC number. By contrast, miR-300 inhibition did not alter LSC survival/self-renewal, further supporting a role for TUG1 as an miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low levels of ectopic miR-300 induced growth arrest (decreased LTC-IC and CFC/replating activity) without affecting quiescent LSC number. By contrast, high doses of miR-300 but not scramble CpG-ODN impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced marked killing of quiescent LSCs and dividing progenitors, and impaired CML engraftment in NRG-SGM3 mice. Such effects were further enhanced when CPG-miR-300 and CPG-anti-TUG1 were combined. By contrast, high CpG-miR-300 levels did not affect normal CD34+ cell survival/self-renewal likely because of high TUG1 expression. Altogether our results indicate that while miR-300 loss is essential for survival/proliferation of leukemic progenitors, increased miR-300 levels are required for LSC maintenance. Thus, induction of TUG1 may occur to preserve LSC survival in the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through abnormal miR-300 induction. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias. This work is supported in part by NIH-NCI R01CA163800. Citation Format: Giovannino Silvestri, Lorenzo Stramucci, Justin Ellis, Jason Harb, Paolo Neviani, Bin Zhang, Klara Srutova, Gabriel Pineda, Catriona Jamieson, Bruno Calabretta, Fabio Stagno, Paolo Vigneri, Georgios Nteliopoulos, Philippa May, Alistar Reid, Ramiro Garzon, Denis-Claude Roy, Martin Guimond, Peter Hokland, Michael Deininger, Garrett Fitzgerald, Chris Harman, Francesco Dazzi, Dragana Milojkovic, Jane Apperley, Guido Marcucci, Jianfei Qi, Katerina Machova-Polakova, Xiaoxuan Fan, Maria Baer, Rossana Trotta, Danilo Perrotti. The tumor suppressor activity of miR-300 is detrimental for leukemia development but required for leukemia stem cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1134.

Dual inhibition of Bcr-Abl and Hsp90 by C086 potently inhibits the proliferation of imatinib-resistant CML cells.

Although tyrosine kinase inhibitors (TKI) such as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of patients with chronic myelogenous leukemia (CML), TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation, and immunoblotting were applied to examine Hsp90 function. Colony-forming unit, long-term culture-initiating cells (LTC-IC), and flow cytometry were used to test CML progenitor and stem cells. Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, it inhibited the growth of both imatinib-sensitive and -resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34(+)CD38(+) and CD34(+)CD38(-) cells in vitro. Moreover, C086 could decrease the number of CD45(+), CD45(+)CD34(+)CD38(+), and CD45(+)CD34(+)CD38(-) cells in CML NOD-SCID mice. Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs.

MiR-300 Acts As a Tumor Supressor in Ph+ Progenitors By Modulating the JAK2-SET/PP2A/β-Catenin Interplay

The persistence of tyrosine kinase inhibitor (TKI)-resistant malignant Philadelphia-positive (Ph+) hematopoietic stem cells (HSC) in chronic myelogenous leukemia (CML) TKI-treated patients in complete molecular remission, and the dismal prognosis of blast crisis CML indicate that the molecular mechanisms underlying its emergence, maintenance and progression are not totally dependent on the unrestrained kinase activity of BCR-ABL1 and might rely on other cell autonomous and/or microenvironmental signal capable of sustaining survival and self-renewal of the chronic phase and blast crisis Ph+ HSC compartment(s).We recently demonstrated that the Jak2/SET-PP2A/β-catenin pathway is essential for survival and self-renewal of quiescent TKI-resistant CD34+CD38- Ph+ HSC and that activation of such oncogenic signals requires the expression but not the activity of BCR-ABL1. Because microRNAs (miRNAs) are likely to control in a canonical and/or decoy manner the expression of different components of the Jak2 signalosome, this makes them an attractive target for further understanding the mechanisms of leukemogenesis and, perhaps, for developing new alternative therapies that selectively eradicate quiescent leukemic HSCs.In silico analysis revealed that a specific miR subset shares multiple targets of the BCR-ABL1/Jak2/SET-PP2A signalosome. Nanostring Array analysis performed on primary bone marrow cells from normal individuals and chronic phase or blast crisis CML patients revealed that expression of some of these miRNA is altered in CML. For example, expression of miR-300 and miR-101, which are predicted to simultaneously modulate directly Jak2, hnRNP-A1 and β-catenin and, indirectly, other molecules of the BCR-ABL1/Jak2/SET-PP2A/β-catenin pathway, is significantly inhibited in chronic phase CML and further decreases in advanced CML samples. Additionally, miR-300 expression is several folds lower in dividing (div. 1) compared to quiescent (CFSEMAX) CD34+ CML cells.Lentiviral-transduction of miR-300 in human BCR-ABL+ cell lines resulted in marked downmodulation of JAK2, β-Catenin hnRNPA1 and SET and, as expected, in increased PP2A activity. Moreover, ectopic miR-300 expression decreased reduced clonogenic potential and proliferation, and increased susceptibility to TKI (e.g. Imatinib) induced apoptosis. Interestingly, it appears that forced BCR-ABL1 expression in TF-1 leukemic cells decreases miR-300, consistent with the reported activation in these cells of the Jak2-SET-PP2A-β-catenin pathway.Altogether our results suggest that miR-300 expression and, potentially, that of other deregulated non-coding RNAs might be dispensable or deleterious for the phenotype of Ph+ progenitors and/or indispensable for that of Ph+ HSCs. Thus, experiments in BCR-ABL1 cell lines as well as primary stem and progenitor cell fractions are currently ongoing to assess the role of this and other miRNAs in survival, self-renewal/proliferation of CML stem and progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

