CML Leukemia Stem Cells Research Articles

Molecular and Structural Characterization of the SH3 Domain of AHI-1 in Regulation of Cellular Resistance of BCR-ABL+ Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitors

Abstract 966Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disorder characterized by the presence of the fusion gene BCR-ABL with increased tyrosine kinase activity. Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (TKIs), including dasatinib (DA) and nilotinib (NL), have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells remain problematic. Improved treatment approaches to target other key molecular elements active in CML stem/progenitor cells are needed. One candidate is AHI-1 (Abelson helper integration site 1), an oncogene that is highly deregulated in CML leukemic stem cells. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. We recently demonstrated that AHI-1 physically interacts with BCR-ABL and JAK2 in CML cells and this interaction complex mediates transforming activity and TKI response/resistance of CML stem/progenitor cells. We have also shown that AHI-1 interacts independently with JAK2 and BCR-ABL via different binding sites to mediate their activities. In this study, we have characterized the biological and structural functions of the SH3 domain of AHI-1. To determine roles of the SH3 domain in regulation of cell proliferation and TKI response/resistance, several mutant forms, including SH3 domain deletion (SH3Δ), double WD40-repeat and SH3 domain deletion (SH3WD40Δ) and N-terminal deletion (N-terΔ, containing SH3 and WD40-repeat domains) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells with doxycyclin (suppression of BCR-ABL) compared to BCR-ABL inducible cells (3 and 29% v.s.10 and 60% after 24 or 48 hours). Cells expressing the SH3Δ mutant and the SH3WD40D mutant displayed dramatically increased Annexin V+ cells (10, 77% and 34, 90% v.s.3 and 29%), while cells expressing the N-terΔ mutant had similar numbers of Annexin V+ cells compared to BCR-ABL inducible cells (6 and 41% v.s.10 and 60%). Similarly, BCR-ABL+ cells transduced with SH3Δ and SH3WD40D mutants displayed significantly increased apoptotic cells compared to cells transduced with full-length Ahi-1 in the presence of 2 μM IM (57, 87 vs. 26%), 2μM NL (65, 87 vs. 25%) and 150 nM DA (63, 96 vs. 34%) after 24 hour treatment. BCR-ABL+ cells transduced with the N-terδ mutant also showed more sensitivity to the drug treatments compared to the cells with the full-length Ahi-1(36% for IM, 40% for NL and 40% for DA), but with lower sensitivity than cells carrying the Ahi-1 SH3 domain deletion mutants, indicating that the SH3 domain of Ahi-1 plays a role in the mediation of TKI resistance. The crystal structure of the AHI-1 SH3 domain at 1.32-Å resolution revealed that the AHI-1SH3 domain adopts a canonical SH3 folding, but with an unusual C-terminal α helix. There are three large negatively charged patches, which are constructed by the n-Src loop, the end of the RT loop and the C-terminal helix, and this special feature may be involved in binding selectivity and specificity. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between AHI-1 SH3 domain and its ligands, and there may be formation of an “Arg-Arg-Trp” stack within the binding interface, which could be a targeting site for designing specific drugs. Moreover, using the AHI-1 SH3 domain as protein ‘bait’ in immunoprecipitation/mass spectrometry, Dynamin-2 was identified as a potential interacting partner of AHI-1; both AHI-1 and Dynamin-2 are involved in trafficking and signaling processes. In conclusion, the investigation of the structure of AHI-1 SH3 domain and its interacting proteins will thus provide invaluable insight in identification of key interaction sites in regulation of drug resistance and may be utilized for development of small molecule inhibitors for CML. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

AbstractAbstract 514 Background:Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results:1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions:We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures:Buonamici:Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

Notch-Mediated Suppression of PKCθ Reduces Reactive Oxygen Species and Promotes Leukemic Stem Cell Activity In T-Cell Acute Lymphoblastic Leukemia (T-ALL)

