The Hematopoietic Oxidase NOX2 Regulates Self-Renewal of Leukemic Stem Cells

Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML.

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Targeting Tetramer-Forming GABPβ Isoforms Impairs Self-Renewal of Hematopoietic and Leukemic Stem Cells

Cell Stem Cell ◽

10.1016/j.stem.2012.05.021 ◽

2012 ◽

Vol 11 (2) ◽

pp. 207-219 ◽

Cited By ~ 20

Author(s):

Shuyang Yu ◽

Xuefang Jing ◽

John D. Colgan ◽

Dong-Mei Zhao ◽

Hai-Hui Xue

Keyword(s):

Stem Cells ◽

Leukemic Stem Cells ◽

Self Renewal

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MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v104.11.62.62 ◽

2004 ◽

Vol 104 (11) ◽

pp. 62-62

Author(s):

Brian J.P. Huntly ◽

Hirokazu Shigematzu ◽

Kenji Deguchi ◽

Benjamin Lee ◽

Shinichi Mizuno ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mononuclear Cells ◽

Hierarchical Organization ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Acute Myeloid ◽

Myeloid Progenitors

Abstract The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

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BMI-1 Overexpression in 32Dcl3 Cells Suppresses Granulocytic Differentiation and Ameliorates Their Growth under IL-3 Diminished Conditions.

Blood ◽

10.1182/blood.v106.11.4211.4211 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4211-4211

Author(s):

Asahi Hishida ◽

Kazuhito Yamamoto ◽

Masashi Sawa ◽

Tomoki Naoe

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Real Time Pcr ◽

Bone Marrow Cells ◽

Polycomb Group ◽

The Self ◽

Leukemic Stem Cells ◽

Granulocytic Differentiation ◽

Self Renewal ◽

Mtt Assays

Abstract The BMI-1 gene is one of the polycomb group (PcG) genes that have been shown to play essential roles in the self-renewal of both normal and leukemic stem cells. BMI-1 is reported to be highly expressed in primitive cells and its expression level decreases as bone marrow cells differentiate. We hypothesized that overexpression of BMI-1 might modulate the growth, differentiation and survival of 32D cells, and examined this hypothesis by generating BMI-1 overexpressing 32D cells. 32D cells were infected with MIG-BMI-1-IRES-EGFP vector or MIG vector, and the GFP positive rates of BMI-1 overexpressing 32D cells gradually increased under IL-3 diminished conditions, and the consecutive MTT assays using cells sorted by GFP positivity confirmed this finding, suggesting that BMI-1 could confer growth advantages on 32D cells under IL-3 diminished conditions. Exposure of the GFP sorted 32D cells to G-CSF revealed that BMI-1 overexpression suppressed the granulocytic differentiation of 32D cells by G-CSF. The expressions of CD11b and Gr-1 were both suppressed by the overexpression of BMI-1, and real-time PCR analyses indicated that the expression levels of MPO was remarkably suppressed, while those of C/EBPα were not significantly changed. Our experiments revealed that BMI-1 delayed granulocytic differentiation and ameliorated the growth of 32D cells under IL-3 diminished conditions.

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Identification and Purification of Leukemic Stem-Like Cells in Acute Myeloid Leukemia Cell Line

Blood ◽

10.1182/blood.v112.11.4734.4734 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4734-4734

Author(s):

Miaorong She ◽

Xingqing Niu ◽

Xilin Chen ◽

Guo Kunyuan ◽

Maohua Zhou ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Cell Line ◽

Myeloid Leukemia ◽

Critical Role ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is initiated and maintained by a rare population of (leukemic stem cells) LSCs. LSCs play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population for ultimately curing leukemia which is it is important to identify and study LSCs. However, relatively little is known about the unique molecular mechanisms of survival and self-renewal of LSCs because of very small number of LSCs in bone marrow. In this study, we investigated whether established leukemia cell lines contain LSCs. We showed that leukemia cell line contain leukemic stem-like cells which have been phenotypically restricted within the CD34+CD38− fraction. We demonstrated that CD34+CD38− cells could generate CD34+CD38+ cells in culture medium and had proliferation function. Moreover, CD34+CD38− cells had self-renewal potential both in vitro soft agar colonies formation assay and in vivo NOD/SCID mouse xenotransplant model serial transplantation. Furthermore, CD34+CD38− cells isolated from leukemia cell line were found resistant to conventional chemotherapy and NK cells-mediated cytotoxicity and these were related to up-regulation of ABCG2 and MRP-1 and antiapoptotic proteins of Bcl2. Down-regulation of NKG2D ligand also played a critical role in NK cytotoxicity resistance. Taken together, our studies provide a novel cell model for leukemic stem cells research. Our data also shed light on mechanism of double resistant to resistant to chemotherapy and NK cell immunotherapy, which was helpful for developing novel effective strategies for LSCs.

