Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Abstract Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors. Abstract 4508. Table CML CP Normal CML BC Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Apoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+) 3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 ** 3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 * 3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 ** CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+) 71.3± 7.8 7± 3.2 ** 21± 7.3 ** 5 ± 2.3 ** 121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 *** 82.7±33.1 ** 8 ± 3.6 *** NSG engraftment (CD34+) 1.8± 0.3 0.4± 0.1 *** 0.8± 0.3 ** 0.4± 0.04 *** 68.2± 4.9 61± 2.2 68.1± 2.9 64.2± 3.9 Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 Disclosures No relevant conflicts of interest to declare.

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The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽

10.1182/blood.v116.21.516.516 ◽

2010 ◽

Vol 116 (21) ◽

pp. 516-516 ◽

Cited By ~ 1

Author(s):

Daniel Goff ◽

Alice Shih ◽

Angela Court Recart ◽

Larisa Balaian ◽

Ryan Chuang ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Blast Crisis ◽

Chemotherapy Resistance ◽

Chronic Phase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

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Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Blood ◽

10.1182/blood.v118.21.448.448 ◽

2011 ◽

Vol 118 (21) ◽

pp. 448-448

Author(s):

Ling Li ◽

Lisheng Wang ◽

Liang Li ◽

Tinisha McDonald ◽

Yin Wei Ho ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Blast Crisis ◽

Fish Analysis ◽

Pharmacological Inhibition ◽

Knock Down ◽

Cd34 Cells

Abstract Abstract 448 BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSEhigh cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*105±0.1*105 human CD45+ cells in murine BM vs. Control 1.8*105±0.6*105, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*106±0.7*106 human CD45+ cells in murine BM, vs. Control 2.4*106±0.8*106, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML. Disclosures: No relevant conflicts of interest to declare.

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Antibody-Targeting of IL-3 Receptor-α Increases the Susceptibility of CD34+ CML Progenitors to Dasatinib-Induced Cell Death,

Blood ◽

10.1182/blood.v118.21.3745.3745 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3745-3745

Author(s):

Eva Nievergall ◽

Deborah L. White ◽

Hayley Ramshaw ◽

Angel F. Lopez ◽

Timothy P. Hughes ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Progenitor Cells ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Chronic Phase ◽