AbstractAbstract 12T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors which affects both children and adults. Current therapies achieve cure in about 80% of pediatric patients, but only 40% of adults survive beyond 5 years. Disease relapse may be attributed to ineffective targeting of leukemia stem cells (LSCs) due to their presumed quiescence, resistance to apoptosis, enhanced expression of DNA repair enzymes and drug efflux pumps, and localization to protective or inaccessible niches. More efficient targeting of LSCs in this disease thus could offer great potential for improving patient outcomes.Recent interest in the role of reactive oxygen species (ROS) in cancer stem cell biology has been sparked by studies demonstrating that low ROS levels promote clonogenicity and radioresistance of breast cancer stem cells and that parthenolide specifically induces apoptosis of LSCs in AML and blast crisis CML by increasing levels of ROS in these cells. In T-ALL, activation of NOTCH1 signaling, either by mutation of NOTCH1 itself (50-60% of cases) or its negative regulator FBW7 (15% of cases), contributes to malignant behavior by blocking differentiation, promoting cellular proliferation/survival, and enhancing self-renewal. Similar gain-of-function Notch1 mutations are also observed to occur spontaneously in several murine T-ALL models at high frequency. To examine ROS levels in T-ALL LSCs, we first generated murine disease by retroviral transduction of bone marrow with a constitutively activated form of NOTCH1 termed “ΔE”. We utilized limiting dilution transplantation analyses of different cellular phenotypes isolated from primary ΔE leukemias to demonstrate that the CD44+ subset of CD4+ CD8+ (DP) cells were enriched for LSCs and exhibited lower ROS levels as compared to the CD44- DP subset and bulk cells. Since ROS levels were heterogeneous within the CD44+ DP subset, we combined these markers with DC-FDA staining for ROS and demonstrated as few as 100 ROSlow CD44+ DP cells are able to reproduce disease in 100% of recipients whereas 100,000 bulk cells are required to achieve the same effect.Because protein kinase C theta (PKCθ) was recently identified as an important mediator of ROS production in T-cells during activation-induced cell death and expression of PKCθ mRNA could be detected broadly in primary human and mouse T-ALL samples, we reasoned that this signaling intermediate might be a regulator of ROS levels in malignant T-ALL LSCs. In fact, using a combination of viral expression and knock-down approaches in both human and mouse T-ALLs as well as mouse PKCθnull ΔE leukemias, we demonstrate that PKCθ is a primary regulator ROS production in T-ALL. Moreover, low PKCθ/ROS levels correlate with resistance to chemotherapy and irradiation, and promote the competitive in vivo growth of T-ALL cells. Interestingly, we also find that Notch signaling represses PKCθ expression which in turn results in reduced ROS levels. Our results identify PKCθ as a novel target of Notch signaling in LSCs whose key malignant properties are highly dependent on low ROS levels. These findings provide mechanistic basis for rational design of LSC-targeted therapies in T-ALL. Disclosures:No relevant conflicts of interest to declare.

β-Catenin Suppression Targets Imatinib Resistant Leukemia Stem Cells In Mice with BCR-ABL Induced Myeloproliferative Disease