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BCR-ABL-transformed GMP as myeloid leukemic stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0808303105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17967-17972 ◽

Cited By ~ 64

Author(s):

Yosuke Minami ◽

Scott A. Stuart ◽

Tomokatsu Ikawa ◽

Yong Jiang ◽

Asoka Banno ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Knockout Mouse ◽

Ex Vivo ◽

Blast Crisis ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Established Line ◽

Ex Vivo Culture

During blast crisis of chronic myelogenous leukemia (CML), abnormal granulocyte macrophage progenitors (GMP) with nuclear β-catenin acquire self-renewal potential and may function as leukemic stem cells (Jamieson et al. N Engl J Med, 2004). To develop a mouse model for CML-initiating GMP, we expressed p210BCR-ABL in an established line of E2A-knockout mouse BM cells that retain pluripotency in ex vivo culture. Expression of BCR-ABL in these cells reproducibly stimulated myeloid expansion in culture and generated leukemia-initiating cells specifically in the GMP compartment. The leukemogenic GMP displayed higher levels of β-catenin activity than either the nontransformed GMP or the transformed nonGMP, both in culture and in transplanted mouse BM. Although E2A-deficiency may have contributed to the formation of leukemogenic GMP, restoration of E2A-function did not reverse BCR-ABL-induced transformation. These results provide further evidence that BCR-ABL-transformed GMP with abnormal β-catenin activity can function as leukemic stem cells.

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439 Dual modes of action for anti-TIM-3 antibody MBG453 in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML): preclinical evidence for immune-mediated and anti-leukemic activity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0439 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A465-A465

Author(s):

Catherine Sabatos-Peyton ◽

Tyler Longmire ◽

Lisa Baker ◽

Nidhi Patel ◽

Anne-Sophie Wavreille ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

High Risk ◽

Myeloid Leukemia ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Immune Mediated ◽