Advisory Committees ◽

Cd34 Cells

Abstract Abstract 3745 Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML), Ph+ CD34+ progenitor cells remain detectable even in patients with stable complete cytogenetic response. Over 40% of patients in stable complete molecular remission will develop molecular relapse within 6 months of stopping imatinib. While the exact causes are largely unknown, one of the proposed mechanisms is the protection of leukemic stem and early progenitor cells by the paracrine or autocrine production of cytokines, such as IL-3, GM-CSF and G-CSF, which activate survival pathways that bypass TKI-induced cytocidal effects. In acute myeloid leukemia (AML), the IL-3 receptor α chain (CD123) is recognized as a specific marker for CD34+/CD38− stem cells and therefore is attracting increasing interest as a therapeutic target. However, the function of CD123 in CML remains to date mostly unexplored. The aim of this study is to investigate potential synergy between TKIs and CSL362 (a humanized antibody version of 7G3 against CD123) in targeting CML progenitor and stem cells. CD34+ and CD34+/CD38− cells were isolated from mononuclear cells of newly diagnosed CML chronic phase and blast crisis patients. Flow cytometry studies indicated significantly increased CD123 expression on CD34+/CD38− cells of CML patients in both chronic phase and blast crisis when compared to normal hematopoietic stem cells (p<0.01 and p<0.001 for chronic phase and blast crisis, respectively; Figure A). A functional relevance of increased CD123 expression was demonstrated by IL-3-dependent increase in STAT5 phosphorylation (260.5% of baseline with 20 ng/ml IL-3; n=12; p<0.001) in CML CD34+ cells. Dasatinib inhibits STAT5 phosphorylation by blocking BCR-ABL signaling but only in the absence of IL-3 (62.5% of baseline for dasatinib alone vs. 130.8% for dasatinib + IL-3; n=3; p<0.01). In agreement, IL-3 effectively rescues dasatinib-induced cell death, as evaluated by AnnexinV/7-AAD staining (103.3% vs. 72.45%, n=5; p<0.01) and CFU-GM colony forming assays (69.39% vs. 46.13% relative to no treatment control; n=4; p<0.05). CSL362, in turn, revokes IL-3-mediated STAT5 phosphorylation (37.12% vs. 130.8%; n=3; p<0.001) and cytoprotection (45.05% vs. 69.39% CFC; n=4; p<0.01). In order to further elucidate the role of CSL362, CML CD34+ cells were cultured with increasing concentrations of dasatinib in the presence of IL-3 and CSL362 or BM4 isotype-matched control antibody. Even at very low dasatinib concentrations, CSL362 significantly reduces CML CD34+ colony forming cells (p<0.05; Figure B). Together these results substantiate a relevant role for IL-3-mediated resistance in CML progenitor cells and additionally confirming the ability of CSL362 to effectively bind to CD123 and impede IL-3 function. CSL362 furthermore has been optimized to mediate antibody dependent cell cytotoxicity (ADCC). CSL362 causes specific cell lysis of CML CD34+ progenitor cells in co-culture with allogeneic Natural killer cells as determined by increased lactate dehydrogenase release (ADCC activity of 42.4% ± 8.1%; n=3) and a decrease in the number of CFU-GM colonies by 74.1 % ± 12.2% (n=3). Collectively, our results indicate that a combination of dasatinib and CSL362 inhibits CML progenitor cell survival more effectively in vitro. Therefore, targeting IL-3 receptor α with CSL362 in chronic phase and blast crisis CML patients might provide a novel specific treatment approach aiding the elimination of refractory chronic myeloid leukemic stem and progenitor cells. A: Flow cytometry analysis reveals that CD123 expression is significantly higher in CD34+/CD38− cells of CML patients in chronic phase (CML-CP) and blast crisis (BC-CML) as compared to normal patients (NP), as previously documented for AML patients. ** p<0.01, *** p<0.001 by unpaired, two-tailed Student's t-test. B: In the presence of IL-3, CSL362 significantly reduces the number of colony forming cells. CD34+ cells of de novo CML-CP patients were cultured with dasatinib (0 to 10 nM) +IL-3 (1 ng/ml) ± CSL362 or BM4 (isotype control for CSL362). After 72 hours of culture live cells were plated for CFU-GM assay and colonies were counted after 2 weeks. Mean ± SE of three independent experiments is shown, n=4, p<0.05 by two-way ANOVA. Disclosures: Nievergall: CSL: Research Funding. White:CSL: Research Funding. Lopez:CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:CSL: Research Funding.

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Cooperative Targeting of Bcl-2 Family Proteins By ABT-199 (GDC-0199) and Tyrosine Kinase Inhibitors to Eradicate Blast Crisis CML and CML Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.512.512 ◽

2014 ◽

Vol 124 (21) ◽

pp. 512-512 ◽

Cited By ~ 2

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Hong Mu ◽

Hongsheng Zhou ◽

Duncan H Mak ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Single Agent ◽

Specific Inhibitor ◽

Chronic Phase

Abstract Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy. To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation. To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing. Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

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In Vivo Analyses of Leukemia Stem Cells in Genetically Defined Murine Models of Chronic and Blast Crisis CML.

Blood ◽

10.1182/blood.v108.11.236.236 ◽

2006 ◽

Vol 108 (11) ◽

pp. 236-236

Author(s):

Craig T. Jordan ◽

Sarah J. Neering ◽

Pin-Yi Wang ◽

Randall M. Rossi ◽

Timothy Bushnell

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Blast Crisis ◽

Chronic Phase ◽

Leukemic Cells ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Normal Controls