AbstractAbstract 93CML is driven by the oncogenic fusion kinase BCR-ABL which is capable of corrupting and transforming hematopoietic stem cells (HSC) and thus inititating myeloproliferative disease (MPD). While imatinib (IM) has become an effective treatment for CML, the leukemia stem cells (LSC) which reside in the bone marrow (BM) remain largely unaffected by imatinib treatment. As LSC rely on their ability to self-renew, several genes/pathways responsible for self-renewal properties have been investigated as potential targets of LSCs in myeloid neoplasia. Recent studies clearly indicate that canonical Wnt/β-catenin-signaling plays a pivotal role in initiation and development of BCR-ABL induced MPD (Zhao, Cancer Cell 2009; Hu, Leukemia 2009). However, the impact of β-catenin (Ctnnb1) on the maintenance of LSC in established disease, which more accurately models the clinical scenario, has not been extensively studied.To address the impact of targeting Ctnnb1 in CML-LSC, we used a conditional Ctnnb1 knockout mouse with an estrogen-receptor driven Cre-recombinase as BM-donors. CML was induced by retroviral transduction of either Ctnnb1flox/flox ER-Cre+, Ctnnb1flox/wt ER-Cre+, or Ctnnb1wt/wt ER-Cre+ murine bone marrow with a virus encoding BCR-ABL (MSCV-BCR-ABLp210-GFP), and injection of transduced cells into wildtype syngeneic recipient mice. Administration of tamoxifen to recipient mice with CML led to excision of Ctnnb1 in the Ctnnb1flox/flox homozygous or Ctnnb1flox/wt heterozygous cells.Interestingly genetic inactivation of Ctnnb1 after onset of CML in primary recipient mice resulted only in minor prolongation of survival. The animals died due to organ infiltration (primarily lung) by differentiated progenitor cells that were presumably not affected by Ctnnb1 deletion. However, in the BM and peripheral blood, a clear reduction of GFP+ cells was evident in heterozygous - and even more pronounced - in homozygous animals, indicating a potential effect on more immature cells and perhaps LSC. To determine if LSC were compromised and to investigate the efficacy of a combination treatment with IM, we used a serial BM-transplantation assay. Consistent with previously published data, we found that leukemia cells in the Sca-1/GFP+ fraction are able to re-establish CML in secondary recipient mice and thus contain LSC. IM treatment alone led to a relative increase of Sca-1/GFP+ LSC over time. However, we found a significant reduction of Sca-1/GFP+ bone marrow cells after inactivation of Ctnnb1 during IM-treatment. Mice transplanted with Ctnnb1flox/flox ER-Cre+ CML cells had on average 0.025% Sca-1/GFP+ (total of 7,375 cells/mouse) cells in the bone marrow after treatment of the recipient mice with IM and tamoxifen, mice that received Ctnnb1flox/wt ER-Cre+ CML had 0.036% Sca-1/GFP+ cells (total of 10,584 cells/mouse) and mice that received Ctnnb1wt/wt ER-Cre+ CML had 0.075% Sca-1/GFP+ cells (total of 24,375 cells/mouse). As indomethacin has recently been shown to inhibit Ctnnb1 in hematopoietic and AML stem cells (Goessling, Cell 2009; Wang, Science 2010), we aimed to investigate the efficacy of indomethacin combined with IM. We were able to demonstrate a decrease in Ctnnb1 protein after indomethacin treatment of human cell lines and transformed murine BM cells by either immunoblot, flow-cytometry or immunofluorescence. Moreover, we were able to show synergy between indomethacin and IM co-treatment which produced almost complete abrogation of Ctnnb1 protein. IM/Indomethacin co-treatment of mice with CML decreased the Kit+Sca1+ fraction of the Lin-GFP+ bone marrow population to 0.92% (1799 cells/mouse) whereas treatment with IM alone was similar to untreated mice with 2.8% of the Lin-GFP+ population being Kit+Sca1+ (22,601 cells/mouse). Importantly, transplantation of the bone marrow from treated mice into tertiary recipients showed significantly prolonged survival of tertiary recipient mice that received bone marrow from donors treated with the combination therapy (IM/INDO) compared to IM/DMSO controls (p=0.0038*).In summary, we demonstrate that inhibiting Ctnnb1 by genetic inactivation or drug treatment is an effective combination therapy with imatinib mesylate and targets CML leukemia stem cells. This provides strong evidence for the development and clinical use of β-catenin-targeted therapies in CML. Disclosures:No relevant conflicts of interest to declare.

Effective Targeting of Quiescent CML Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate.

