Proliferation Of Leukemic Stem Cells Research Articles

The IGF2/IGF1R/Nanog Signaling Pathway Regulates the Proliferation of Acute Myeloid Leukemia Stem Cells.

Acute myeloid leukemia is an aggressive disease characterized by clonal proliferation and differentiation into immature hematopoietic cells of dysfunctional myeloid precursors. Accumulating evidence shows that CD34+CD38- leukemia stem cells (LSCs) are responsible for drug resistance, metastasis, and relapse of leukemia. In this study, we found that Nanog, a transcription factor in stem cells, is significantly overexpressed in CD34+ populations from patients with acute myeloid leukemia and in LSCs from leukemia cell lines. Our data demonstrate that the knockdown of Nanog inhibited proliferation and induced cell cycle arrest and cell apoptosis. Moreover, Nanog silencing suppressed the leukemogenesis of LSCs in mice. In addition, we found that these functions of Nanog were regulated by the insulin-like growth factor receptor (IGF1R) signaling pathway. Nanog overexpression rescued the colony formation ability of LSCs treated with picropodophyllin (PPP), an IGF1R inhibitor. By contrast, knockdown of Nanog abolished the effects of IGF2 on the colony formation ability of these LSCs. These findings suggest that the IGF2/IGF1R/Nanog signaling pathway plays a critical role in LSC proliferation.

Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

Early and Late Leukemic Stem Cells in Murine Leukemia Models

Acute leukemia is uncontrolled proliferation of leukemic stem cells (LSCs). Murine models of leukemia suggest that LSCs arise from lineage-committed progenitor cells. However, whether LSCs also directly arise from long-term (LT) and short-term (ST) hematopoietic stem cells (HSCs) and other hematopoietic progenitor cells (HPCs) and whether differentiation potentials influence the leukemia types are poorly understood.In this study, we used two murine leukemia models (AML with MLL-AF9 fusion protein and T-ALL with Notch-1 intracellular domain, ICN-1). Two HSC and three HPC populations were sorted from B6 (CD45.1) mouse bone marrow (BM) by flow cytometry: HSC1, CD150+CD41-CD34-Lin-Sca-1+c-Kit+ (LSK) cells; HSC2, CD150-CD41-CD34-LSK cells; HPC1, CD150+CD41+CD34-LSK cells; HPC2, CD150+CD41+CD34+LSK cells; HPC3, CD150-CD41-CD34+LSK cells. HSC1, HSC2, HPC1, and HPC3 are enriched in LT-HSCs, ST-HSCs, repopulating common myeloid progenitors, and lymphoid-primed multipotent progenitors, respectively. 400-600 cells were sorted from each population, pre-stimulated for 24 hrs, and transduced with MLL-AF9 or ICN-1 retrovirus for another 24 hrs. Cells were mixed with 3-5 x 105 BM cells (CD45.2), and injected into the lethally irradiated mice (CD45.2). The recipient mice were monitored by detection of GFP+ cells in the peripheral blood (PB). When the recipient mice showed > 50% GFP+ cells in the PB or became ill, mice were killed and analyzed for leukemia.In the MLL-AF9 AML model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 6 weeks after transplantation. Leukemic cells in PB and BM appeared positive for Mac-1/Gr-1, but negative for CD3 and B220. We detected a new LSC population in the BM: Lin-Mac-1+c-Kit+Sca-1+CD150-CD16/32+ cells. Both CD34-negative and CD34-positive cells were detected in this population. We named this population as "early LSCs" because it was phenotypically similar to ST-HSCs and/or LMPPs in normal mouse BM cells except Mac-1 expression. Early LSCs differed from previously identified LSCs (L-GMP) because they are Sca-1-positive. We now consider L-GMP as one of "late LSC" types. Early LSCs formed leukemic colonies in vitro and initiated the AML in serial transplantation. These results show the similar AML develops regardless of HSC and HPC types transduced. Both early and late LSCs likely play a role in establishment of AML.In the ICN-1 T-ALL model, leukemia developed in all recipient mice injected with HSC1, 2, HPC1, 2, or 3 around 4 weeks after transplantation. Most leukemic cells in PB and BM appeared positive for CD4/CD8, but negative for Mac-1/Gr-1 and B220. We did not detect early LSCs in BM. To identify late LSCs in BM, we injected the CD3+CD8+CD4- and CD3+CD8+CD4+ cells into the sub-lethally irradiated mice. T-ALL developed from both populations (median latency, 23 vs 35 days). These results show the similar T-ALL develops regardless of HSC and HPC types transduced. In this case, late LSCs can be directly generated without early LSCs from HSC or HPC populations.Taken together, we found that the similar types of leukemia develop regardless of different types of initiating cells in both models. We also found new LSC populations, early LSCs in the AML model and late LSCs in the T-ALL model. Both early and late LSCs were able to re-initiate leukemia after secondary transplantation.In conclusion, highly purified murine HSCs and HPCs were used for the first time to initiate leukemia. Both MLL-AF9 AML and ICN-1 T-ALL models suggest that gene mutations at all differentiation stages from HSCs to HPCs potentially induce the similar types of leukemia. Of note is that distinct lineage differentiation potentials of HSCs and HPCs do not affect leukemia types. More importantly, early LSCs may serve as the earliest event in leukemogenesis in AML. In the case of ALL, gene mutations seem carried over until reaching the developmental stage of late LSCs. Our results suggest that both early and late LSCs should be eradicated to achieve complete remission and prevent relapse. DisclosuresNo relevant conflicts of interest to declare.