Abstract Studies to date have shown that primary human leukemia stem cells (LSC) are resistant to standard chemotherapy agents and are likely to be a major cause of drug refractory disease and relapse. Therefore, elucidating the in vivo biology of LSC is critical in order to develop more effective therapeutic regimens. To this end, we report the first genetically defined model of LSC, using syngeneic murine systems in which the biological features of human LSC are recapitulated. The approach employs retroviral vectors to transduce normal murine hematopoietic stem cells with either BCR/ABL-GFP alone, or in combination with Nup98/HoxA9-YFP. Expression of BCR/ABL creates a well-described model of chronic phase CML, whereas expression of BCR/ABL in combination with Nup98/HoxA9 induces acute disease that mimics blast crisis CML. Analysis of the normal cell competent to generate LSC indicates that the BCR/ABL mutation must occur in primitive HSC in order to manifest disease, however, subsequent progression to blast crisis can occur through mutation in cells at the myeloid progenitor stage. Characterization of stem cells in these models revealed several striking features. First, chronic phase stem cells are1 phenotypically identical to normal hematopoietic stem cells (lin−, Sca-1+, c-kit+) and display cell cycle rates (percentage of cells in S or G2 phase) that are nearly double normal controls. However, the overall frequency of such cells is not elevated. In contrast, blast crisis stem cells show a distinct immunophenotype (lin−, Sca-1+, c-kit-lo, Flt3+, CD150−) and cycle rates nearly identical to normal controls, but are approximately 10-fold increased numbers. These data indicate that BCR/ABL alone functions as a stem cell mitogen, but does not enhance self-renewal, whereas added expression of Nup98/HoxA9 is sufficient to increase self-renewal, but return cell cycle regulation to normal levels. Furthermore, analysis of co-resident non-leukemic cells in each model shows that while the cycle activity of normal stem cells (HSC) was not affected, the cycle rates of normal progenitors (lin−, c-kit+) were substantially reduced. Thus, in either disease, active suppression of normal progenitors is evident and thereby increases the growth advantage of malignant populations. To test methods for modulation of normal vs. leukemic cells in vivo, we challenged blast crisis animals with ara-C (single dose, 100mg/kg) or imatinib mesylate (200mg/kg/day for 3 consecutive days) and assessed the consequences in primitive populations. The data indicate that ara-C reduced frequency and cycle rate of progenitor cells in vivo, but that the effects were identical between normal and malignant populations. Thus, at least for short-term studies there was no therapeutic index for ara-C at the level of primitive cells. In contrast, treatment with imatinib induced a 50% increase in the cycle rate and a 2–4 fold increase in numbers of progenitor cells. These findings imply a homeostatic mechanism in blast crisis leukemia, where pressure towards the malignant population may induce increased activity of stem and progenitor cells. In summary, this model provides a novel means by which the biology of LSCs may be directly characterized and the consequences of candidate treatment regimens can be assessed with regard to normal vs. leukemia stem cells in vivo.

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Leukemic Stem Cells of Chronic Phase CML Patients Consistently Display Very High BCR-ABL Transcript Levels and Reduced Responsiveness to Imatinib Mesylate in Addition to Generating a Rare Subset That Produce Imatinib Mesylate-Resistant Differentiated Progeny.

Blood ◽

10.1182/blood.v104.11.711.711 ◽

2004 ◽

Vol 104 (11) ◽

pp. 711-711 ◽

Cited By ~ 7

Author(s):

Xiaoyan Jiang ◽

Yun Zhao ◽

Wing Yiu Chan ◽

Emily Pang ◽

Allen Eaves ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Imatinib Mesylate ◽