Abstract 190▪▪This icon denotes an abstract that is clinically relevant.The BCR-ABL tyrosine kinase inhibitor imatinib mesylate (IM) is highly effective in inducing remissions and improving survival in CML patients but fails to eliminate leukemia stem cells, which remain a potential source of relapse. Quiescent leukemia stem cells resist apoptosis following BCR-ABL kinase inhibition, and other strategies are required for their elimination. Histone deacetylase inhibitors (HDACi) have shown promise in the treatment of several cancers, and it is of particular interest that reports suggest that they are also capable of inducing apoptosis in non-proliferating cells. It is known that the potent pan-HDACi LAQ824 (LAQ) and LBH589 (LBH) can induce apoptosis in CML cell lines and blast crisis CML cells. However, the effect of HDACi on quiescent leukemia stem cells from chronic phase CML patients is not known. Here we investigated the effects of LAQ and LBH, alone and in combination with IM, on CML stem and progenitor cells. CML and normal CD34+ cells were cultured with LAQ (10–100nM) or LBH (25–50nM) alone, IM (1mM) alone, and LAQ or LBH with IM for 96 hours. HDACi treatment effectively enhanced Histone H3 and H4 acetylation in CML CD34+ cells. HDACi treatment by itself induced less apoptosis in CML compared to normal CD34+CD38- primitive and CD34+CD38+ committed progenitors, but was highly effective in inducing apoptosis in CML progenitors when combined with IM. In addition, the combination induced significantly higher apoptosis in CML compared with normal CD34+ cells, and unlike IM, also induced apoptosis in non-dividing cells. Combination treatment also inhibited the proliferation of CML progenitor cells as measured by CFSE and growth of CML CFC in methylcellulose progenitor assays. Treatment of CML CD34+ cells with IM resulted in modest reduction in levels of engraftment in NSG mice. In contrast treatment with LAQ824 plus IM resulted in abrogation of engraftment of BCR-ABL+ CML cells (p<0.001). Significantly less inhibition of normal compared to CML cell engraftment was seen following LAQ824 and IM treatment (p=0.006). We used a transgenic Scl-tTa-BCR-ABL mouse model to investigate the effect of HDACi treatment on CML stem cells in vivo. SCLtTA/BCR-ABL transgenic mice were crossed with GFP transgenic mice to allow tracking of transplanted cells. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Mice were treated with IM (200mg/kg daily by gavage), LBH (30 mg/kg IP 3 times a week), LBH with IM, or vehicle alone (control) for 4 weeks. LBH combined with IM resulted in greater reduction in WBC, neutrophils and GFP+ cells than LBH or IM alone. Significantly increased apoptosis and a profound reduction of Lin- Sca-1+ Kit+ (LSK) stem cells were seen in mice treated with IM plus LBH (p<0.001) but not IM or LBH alone. LBH plus IM treatment did not significantly inhibit LSK cells in normal mice. BCR-ABL-transgenic mice treated with IM plus LBH demonstrated prolonged leukemia-free survival after discontinuing treatment compared with mice treated with IM or LBH alone (p<0.05). Combined IM and LAQ824 treatment resulted in marked inhibition of the anti-apoptotic protein MCL-1 in CML CD34+ cells (p<0.001). RNAi mediated inhibition of MCL-1 expression resulted in increased apoptosis in CML compared with normal CD34+ cells that was further enhanced by IM treatment. Our results indicate that treatment with HDACi plus IM effectively and selectively targets leukemia stem cells in CML, suggest a potential role for MCL-1 inhibition in HDACi induced apoptosis, and support ongoing clinical trials of LBH combined with IM to eliminate residual leukemia stem cells in IM-treated CML patients. Disclosures:Bhatia:Novartis: Consultancy.

The Broad Spectrum Bcl-2 Inhibitor Apogossypol Induces Apoptosis and Differentiation of Blast Crisis Chronic Myeloid Leukemia Stem Cells.