Potential role of Wnt/β-catenin signaling in blastic transformation of chronic myeloid leukemia: cross talk between β-catenin and BCR-ABL.

Chronic myeloid leukemia (CML) results from malignant transformation of hematopoietic stem cells induced by the BCR-ABL oncogene. Transformation from chronic to blastic phase is the lethal step in CML. Leukemic stem cells (LSCs) are the basic reason for blastic transformation. It has been shown that Wnt/β-catenin signaling contributes to the self-renewal capacity and proliferation of LSCs in CML. However, the role of Wnt/β-catenin signaling in blastic transformation of CML is still obscure. Here, we explored the relationship between BCR-ABL and β-catenin signaling in vitro and in vivo. We found that BCR-ABL stimulated β-catenin via activation of PI3K/AKT signaling in blastic phase CML cells. Inhibition of the kinase activity of BCR-ABL, PI3K, or AKT decreased the level of β-catenin in both K562 cells and a CML mouse model and suppressed the transcription of downstream target genes (c-myc and cyclin D1). In addition, inhibition of the BCR-ABL/PI3K/AKT pathway delayed the disease progression in the CML mouse model. To further explore the role of β-catenin in the self-renewal and survival of CML LSCs, we established a secondary transplantation CML mouse model. Our data revealed that inhibition of the BCR-ABL/PI3K/AKT pathway reduced the tumor-initiating ability of K562 cells, decreased leukemia cell infiltration into peripheral blood and bone marrow, and prolonged the survival of mice. In conclusion, our data indicate a close relationship between β-catenin and BCR-ABL/PI3K/AKT in blastic phase CML. β-Catenin inhibition may be of therapeutic value by targeting LSCs in combination with a tyrosine kinase inhibitor, which may delay blastic transformation of CML.

Abstract 3120: B-Catenin gene expression and its mutations: promising and key component in the pathogenesis of acute myeloid leukemia (AML)