BCR-ABL–Induced Deregulation of the IL-33/ST2 Pathway in CD34(+) Progenitors from Chronic Myeloid Leukemia Patients

Although it is generally acknowledged that cytokines regulate normal hematopoiesis in an autocrine/paracrine fashion, their possible role in chronic myelogenous leukemia (CML) and resistance to imatinib mesylate treatment remain poorly investigated. Here, we report that CD34(+) progenitors from patients with CML at diagnosis are selectively targeted by the cytokine/alarmin interleukin (IL)-33. Indeed, CML CD34(+) progenitors upregulate their cell surface expression of the IL-33-specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34(+) cells from healthy individuals. Moreover, ST2 overexpression is normalized following imatinib mesylate therapy, whereas IL-33 counteracts in vitro imatinib mesylate-induced growth arrest in CML CD34(+) progenitors via reactivation of the STAT5 pathway, thus supporting the notion that IL-33 may impede the antiproliferative effects of imatinib mesylate on CD34(+) progenitors in CML. Clinically, the levels of circulating soluble ST2, commonly considered a functional signature of IL-33 signaling in vivo, correlate with disease burden. Indeed, these elevated peripheral concentrations associated with a high Sokal score predictive of therapeutic outcome are normalized in patients in molecular remission. Finally, we evidenced a facilitating effect of IL-33 on in vivo maintenance of CD34(+) progenitors from patients with CML by using xenotransplant experiments in immunodeficient NOG mice, and we showed that engraftment of mouse BCR-ABL-transfected bone marrow progenitors was less efficient in IL-33-deficient mice compared with wild-type recipients. Taken together, our results provide evidence that IL-33/ST2 signaling may represent a novel cytokine-mediated mechanism contributing to CML progenitor growth and support a role for this pathway in CML maintenance and imatinib mesylate resistance.

KIT Signaling Governs Differential Sensitivity of Mature and Primitive CML Progenitors to Tyrosine Kinase Inhibitors

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34(+)38(-) cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34(+)38(+) cells, SCF strongly induced pAKT(S473) in a phosphoinositide 3-kinase (PI3K)-dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKT(S473) levels remained low in CD34(+)38(-) cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34(+)38(-) compared with CD34(+)38(+) CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells.

Chronic myelogenous leukemia stem and progenitor cells demonstrate chromosomal instability related to repeated breakage-fusion-bridge cycles mediated by increased nonhomologous end joining