Abstract 3275Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant. Introduction:Leukemia stem cells (LSC) are rare, self-renewing cells capable of proliferation and differentiation into the bulk of cells that make up a leukemia. LSC, like normal tissue-specific stem cells, can be highly quiescent and resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. While traditional chemotherapy may successfully eradicate the bulk of tumor cells, it often fails to kill LSC resulting in their reactivation and eventual relapse of disease.Apoptosis resistance in cancer often involves deregulation of Bcl-2 family proteins. Bcl-2 family expression also seems to have an important role in normal stem cell function as expression changes of Mcl-1, Bcl-2, and Bcl-XL in mouse hematopoietic stem and progenitors characterize the differentiation of hematopoietic cells to different lineages.Several studies have linked expression of Bcl-2 family members to the development of blast crisis chronic myeloid leukemia (BC CML) in vitro, however there has been little data on the role of Bcl-2 expression in apoptosis resistance in human LSC. Also, various data has shown that CML cells become increasingly resistant to BCR-ABL inhibition with progression to blast crisis. In BC CML, LSC are enriched in the progenitor population and can be serially transplanted in immunodeficient mice leading to BC CML in vivo. We hypothesize that human BC CML LSC may resist chemotherapy by overexpression of Bcl-2 family proteins and maintenance of quiescence. We analyzed Bcl-2 family protein expression in BC CML LSC and analyzed whether these cells were sensitive to chemotherapy treatment in vitro. We further analyzed whether BC CML LSC could maintain quiescence in vivo. Finally, we tested the efficacy of the broad spectrum Bcl-2 antagonist apogossypol on BC CML LSC in vitro and in vivo. Methods:Bcl-2 and Mcl-1 protein expression was measured in 1° BC CML LSC by intracellular FACS analysis and compared to expression in normal and chronic phase CML cells. CD34+ cells isolated from serially transplanted BC CML LSC were used for all subsequent studies. To assess whether BC CML cells maintained quiescence in vivo, they were stained with DiR, an infrared fluorescent cell membrane dye, transplanted into neonatal mice, and analyzed for DiR fluorescence 18 weeks later. Dividing cells will distribute DiR to all daughter cells leading to dilution of the dye and a decrease in fluorescent signal per cell. LSC drug resistance was tested in vitro by culturing the cells with etoposide, dasatinib, and apogossypol and by measuring apoptosis by FACS using annexin-V/7-AAD staining. Finally, apogossypol efficacy was tested in vivo in LSC transplanted mice. At 8 weeks post-transplantation, mice were treated for 3 weeks with apogossypol or vehicle and then analyzed for human hematopoietic cell engraftment and apoptosis by FACS. Results:1° BC CML progenitors expressed significantly higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. LSC cultured in vitro were also resistant to etoposide and dasatinib-induced apoptosis. Apogossypol treatment in vitro however led to a dose-dependent increase in cell death and apoptosis and resulted in a significant increase in the frequency of lin+ staining cells along with a significant shift in the frequency of the common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) populations compared to vehicle treated controls.Mice transplanted with BC CML LSC developed diffuse myeloid sarcomas and had high levels of human engraftment in the liver, spleen, and bone marrow. Human cells in tumors and the liver were uniformly DiR- while cells engrafted in the spleen and bone marrow retained DiR fluorescence. In vivo treatment with apogossypol led to a significant reduction in human cell engraftment in mouse bone marrow compared to vehicle controls. Consistent with the results in vitro, there was also a significant increase in the frequency of lin+ staining cells in engrafted mouse spleens as well as a significant decrease in engrafted GMP in the mouse bone marrow. Conclusions:Our results demonstrate that BC CML LSC are highly resistant to conventional chemotherapy but are sensitive to apogossypol in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC by inducing apoptosis as well as by inducing differentiation. Disclosures:Goff:Coronado Biosciences: Research Funding. Tesi:Coronado Biosciences: President and CEO. Jamieson:Coronado Biosciences: Research Funding. Jamieson:Coronado Biosciences: Research Funding.