Abstract INTRODUCTION AND AIM: Wnt signaling pathway plays a critical role in stem cell self-renewal and proliferation of leukemic stem cells and its association with the pathogenesis of acute myeloid leukemia (AML) has also been described. Beta-Catenin, a key regulator of Wnt signaling is highly expressed in cancers. Its role has been recently documented in AML however its prognostic significance in AML is not clear. The present study was carried to determine the potential prognostic implications of B-catenin expression and mutations in de novo AML patients. METHODS: We performed the gene expression of B-Catenin and its mutations analysis in exon 3. We investigated the expression of B-Catenin gene in bone marrow and/or blood samples by quantitative PCR using SYBR-Green chemistry and analysis was done by ΔΔCT Method. 18S RNA and B2M genes were used as reference genes. B-Catenin mutations analysis was by done direct DNA Sanger sequencing. RESULTS: Total 117 de novo non M3 AML patients and 40 healthy controls were included for the gene expression and analyzed for their clinicopathologic and prognostic significance. Median age for entire cohort (n = 117) was 17.0 years (Range 1-60 years) with male:female ratio of (1.8:1). FLT3-ITD mutation was positive in 11 (9.5%) patients. Only 4% of AML patients showed genetic alterations in exon 3 of B-catenin and no significant correlation with base line characteristics. B-catenin showed a significantly increased mRNA expression with a 5 fold rise as compared to healthy controls (p<0.05). B-catenin over expression was significantly associated with poor survival at median followup of 17 months and having less CR rates (p = 0.03). There was no significant correlation of B-catenin over expression vs baseline clinical characteristics like Hb, TLC, Platelets, Cyto group (p = 0.11), blast percentage (p = 0.28), Flt3-itd mutations (p = 0.42). CONCLUSION: These results provide the evidence for WNT signaling activation existence in AML patients and suggest that B-Catenin could be the key component in the pathogenesis and can be prognostically important marker in AML. However B-catenin expression in larger cohort of samples and with longer follow-up is required to confirm our findings. Citation Format: Mohsin Maqbool, Sobuhi Iqbal, Atul Sharma, Sameer Bakhshi, Sreenivas Vishnubhatla, Ashish Datt Upadhyay, Lalit Kumar. B-Catenin gene expression and its mutations: promising and key component in the pathogenesis of acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3120.

Leukemia Stem Cell-Released Microvesicles Promote the Survival and Migration of Myeloid Leukemia Cells and These Effects Can Be Inhibited by MicroRNA34a Overexpression.

Leukemia stem cells (LSCs) play the major role in relapse of acute myeloid leukemia (AML). Recent evidence indicates that microvesicles (MVs) released from cancer stem cells can promote tumor growth and invasion. In this study, we investigated whether LSCs-released MVs (LMVs) can regulate the malignance of AML cells and whether overexpression of tumor suppressive microRNA (miR), miR34a, is able to interrupt this process. LSCs were transfected with miRNA control (miRCtrl) or miR34a mimic for producing LMVs, respectively, defined as LMVsmiRCtrl and LMVsmiR34a. The effect of miR34a transfection on LSC proliferation and the effects of LMVsmiRCtrl or LMVsmiR34a on the proliferation, migration, and apoptosis of AML cells (after LSC depletion) were determined. The levels of miR34a targets, caspase-3 and T cell immunoglobulin mucin-3 (Tim-3), were analyzed. Results showed that (1) LMVsmiRCtrl promoted proliferation and migration and inhibited apoptosis of AML cells, which were associated with miR34a deficit; (2) transfection of miR34a mimic inhibited LSC proliferation and increased miR34a level in LMVsmiR34a; (3) LMVsmiR34a produced opposite effects as compared with LMVsmiRCtrl, which were associated with the changes of caspase-3 and Tim-3 levels. In summary, LMVs support AML cell malignance and modulating miR34a could offer a new approach for the management of AML.

Survival regulation of leukemia stem cells.

Leukemia stem cells (LSCs) are a subpopulation cells at the apex of hierarchies in leukemia cells and responsible for disease continuous propagation. In this article, we discuss some cellular and molecular components, which are critical for LSC survival. These components include intrinsic signaling pathways and extrinsic microenvironments. The intrinsic signaling pathways to be discussed include Wnt/β-catenin signaling, Hox genes, Hh pathway, Alox5, and some miRNAs, which have been shown to play important roles in regulating LSC survival and proliferation. The extrinsic components to be discussed include selectins, CXCL12/CXCR4, and CD44, which involve in LSC homing, survival, and proliferation by affecting bone marrow microenvironment. Potential strategies for eradicating LSCs will also discuss.