Chromosomal aberrations are an important consequence of genotoxic exposure and contribute to pathogenesis and progression of several malignancies. We investigated the susceptibility to chromosomal aberrations in chronic myelogenous leukemia (CML) progenitors after exposure to ionizing radiation. In normal progenitors, ionizing radiation induced both stable and unstable chromosomal lesions, but only stable aberrations persisted after multiple divisions. In contrast, radiation of chronic phase CML progenitors resulted in enhanced generation of unstable lesions that persisted after multiple divisions. CML progenitors demonstrated active cell cycle checkpoints and increased nonhomologous end joining DNA repair, suggesting that persistence of unstable aberrations was the result of continued generation of these lesions. CML progenitors demonstrated enhanced susceptibility to repeated cycles of chromosome damage, repair, and damage through a breakage-fusion-bridge mechanism. Perpetuation of breakage-fusion-bridge cycles in CML progenitors was mediated by classic nonhomologous end joining repair. These studies reveal a previously unrecognized mechanism of chromosomal instability in leukemia progenitors because of continued generation of unstable chromosomal lesions through repeated cycles of breakage and repair of such lesions.

Activation of p53 by SIRT1 Inhibition Enhances Elimination of CML Leukemia Stem Cells in Combination with Imatinib

BCR-ABL tyrosine kinase inhibitors (TKI) fail to eliminate quiescent leukemia stem cells (LSC) in chronic myelogenous leukemia (CML). Thus, strategies targeting LSC are required to achieve cure. We show that the NAD(+)-dependent deacetylase SIRT1 is overexpressed in human CML LSC. Pharmacological inhibition of SIRT1 or SIRT1 knockdown increased apoptosis in LSC of chronic phase and blast crisis CML and reduced their growth in vitro and in vivo. SIRT1 effects were enhanced in combination with the BCR-ABL TKI imatinib. SIRT1 inhibition increased p53 acetylation and transcriptional activity in CML progenitors, and the inhibitory effects of SIRT1 targeting on CML cells depended on p53 expression and acetylation. Activation of p53 via SIRT1 inhibition represents a potential approach to target CML LSC.

Leukemia-Induced Alterations in Bone Marrow Cytokine and Chemokine Levels Contribute to Altered Stem Cell Lodgment and Impairment of Normal Stem Cell Growth in CML

Abstract 962Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However microenvironmental interactions of leukemia stem cells (LSC) are poorly understood. Although chronic myelogenous leukemia (CML) results from HSC transformation by the BCR-ABL gene, the role of the microenvironment in modulating leukemia development is not known. We employed the SCL-tTA-BCR/ABL mouse model of CML to investigate the LSC interactions with the BM microenvironment. In this model, targeted expression of the BCR-ABL gene in murine HSC via a tet-regulated SCL promoter results in development of a chronic phase CML-like disorder. We have reported that LSC capacity is restricted to BCR-ABL+ cells with long-term hematopoietic stem cell (LTHSC) phenotype(LSK Flt3-CD150+CD48-) (Blood 2010 116:1212A). LSC numbers are reduced in the BM but increased in the spleen of CML mice compared with LTHSC from control mice, suggesting that LSC have altered niche interactions. LSC also demonstrate altered trafficking with significant reduction in homing of IV injected LSC to BM, and markedly increased egress of intrafemorally injected LSC to the spleen, potentially related to reduced CXCL12 levels in the BM of CML mice. In addition, levels of several chemokines and cytokines, including MIP1α, MIP1β, MIP2, IL-1α, IL-1β, TNF-α, G-CSF and IL-6, were increased in CML BM, related to increased production by malignant hematopoietic cells. We investigated whether altered chemokine and cytokine expression was associated with altered capacity of the CML BM microenvironment to support LTHSC engraftment. LTHSC from control mice or LSC from CML mice were transplanted into irradiated CML or control recipients. There was reduced engraftment of both control LTHSC and CML LSC in the BM of CML compared to control recipients at 2 weeks after transplantation, associated with reduced homing to CML BM, potentially related to low BM CXCL12 levels. The numbers of control LTHSC in the BM of CML recipient mice remained low at 4 weeks post-transplantation, whereas the numbers of CML LSC increased to numbers similar to those seen in the BM of control recipients. Culture with CML BM supernatants (SN) resulted in impaired growth of control LTHSC compared to control BM SN. In contrast the growth of CML LSC was similar following culture with CML and control BM SN. Culture with individual factors at concentrations similar to those observed in CML BM (16ng/ml MIP1α, 8ng/ml MIP1β, 2.5ng/ml IL-1α, 3.5ng/ml IL-1β, 0.05ng/ml TNF-α) resulted in significantly reduced growth of normal LTHSC compared with CML LSC. These results indicate that diffusible factors produced by leukemic cells in the CML BM environment selectively inhibit normal LTHSC compared to CML LSC growth. Exposure of a murine stromal cell line to CML BM SN resulted in reduced CXCL12 mRNA levels compared to BM SN from control mice. The cytokine G-CSF, which was increased in CML BM SN, has been reported to reduce CXCL12 transcription. We observed significant reduction of CXCL12 mRNA levels in stromal cells cultured with G-CSF (0.2ng/ml), supporting a potential role for increased G-CSF production by leukemia cells in reduced CXCL12 production by CML BM stromal cells and reduced LSC retention in the BM. We evaluated whether defects in microenvironmental function in CML were affected by imatinib treatment. Treatment of CML mice with imatinib (200mg/kg/day, 2 weeks) led to reduction in MIP1α, MIP1β, IL-1β, and IL-6 levels in BM cells. Engraftment of normal LTHSC was significantly enhanced in BM of CML recipients pre-treated with imatinib. Results obtained with the mouse model were validated using specimens obtained from CML patients. CXCL12 mRNA levels were significantly reduced in human CML compared to normal MNCs, whereas expression of MIP1α, MIP-2, IL-1α and IL-1β were increased in CML MNCs, consistent with results obtained with the mouse model. Coculture with CML MNC conditioned medium (CM) resulted in selective impairment of growth of normal CD34+CD38- primitive progenitors compared to CM from normal MNC, but did not inhibit growth of CML progenitors. We conclude that leukemia-induced alterations in BM cytokine and chemokine levels contribute to altered LSC lodgment and to selective impairment of growth of normal LTHSC in the CML BM microenvironment, leading to a relative growth advantage for CML LSC over normal LTHSC and expansion of the leukemic clone. Disclosures:Holyoake:Novartis: Research Funding; Bristol Myers Squibb: Research Funding.