Twist-1 Expression Represents a Novel Prognostic Factor in Chronic Myelogenous Leukemia.

Abstract 2592Poster Board II-568Chronic Myelogenous Leukemia (CML) represents one of the best characterized models for studying cancer stem cell properties and resistance to tyrosine kinase inhibitors (TKIs). A substantial proportion of CML patients develop primary or secondary resistance to such agents, and in approximately ∼30% of the cases the resistance mechanism(s) remain(s) obscure. It is known that CML leukemic stem cells could remain viable and quiescent despite the presence of growth factors that seem to protect them from apoptosis, or drugs. By performing genome-wide microarray analysis of CML sensitive and resistant cell lines, a novel oncogene Twist-1 has been shown to be deregulated in CML cells innately resistant to imatinib through, an unknown mechanism (Tipping AJ et al. Exp Hematol 2003; 31(11): 1073–1080). In this study, we have observed similar results in the same BCR-ABL+ KCL22 cell line resistant and sensitive to imatinib by quantitative RT-PCR (qRT-PCR) using specific primers and at the protein level. Twist-1 is a key player during embryogenesis that represses mesoderm differentiation and retains undifferentiated cells into a proliferative state. In adult tissues, its expression has been reported in intestinal crypts where adult gut stem cells reside. In cancer, Twist-1 overexpression has been reported in a variety of solid tumors and hematopietic malignacies (Sezary syndrome, B-Cell leukemias) and its deregulation is always associated with poor prognosis and resistance to reagents targeting microtubules (Puisieux A et al. Br J Cancer 2006; 94 (1):13–17). In addition, functional studies show that Twist-1 plays a major role in tumor promotion and progression, by inhibiting differentiation, and interfering with the p53 tumor suppressor pathway, thereby favoring cell survival (Ansieau S et al. Cancer Cell 2008; 14(1):79–89). In this context, we have analyzed Twist-1 gene expression by qRT-PCR in both CD34+ (immature cells) and CD34− (mature compartment) blood or marrow cells from CML patients at diagnosis, at different disease phases, and once TKIs resistance has occur. Results are expressed as ratio to normal CD34− cells values. We observed a 74- and 39-fold increase in CD34+ and CD34− CML cells (n= 30 patients), respectively. When Twist-1 expression could be monitored during CML progression in a same patient, a regular expression increase in the CD34+ compartment was observed [8.5 fold (n=17), 18 fold (n=4) and ≥105 fold (n=8) in chronic, accelerated and blastic phases respectively]. Twist-1 was also overexpressed in the CD34− compartment along with disease progression [between chronic and later stages (11 fold), but with no significant differences between accelerated (88 fold) and blastic (73 fold) phases]. Our results then clearly identify Twist-1 gene expression as a unique predictive molecular marker of resistance, especially in patients with unidentified mechanism of resistance as we were able at the time of diagnosis to point out 2 patients/13 that present a higher Twist-1 level relatively to the mean value and which have develop a cytogenetic resistance within 6 months (ELN criteria) without any mutation, BCR-ABL overexpression or clonal evolution. The overexpression of Twist-1 is predominant in the immature compartment during CML progression and resistance, supporting the hypothesis that resistant cells that overexpress Twist-1 might emerge from CML stem cells. In samples from patients who became resistant to imatinib within the course of the disease, Twist-1 expression follow-up at the time of cytogenetic remission, and later resistance, indicate that Twist-1 expression was transiently down regulated by TKIs. Finally, using Twist-1 specific siRNA we confirmed in CML cell lines the direct involvement of this protein in CML resistance to imatinib. Altogether, our results suggest for the first time that Twist-1 may represent a useful molecular marker to identify at diagnosis patients prone to become resistant to imatinib. The exact interactions between Twist-1 and BCR-ABL proteins pathways remain to be determined yet. Disclosures:No relevant conflicts of interest to declare.