Chronic Phase ◽

Stem Cell Differentiation ◽

Epigenetic Mechanism ◽

Transcript Levels ◽

Viable Cells ◽

Cd34 Cells

Abstract Imatinib mesylate (IM) is an inhibitor of the BCR-ABL oncoprotein associated with human chronic myeloid leukemia (CML). IM therapy has shown remarkable effects in initial clinical trials, but both clinical and laboratory studies increasingly suggest that, on its own, IM may have limited curative potential, due to a reduced IM sensitivity of the more primitive, slowly proliferating CD34+ CML cells thought to be responsible for sustaining the disease in vivo. To investigate the basis of this unresponsiveness, we compared the IM sensitivity and BCR-ABL expression of FACS-purified subsets of lin−CD34+ cells from 4 CML chronic phase patients. None of these had been treated with IM and their cells at all stages of differentiation were exclusively leukemic; i.e., >95% of the lin−CD34+CD38−, lin−CD34+CD38+ and lin+CD34− cells were BCR-ABL+ (by direct FISH) and all longterm culture-initiating cell (LTC-IC) -derived CFCs were Ph+. In the absence of IM, suspension cultures initiated with these lin−CD34+CD38− CML cells (0.5–5% of the lin−CD34+ cells) showed a net expansion of viable cells after 3 weeks; 100x with and 10x without added growth factors (GFs). Addition of 0.1–10 μM/ml IM reduced the yield of viable cells in a dose-dependent fashion, particularly when GFs were not added (100-fold decrease with 10 μM/ml IM). Parallel cultures of the corresponding lin−CD34+CD38+ CML cells showed these did not expanded as much (~8x +GFs, 2x -GFs) and were more sensitive to IM (1000-fold decrease after 3 weeks in 10 μM/ml IM -GFs). Quantitative real-time RT-PCR analysis revealed BCR-ABL transcripts to be present in the most primitive, freshly isolated lin−CD34+CD38− cells (n=12) at >300-fold higher levels than in the terminally differentiating lin+CD34− CML cells (n=21), at >10-fold higher levels than the normal BCR transcripts in the same lin−CD34+CD38− cells, and at 40-fold higher levels than in the less primitive lin−CD34+CD38+ cells (n=12), indicating a correlation between decreasing BCR-ABL transcripts and increasing IM sensitivity during CML stem cell differentiation in vivo. Interestingly, maintenance of the lin−CD34+CD38− CML cells for 3 weeks in vitro with 10 μM/ml IM (±GFs) consistently selected for a subset of leukemic cells (80–100% BCR-ABL+ by FISH) that showed complete resistance to 5 μM/ml IM in CFC assays, in marked contrast to the CFCs in the starting lin−CD34+CD38− cells that were inhibited 5–10-fold by 5 μM/ml IM. Moreover, although the Ph was the sole abnormality present in all direct metaphases, initial CFCs and LTC-IC-derived CFCs from all samples, a 17p+ abnormality was seen in 4/4 metaphases obtained from one colony generated from the cells present in one of the 3-week IM-containing cultures, suggesting the selective survival of differentiating progeny of rare, pre-existing, IM-resistant stem cells. Consistent with this possibility was the finding that BCR-ABL transcript levels in the cells present in the 3 week cultures were reduced 50-fold relative to the input lin−CD34+CD38− cells. Taken together, these findings suggest a previously undescribed epigenetic mechanism of IM unresponsiveness characteristic of chronic phase CML stem cells, in addition to the silent accumulation of genetically-determined IM-resistant members as the CML stem cell population expands during the development of the chronic phase of the disease.

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Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v110.11.324.324 ◽

2007 ◽

Vol 110 (11) ◽

pp. 324-324

Author(s):

Xiaoyan Jiang ◽

Donna Forrest ◽

Franck Nicolini ◽

Karen Lambie ◽

Kyi Min Saw ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Blast Crisis ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase ◽

Cd34 Cells ◽

Clinical Responses ◽

Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

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Combined Pharmacologic Inhibition of Bcl-Xl/Bcl-2 and mTORC1/2 Survival Signals Trigger Apoptosis in BCR-ABL1+in Vitro Models of Blast Crisis Chronic Myelogenous Leukemia (CML-BC), and Primary CD34+/CD38− Stem and CD34+ progenitor Cells From CML-BC Patients

Blood ◽

10.1182/blood.v118.21.2738.2738 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2738-2738

Author(s):

Jason G Harb ◽

Paolo Neviani ◽

Claudia S Huettner ◽

Guido Marcucci ◽

Danilo Perrotti

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Colony Formation ◽

Mitochondrial Membrane ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Combined Treatment ◽