Enhanced ABL-inhibitor-induced MAPK-activation in T315I-BCR-ABL-expressing cells: a potential mechanism of altered leukemogenicity

Targeted treatment of chronic myelogenous leukemia using imatinib has dramatically improved patient outcome. However, residual disease can be detected in the majority of patients treated with imatinib. Compensatory activation of MAP kinases (MAPK1/2) in response to BCR-ABL-inhibitors has been reported as a potential cytokine-dependent resistance mechanism leading to the rescue of leukemic progenitor cells. Differential MAPK-modulating activity of clinically approved tyrosine kinase inhibitors was assessed in vitro using BCR-ABL-transformed cells. CD34+-enriched progenitors of newly diagnosed chronic myelogenous leukemia patients were exposed to tyrosine kinase inhibitors. MAPK-signaling was studied by Western blot technique. Proliferation assays were used to analyze response to antileukemic treatment. The ABL-inhibitors imatinib and nilotinib activate MAPKs in CD34+ chronic myelogenous leukemia progenitor cells, whereas treatment with the SRC/ABL-inhibitor dasatinib does not affect MAPK-activation at clinically relevant concentrations. Similar results are seen in BCR-ABL-transformed cells in the presence of interleukin-3 (IL-3). Experiments using BCR-ABL-mutant T315I, a resistance mutation not amenable to tyrosine kinase inhibitor binding, demonstrate that ABL-inhibitor-induced MAPK-activation does not depend on BCR-ABL-inhibition and cannot be prevented by selective SRC-inhibition. However, BCR-ABL-T315I enhances MAPK-activation, suggesting a T315I-dependent positive feedback of MAPK-activation. An autocrine IL-3-loop as trigger for aberrant T315I-dependent MAPK-activation was excluded. Aberrant MAPK-activation triggered by ABL-inhibitors and positively regulated by BCR-ABL kinase mutation T315I might be an experimental explanation for the clinical observation that patients carrying high-resistance mutations show a highly aggressive course of their disease when tyrosine kinase inhibitor treatment is not discontinued in time.