Targeting F2r/Beta-Catenin/JNK Signaling for Leukemia Stem Cell Eradication

G-protein coupled receptors (GPCRs) are the most successful drug targets with 36% of currently marketed drugs targeting human GPCRs. F2r, a GPCR that is overexpressed in various human cancers (Ribeiro et al., Oncol Rep 2009), is a positive regulator of the β-catenin pathway and an inhibitor of the JNK pathway in mammalian cells (Sun et al., Nat Cell Biol 2001). The expression of F2r is significantly elevated in aggressive leukemias including blast phase of CML and AML (Veiga et al., Blood Cells Mol Dis 2011). While these observations implicate an involvement of F2r in human leukemia, its function in AML remains unknown.Our preliminary data showed that shRNA-mediated knockdown of F2r markedly decreased active β-catenin in MLL-AF9 mediated pre-leukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs), confirming that F2r is a positive regulator of β-catenin. F2r deficiency decreased LSC colony formation (p=0.0002) in a serial replating assay, indicating a reduction in self-renewal capacity. Furthermore, LSCs exhibited a significant enhancement in apoptotic activity in response to F2r deficiency, displaying a 12-fold increase in apoptosis. Our microarray expression analysis revealed that F2r inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). To investigate this further we used a mitochondria-specific fluorogenic probe to measure reactive oxygen species (ROS) production. A significant increase in ROS production (p=0.0003) indicated that F2r inhibition destabilizes the mitochondrial membrane. This was accompanied by a marked increase, as observed by Western blot analysis, in the proapoptotic proteins Bcl-2-interacting mediator of cell death (Bim) and thioredoxin-interacting protein (Txnip) which permeabilize the mitochondrial membrane releasing cytochrome c and inducing apoptosis. Through oxidative phosphorylation, mitochondria play an essential role in the supply of metabolic energy (ATP) to the cell. F2r deficient LSCs had a significantly reduced rate of oxygen consumption measured using a phosphorescent oxygen probe (p=0.0066) and a significantly lower concentration of basal ATP (p=0.012) compared to control LSCs. F2r inhibition, therefore, induces substantial oxidative stress which triggers the intrinsic apoptotic pathway.To assess the therapeutic value of F2r inhibition, we used the selective non-peptide F2r inhibitor SCH79797. Alamar blue-based cell viability assays showed that SCH79797 was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells. F2r inhibitor treatment resulted in a 2-fold reduction in colony forming ability, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labeling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with SCH79797 for 48 hours in culture, transplanted into recipient mice and collected from bone marrow 8 days post-transplantation (p=0.0008). Additional in vivo studies using a mouse model of MLL AML are currently ongoing to further evaluate the therapeutic potential of F2r inhibition. Collectively, our findings suggest that F2r inhibition selectively targets LSC self-renewal, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells.Previous studies suggest that F2r knockdown not only suppresses β-catenin but also activates JNK signaling. Consistently, our Western blot analysis revealed activation of JNK in response to inhibition of F2r. Sustained JNK activation has been reported in many types of AML cells and promotes survival signals during leukemia development (Hess et al., Nat Genet 2002). This suggests that JNK and F2r inhibition could be used in combination to impair LSC self-renewal, with a concurrent increase in cell death. In support of this hypothesis, we have showed that co-inhibition of F2r and JNK induced a potent anti-LSC effect, significantly increasing cell death and ROS production compared to single treatment. The efficacy of this co-treatment is currently being evaluated in primary human AML patient samples in addition to our in vivo mouse model system. Altogether our data suggest a novel LSC-eliminating treatment strategy targeting F2r/β-catenin/JNK signaling for aggressive AML. DisclosuresNo relevant conflicts of interest to declare.

Functional Genomics and Computational Approaches Identify Novel Small Molecules Targeting Quiescent Leukemia Stem Cells

Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients.Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existence of functionally diverse leukemic and preleukemic clones. Accordingly, the hierarchical organization of AML suggests that this may be relevant to current therapies that primarily target proliferating progenitors/blast cells, which lack self-renewal capacity, and not LSCs. In the current study, we rationalized that understanding how LSCs differ from normal HSCs at the molecular level, is an essential first step towards developing novel targeted therapies and achieving permanent disease remission.Despite the identification of novel LSC-specific markers, there is considerable heterogeneity in expression of these markers amongst AML patients. However, in addition to marker-enrichment strategies, LSCs can be identified by virtue of their quiescent and slow-cycling properties. For example, label-retaining cells can be isolated and used in functional assays but significant technical limitations impede broad utility of this approach. To this end, we describe the development and use of novel multi-fluorescent protein markers and DNA bar codes integrated into the cellular genomes by lentivirus, as single-cell tracking devices for monitoring LSCs in vivo. We demonstrate how LSCs can transition between a "proliferation phase" and a "quiescence phase" in vivo. Furthermore, using high-throughput quantitative transcriptome sequencing (Q-RNA-Seq) and RNAi genetic perturbation's focusing on well-defined self-renewal signaling pathways, we develop a differential network-based model to identify LSC-specific genes and subsequently prioritize/rank candidates as potential drug targets.In the current study, we identify several molecular targets deregulated in quiescent versus proliferating LSCs and a mutual set of signaling pathways that facilitate leukemic transformation downstream of diverse initiating mutations/lesions. Remarkably, both quiescent and dividing LSCs but not HSCs, were 'addicted' to SSRP1 - an essential component of the ubiquitous FACT chromatin remodeling complex. Two orally available quinacrine-related DNA-intercalating compounds inhibiting function of FACT (CBL0100 and CBL0175, respectively) suppressed LSC proliferation in vitro and in vivo, as demonstrated by production of leukemic clonogenic cells (CFU) and long-term engraftment of immunodeficient NSG mice, by simultaneous inhibition of NF-kB (stimulated and basal forms) and activation of p53. Furthermore, in a secondary transplantation experiment, leukemic cells obtained from CBL0175 treated mice (primary) failed to engraft into secondary NSG mice in a serial transplantation model by selectively targeting the LSC compartment. Collectively, we present a novel network-based polypharmacology approach that provides unique opportunities to preferentially ablate LSCs (quiescent and dividing types), with potentially profound clinical implications. DisclosuresFrangou:Cellecta: Employment. Portwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

Knockdown (KD) of Mir-126 Expression Enhances Tyrosine Kinase Inhibitor (TKI)-Mediated Targeting of Chronic Myelogenous Leukemia (CML) Stem Cells

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. The long-term goal of our research is to improve the understanding of molecular mechanisms regulating LSC growth and develop effective mechanism-based therapeutic strategies to target LSC in CML. MicroRNAs (miRNAs) are short non-coding RNAs that regulate translation of target protein-coding messenger RNAs (mRNAs) and levels of the corresponding encoded proteins. Among distinct miRNAs, miR-126-3p (miR-126) is expressed in normal hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) and plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo (Cell Stem Cell, 2012). We previously reported that miR-126 expression varies among AML patients; higher level of miR-126 associated with poor outcome, a LSC-associated gene expression signature and increased LSC quiescence; and miR-126 down-regulation decreased LSC self-renewal activity in serial transplant experiments (Leukemia, 2015). Here, we report that the more primitive LSK (Lin-Sca-1+Kit+) from SCL-tTA-BCR/ABL mouse model of CML had higher miR-126 expression compared to more differentiated subpopulations, i.e. common myeloid progenitors (CMP) (3.2 fold) and granulocyte-macrophage progenitors (GMP) (6.4 fold). Among LSK subpopulations, LSC (Flt3-CD150+CD48- LSK) demonstrated the highest miR-126 levels (P<0.01). Furthermore, quiescent (Hoechst-Pyronin-) LSC demonstrated higher miR-126 expression (2.6 fold, p<0.01), generated significantly higher long-term engraftment of donor CML cells in recipient mice (58.6±4.4% vs 33±3.4% in BM at 16 wks after transplanted with 100 LSC/mouse, p=0.003) and enhanced leukemogenic capacity (5 out of 9 mice receiving quiescent LSC developed CML within 14 wks after transplantation vs. none of 10 mice receiving proliferating LSC) compared with proliferating (Hoechst+/-Pyronin+) LSC. Human primary untreated chronic phase (CP) CML CD34+CD38- primitive progenitors also showed higher miR-126 expression compared with CD34+CD38+ committed progenitors. KD of miR-126 in LSK cells of CML mouse model and in primary CML CD34+CD38- cells by using either miRZip-126-3p anti-miR lentivirus (System Biosciences) or a novel, myeloid cell-specific CpG-miR-126 oligonucleotide (ODN) inhibitor increased LSC cell-cycle entry as demonstrated by increased Ki67 staining (p<0.05) and EdU labeling (p<0.05), but affected apoptosis only slightly compared to scramble-treated controls. More importantly, miR-126 KD either by lentivirus or CpG-miR-126 ODN inhibitor combined with Nilotinib (NIL) significantly increased apoptosis of LSC compared with NIL alone (34% vs 19%; p=0.01 for lentivirus KD; and 40% vs 35%; p=0.03 for CpG-miR-126 inhibitor KD), resulting in significant reduction of cell growth and colony forming cell (CFC) frequency in methylcellulose progenitor assays. Conversely, enforced miR-126 expression by miR-126 precursor lentivirus (System Biosciences) impaired cell-cycle entry (28% vs 33%; p=0.05) and led to significantly reduced apoptosis (27% vs 19%; P=0.01) in NIL (5µM) treated CML CD34+CD38- cells compared to cells treated with control lentivirus and NIL. We further investigated the effect of miR-126 KD on growth of primary CML LSC and therapeutic response in vivo. CP CML CD34+ cells were cultured with CpG-scrambled RNA or CpG-miR-126 ODN inhibitor (500nM), in the presence or absence of NIL (5µM) for 4 days, and then transplanted into irradiated NSG mice. While the experiment is still ongoing, we have already observed a significantly reduced engraftment in blood at 4 wks in recipient mice receiving cells treated with CpG-miR-126 inhibitor and NIL compared with those receiving cells treated with CpG-scrambled RNA and NIL (0.16±0.03% vs 0.4±0.04%; P=0.01). Final results of this in vivo experiment and in vivo treatment of SCLtTA/BCR-ABL mice with CpG-miR-126 inhibitor and NIL will be reported at the meeting. Altogether, these observations suggest that miR-126 play a role in CML LSC homeostasis and down-regulation of miR-126 may decrease CML LSC quiescence, increase LSC proliferation and in turn enhance their sensitivity to TKI. miR-126 therefore may represent a novel therapeutic target in CML. DisclosuresStein:Amgen: Speakers Bureau. Snyder:BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.