Acute Myeloid ◽

Cell Stem Cell

BackgroundTIM-3 is expressed on leukemic stem cells (LSCs) and blasts in AML,1 2 and TIM-3 expression on MDS blasts correlates with disease progression.3 Functional evidence for TIM-3 in AML was established with an anti-TIM-3 antibody which inhibited engraftment and development of human AML in immuno-deficient murine hosts.1 TIM-3 promotes an autocrine stimulatory loop via the TIM-3/Galectin-9 interaction, supporting LSC self-renewal.4 In addition to its cell-autonomous role on LSCs/blasts, TIM-3 also has a critical role in immune system regulation, in adaptive (CD4+ and CD8+ T effector cells, regulatory T cells) and innate (macrophages, dendritic cells, NK cells) immune responses.5 MBG453 is a high-affinity, humanized anti-TIM-3 IgG4 antibody (Ab) (stabilized hinge, S228P), which blocks the binding of TIM-3 to phosphatidylserine (PtdSer). Recent results from a multi-center, open label phase Ib dose-escalation study (NCT03066648) in patients with high-risk MDS and no prior hypomethylating agent therapy evaluating MBG453 in combination with decitabine demonstrated encouraging preliminary efficacy with an overall response rate of 58%,6 and MBG453 combined with azacitidine also showed encouraging response rates.7 Preclinical experiments were undertaken to define the mechanism of action of the hypomethylating agent and anti-TIM-3 combination.MethodsTHP-1 cells (a human monocytic AML cell line) were pre-treated with decitabine and co-cultured with anti-CD3 activated healthy human donor peripheral blood mononuclear cells (PBMCs) in an Incucyte-based assay to measure cell killing. The ability of MBG453 to mediate antibody-dependent cellular phagocytosis (ADCP) was measured by determining the phagocytic uptake of an engineered TIM-3-overexpressing Raji cell line in the presence of MBG453 by phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells. Patient-derived AML xenograft studies were undertaken in immune-deficient murine hosts to evaluate the combination of decitabine and MBG453.ResultsMBG453 was determined to partially block the TIM-3/Galectin-9 interaction in a plate-based MSD (Meso Scale Discovery) assay, supported by a crystal structure of human TIM-3.8 Pre-treatment of THP-1 cells with decitabine enhanced sensitivity to immune-mediated killing in the presence of MBG453. MBG453 was determined to mediate modest ADCP, relative to controls. MBG453 did not enhance the anti-leukemic activity of decitabine in patient-derived xenograft studies in immuno-deficient hosts.ConclusionsTaken together, these results support both direct anti-leukemic effects and immune-mediated modulation by MBG453. Further studies are ongoing to determine: (1) whether MBG453 can mediate physiologically relevant ADCP of TIM-3-expressing leukemic cells; and (2) the potential of MBG453 to impact the autocrine feedback loop of TIM-3/Galectin-9.Ethics ApprovalThe human tissue used in these studies was under the Novartis Institutes of BioMedical Research Ethics Board IRB, Approval Number 201252867.ReferencesKikushige Y, Shima T, Takayanagi S, et al. TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem Cell 2010;7(6):708–717.Jan M, Chao MP, Cha AC, et al. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci USA 2011; 108(12): 5009–5014.Asayama T, Tamura H, Ishibashi M, et al. Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes. Oncotarget 2017;8(51): 88904–88917.Kikushige Y, Miyamoto T, Yuda J, et al. A TIM-3/Gal-9 autocrine stimulatory loop drives self-renewal of human myeloid leukemia stem cells and leukemic progression. Cell Stem Cell 2015; 17(3):341–352.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020; 8(1):e000911.Borate U, Esteve J, Porkka K, et al. Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in combination with decitabine in patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Blood 2019;134 (Supplement_1):570.Borate U, Esteve J, Porkka K, et al. Abstract S185: Anti-TIM-3 antibody MBG453 in combination with hypomethylating agents (HMAs) in patients (pts) with high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML): a Phase 1 study. EHA 2020.Sabatos-Peyton C. MBG453: A high affinity, ligand-blocking anti-TIM-3 monoclonal Ab. AACR 2016.

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KLF4 Controls Leukemic Stem Cell Self-Renewal in MLL-AF9-Induced Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129164 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1231-1231

Author(s):

Andrew Lewis ◽

Chun Shik Park ◽

Monica Puppi ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Epigenetic Mechanisms ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Limiting Dilution

Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.

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A STAT5B-CD9 axis determines self-renewal in hematopoietic and leukemic stem cells

Blood ◽

10.1182/blood.2021010980 ◽

2021 ◽

Author(s):

Sebastian Kollmann ◽

Reinhard Grausenburger ◽

Thorsten Klampfl ◽

Michaela Prchal-Murphy ◽

Klavdija Bastl ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Prognostic Marker ◽

Surface Marker ◽

The Self ◽

Leukemic Stem Cells ◽

Self Renewal ◽

Unique Role ◽

Redundant Functions

The transcription factors STAT5A and STAT5B are critical in hematopoiesis and leukemia. They are widely believed to have redundant functions but we describe a unique role for STAT5B in driving the self-renewal of hematopoietic and leukemic stem cells (HSCs/LSCs). We find STAT5B to be specifically activated in HSCs and LSCs, where it induces many genes associated with quiescence and self-renewal, including the surface marker CD9. Levels of CD9 represent a prognostic marker for patients with STAT5-driven leukemia and our findings suggest that anti-CD9 antibodies may be useful in their treatment to target and eliminate LSCs. We show that it is vital to consider STAT5A and STAT5B as distinct entities in normal and malignant hematopoiesis.

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DYRK2 Inhibits the Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Inducing Degradation of c-Myc Downstream of the Reprogramming Factor KLF4

Blood ◽

10.1182/blood.v128.22.1879.1879 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1879-1879

Author(s):

Chun Shik Park ◽

Ye Shen ◽

Koramit Suppipat ◽

Andrew Lewis ◽

Julie Tomolonis ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Parp Cleavage ◽

Leukemic Stem Cells ◽

Vitamin K3 ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+ and CD34+CD38− cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients. Disclosures No relevant conflicts of interest to declare.

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