Small GTPase RAB45-mediated p38 activation in apoptosis of chronic myeloid leukemia progenitor cells

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. BCR-ABL transforming activity is mediated by critical downstream signaling pathways that are aberrantly activated by tyrosine kinases. However, the mechanisms of BCR-ABL anti-apoptotic effects and the signaling pathways by which BCR-ABL influences apoptosis in BCR-ABL-expressing cells are poorly defined. In this study, we found that treatment with ABL kinase inhibitors or depletion of BCR-ABL induced the expression of RAB45 messenger RNA and protein and induced apoptosis via reduction of mitochondrial membrane potential and p38 activation in CML cell lines and BCR-ABL(+) progenitor cells from CML patients. Overexpressed RAB45 induced the activation of caspases-3 and -9 and reduced the expression of Survivin, XIAP, c-IAP1 and c-IAP2 in CML cells. Moreover, in colony-forming cells derived from CML-aldehyde dehydrogenase(hi)/CD34(+) cells, treatment with ABL kinase inhibitors induced RAB45 expression and reduced mitochondrial membrane potential, resulting in inhibited colony formation of Bcr-Abl(+) progenitor cells. The overexpression of RAB45 significantly decreased colony numbers and induced apoptosis through the activation of caspases-3 and -9. Furthermore, the overexpression of RAB45 increased the phosphorylation levels of p38, resulting in the induction of apoptosis and inhibition of proliferation of CML progenitor cells. Our results identify a new signaling molecule involved in BCR-ABL modulation of apoptosis and suggest that RAB45 induction strategies may have therapeutic utility in patients with CML.

Nilotinib: A Novel, Selective Tyrosine Kinase Inhibitor

The development of tyrosine kinase inhibitors (TKIs) for the treatment of chronic myelogenous leukemia (CML) was based on the discovery that CML stem and progenitor cells overexpress the abnormal fusion protein kinase BCR-ABL. The prototype TKI, imatinib, selectively inhibits BCR-ABL, as well as several other kinases, including stem cell factor receptor (KIT), discoidin domain receptor (DDR), platelet-derived growth factor receptor (PDGFR), and colony-stimulating factor receptor-1 (CSF-1R). Although the management of CML improved dramatically with the introduction of imatinib, not all patients benefit from treatment because of resistance or intolerance. Consequently, research efforts have focused on developing more potent TKIs with the ability to circumvent imatinib resistance. Nilotinib, a second-generation oral TKI, was rationally designed based on the crystal structure of imatinib to be highly active against a wide range of imatinib-resistant BCR-ABL mutants and is approved for the treatment of newly diagnosed or imatinib-resistant or -intolerant CML, and has shown superiority over imatinib in first-line treatment for newly diagnosed CML. Furthermore, the activity of nilotinib against KIT and PDGFRα has led to its evaluation in advanced gastrointestinal stromal tumors (GIST). The purpose of this review is to describe the development of nilotinib, providing a structural explanation for the differential activity of nilotinib and imatinib in GIST. Activity of nilotinib against KIT and PDGFR and emerging evidence of differences in cellular uptake between nilotinib and imatinib are discussed.

Efficient gene transfer with pseudotyped recombinant adeno-associated viral vectors into human chronic myelogenous leukemia cells

Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1–ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded self-complementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations. On human CML cell lines, ssAAV2/2 and ssAAV2/6 were most efficient. On primary cells, ssAAV2/6 proved significantly more efficient (4.1 ± 2.5% GFP+ cells, p = 0.011) than the other vectors (<1%). The transduction efficiency could be significantly increased (45.5 ± 13.4%) by using dsAAV2/6 vectors (p < 0.001 vs. ssAAV2/6). In these settings, our data suggest conversion of single- to double-stranded DNA and cell binding/entry as rate-limiting steps. Furthermore, gene transfer was observed in both late and earlier CML (progenitor) populations. For the first time, efficient AAV gene transfer into human CML cells could be shown, with the potential for future clinical application.

BCR-ABL1 Kinase Activity but Not Its Expression Is Dispensable for Ph+ Quiescent Stem Cell Survival Which Depends on the PP2A-Controlled Jak2 Activation and Is Sensitive to FTY720 Treatment