Annexin V ◽

Clonogenic Potential ◽

Induced Apoptosis

Abstract Abstract 2738 Tyrosine kinase inhibitors (TKIs) have become frontline therapy for CML; however, alternative therapies are required, as TKIs do not induce long-term response in CML patients undergoing blastic transformation and are ineffective against Philadelphia-positive (Ph+) quiescent stem cells, which show innate resistance to BCR-ABL1 kinase inhibitors. Therapeutic targets of interest are survival factors conferring resistance to TKI-induced apoptosis and/or those increasing proliferation of leukemic progenitors. We previously reported (Harb JG et al., ASH 2007) that genetic inactivation of Bcl-x did not inhibit BCR-ABL1 leukemogenesis in an inducible mouse model of CML. Thus, we hypothesize that BCR/ABL mediated post-translational modification and inactivation of pro-apoptotic BAD negates the requirement for the anti-apoptotic function of Bcl-xL in stem/progenitor cells from SCLtTA-BCR/ABL1/Bcl-x−/− mice. Following this rationale, we tested if simultaneous pharmacologic BAD activation and Bcl-xL inhibition may be an efficient way of killing CML stem/progenitor cells. To test this, loss of Bcl-xL function with increased levels of active BAD was achieved by expressing Bcl-x shRNA in 32D-BCR/ABL mouse myeloid precursors that were then treated with LY294002 (LY), which suppresses the inhibitory effects of PI-3K/Akt activation on BAD. Flow cytometric analysis of Annexin V+ cells revealed that levels of apoptosis were three times higher in BCR-ABL1+ cells expressing the Bcl-x shRNA when compared with vector-transduced BCR-ABL1+ cells. As expected, increased levels of dephosphorylated (active) BAD at the mitochondrial membrane were found in LY-treated BCR-ABL+ cells. Interestingly, co-treatment of Bcl-x shRNA-expressing BCR-ABL1+ cells with LY and the Bcl-xL/Bcl-2 antagonist ABT-263 (ABT) did not further promote apoptosis, suggesting that decreased survival of BCR-ABL1+ cells was dependent on downregulation of Bcl-xL and not Bcl-2. To determine efficacy of combined pharmacologic Bcl-xL inhibition and BAD activation, 32D-BCR/ABL and K562 cells were treated with compounds expected to activate BAD upon inhibition of PI-3K/Akt/mTOR-generated signals, used alone or in combination with ABT. Individually, at suboptimal doses, LY, Rapamycin (RAP), mTORC1/2 inhibitor PP242, and ABT were tolerated with apoptosis levels lower than 20%. Notably, when combined with ABT, all three efficiently induced apoptosis (∼90% Annexin V+) of BCR-ABL1+ cells. As with LY, increased levels of active BAD were found at the mitochondrial membrane of RAP- and PP242-treated BCR-ABL1+ cells. We found that PP242 downregulated p-Akt (92%), Mcl-1 (67%) and Bcl-xL (51%) more efficiently than RAP or LY. It has been shown that PP242 impairs the clonogenic potential of TKI-resistant mononuclear BM CML-BC cells; however, its effects when used alone or in combination with ABT on survival of normal and leukemic hematopoietic stem (HSCs) and progenitor cells is still unknown. Thus, HSC-enriched (CD34+/CD38-) and progenitor (CD34+) CML-BC cell fractions were isolated from bone marrow and peripheral blood and used in colony forming (CFC) assays with ABT, PP242 or ABT/PP242. ABT alone did not suppress colony formation of Ph+ CD34+/CD38− cells, while PP242 reduced it by nearly 50%. Conversely, ABT/PP242 combination decreased Ph+ stem and progenitor colony formation by ∼80%. Furthermore, the self-renewal of Ph+ CD34+/CD38− cells was markedly impaired by ABT/PP242 as demonstrated by the 80% decrease in replating efficiency. To assess if non-leukemic stem cells would tolerate ABT/PP242, colony assays were performed with LSK from wild type mice treated with ABT, PP242, RAP and ABT/PP242. We did not find a significant effect of ABT or PP242 on clonogenic potential when given as single agents. More importantly, combined treatment decreased CFC output by only 35% while RAP, which has an acceptable toxicity profile as it has been used in clinical trials for patients unresponsive to TKIs, decreased LSK colony forming potential by 50%. In summary, our data showing that combined treatment with the mTORC1/2 inhibitor/BAD activator PP242 and the BCl-xL/Bcl-2 antagonist ABT-263 markedly induces apoptosis of BCR-ABL+ cell lines, in HSCs and in progenitors from CML-BC patients. This approach warrants further pre-clinical investigation aimed at inclusion in clinical protocols for treating blast crisis CML. Disclosures: No relevant conflicts of interest to declare.

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ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.4316.4316 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4316-4316

Author(s):

Hendrik Folkerts ◽

Maria Catalina Gomez Puerto ◽

Albertus T.J. Wierenga ◽

Koen Schepers ◽

Jan Jacob Schuringa ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

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Inhibition of CML Development Following Conditional SIRT1 Deletion in Transgenic BCR-ABL Mice

Blood ◽

10.1182/blood.v128.22.931.931 ◽

2016 ◽

Vol 128 (22) ◽

pp. 931-931

Author(s):

Ajay Abraham ◽

Puneet Agarwal ◽

Hui Li ◽

Andrew Paterson ◽

Jianbo He ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Critical Role ◽

Chronic Phase ◽

Sirtuin 1 ◽

Hematopoietic Stem ◽

Exon 4

Abstract Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. Disclosures No relevant conflicts of interest to declare.

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