AbstractAbstract 515The success of tyrosine kinase inhibitors as first line therapy for t(9;22) Chronic Myelogenous Leukemia (CML) depends on the addiction that Philadelphia-positive (Ph+) hematopoietic progenitors, but not quiescent Ph+ stem cells, have for BCR-ABL1 tyrosine kinase activity. We reported that the activity of the tumor suppressor PP2A is inhibited in a SET-dependent manner in CML progenitors and CD34+/CD38- stem cells from chronic phase (CP) and, to a greater extent, blast crisis (BC) CML patients (Neviani P. et al.: Cancer Cell 2005, J. Clin. Invest 2007, and ASH 2008). Restoration of PP2A activity by the immunosuppressive sphingosine analogue FTY720 markedly decreases the number of Ph+ but not normal long-term culture-initiating cells (LTC-IC) and quiescent stem cells (CFSEMAX/CD34+) by suppressing the BCR/ABL kinase-independent enhancement of b-catenin expression/transcriptional activity (Oaks JJ., et al., ASH 2009).Here we report that FTY720 induces apoptosis of Ph+ CD34+/CD38- cells independent from its phosphorylation as treatment with a phosphorylated FTY720 did not alter the number of Ph+ CFSEMAX/CD34+ cells. By contrast, two non phosphorylatable and non immunosuppressive FTY720 derivatives did significantly affect survival of Ph+ stem/progenitor cells.Interestingly, we also noted that the activity but not expression of BCR-ABL1 is considerably lower in quiescent CFSEMAX/CD34+ than CFSE+/CD34+ cells that underwent at least one division (∼80% decrease; n=3). Conversely, BCR-ABL1 expression is significantly higher in quiescent than proliferating CFSE+/CD34+ cells, suggesting that BCR-ABL1 might serve as a scaffold for other kinase(s) able to sustain survival and quiescence of Ph+ stem cells. Indeed, we found that expression of the K1172R kinase-deficient BCR-ABL1 mutant enhances expression and activity of Jak2, a kinase that is not only associated with BCR-ABL1 but is also capable of inactivating and being inactivated by PP2A. Accordingly, lentiviral shRNA-mediated BCR-ABL1 downregulation in Ph+ CD34+/CD38- stem cells resulted in marked (≥70% inhibition, P<0.05) reduction of Jak2 activity (measured by Jak2pY1007/8 intracellular flow cytometry) and of the PP2A inhibitor SET (≥20% inhibition, P<0.05). Consistent with a role of Jak2 as regulator of Ph+ stem cell survival, pharmacologic Jak2 inhibition (Jak2 inhibitor 1, 1uM; TG101209, 2.5uM; or TG101348, 1uM and 0.5uM) significantly reduces the number of Ph+ CFSEMAX/CD34+ (47% reduction, n=4), impairs LTC-IC Ph+ colony output (60% reduction, n=4), and induces apoptosis of Ph+ CD34+/CD38- cells. Inhibition of Jak2 also resulted in impaired b-catenin dependent transcriptional activity (61% reduction by LET-TCF luciferase assay, n=2), suggesting the existence of an active Jak2-PP2A interplay likely controlling survival and self-renewal of Ph+ stem cells through the Wnt pathway. In this regard, in vivo administration of FTY720 (4 weeks; 10mg/Kg/day) to FVB/N recipient mice transplanted with either GFP-sorted 2×106 whole bone marrow or 3×103 Lin−/Sca-1+/c-Kit+ (LSK) stem cells from leukemic scl-tTA/BCR-ABL/GFP mice not only significantly decreased the number of leukemic common and granulocyte-macrophage (CMP and GMP) progenitors but, more importantly, it resulted in 70–80% reduction in the number of the long-term hematopoietic stem cells (Lin−/Sca-1+/c-Kit+/CD48+/CD150−/Flt3−) compared to untreated leukemic mice. Furthermore, severely impaired engraftment (∼90% untreated vs. ∼37% FTY720-treated) was observed in secondary recipients transplanted with 2×106 bone marrow of FTY720-treated leukemic mice, suggesting that FTY720-induced PP2A activation inhibits the ability of BCR-ABL1-expressing LSK cells to undergo extensive self renewal in primary recipients.Thus, expression but not activity of the oncogenic product of the t(9;22) translocation is important for recruiting and allowing SET-mediated inhibition of PP2A and activation of Jak2; two events important for Ph+ stem cell survival and self renewal. Moreover, the ability of FTY720 and of its non-immunosuppressive derivatives to induce apoptosis of Ph+ progenitors and Ph+ but not normal quiescent stem cells emphasizes the notion that FTY720 and its derivatives represent strong and non-toxic anti-leukemic agents potentially useful not only for the treatment but, perhaps, for eradicating Ph+ leukemias. Disclosures:Holyoake:Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.