Biological mechanisms of human-derived leukemia stem cells senescence regulated by Angelica sinensis polysaccharide

To explore the biological mechanisms underlying Angelica sindsis polysaccharide (ASP) -induced aging of human-derived leukemia stem cells (LSCs) in vitro. Acute myelogenous leukemia stem cells were isolated by magnetic activated cell sorting (MACS). The ability of LSC proliferation treated by various concentration of ASP(20-80 mg · L(-1)) in vitro for 48 hours were tested using cell counting Kit-8 ( CCK8) , colony forming were evaluated by methylcellulose CFU assay. The ultra structure changes of AML CD34+ CD38- cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-β-galactosidase Kit staining. Expression of aging-related p53, p21, p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were detected by quantitative reverse transcription polymerase chain reaction( qRT-PCR) and Western blotting, respectively. The purity of the CD34 + CD38 - cells is (91.15 ± 2.41)% after sorted and showed good morphology. The proliferation of LSC was exhibited significantly concentration-dependent inhibited after exposure to various concentration of ASP. Treated by 40 mg · L(-1) ASP for 48 hours, the percentage of positive cells stained by SA-β-Gal was dramatically increased (P < 0.01) and the colony-formed ability has been weakened (P < 0.01). The observation of ultrastructure showed that cell heterochromatin condensation and fragmentation, mitochondrial swelling, lysosomes increased in number. Aging-related p53, p21, p16, Rb and P16, Rb were up-regulated, protein regulatory cell-cycle CDK4 and Cyclin E were down-regulated. ASP may induce the senescence of LSCs effectively in vitro, P16-Rb cell signaling pathway play a significant role in this process. ASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.

Hypothesis: Tim-3/Galectin-9, A New Pathway for Leukemia Stem Cells Survival by Promoting Expansion of Myeloid-Derived Suppressor Cells and Differentiating into Tumor-Associated Macrophages

Despite the improvements in chemotherapy, about 60 % of acute myeloid leukemia (AML) remission patients still relapse. Leukemic stem cells (LSCs) are the main causes for the relapse and refractory. T cell immunoglobulin mucin-3 (TIM-3), a specific surface molecule expressed on LSCs in most types of AML, is a candidate for AML LSC-targeted therapies. It is important to know how this molecule functions in the maintenance of LSCs and suppression of anti-tumor immunity. Recent data have shown that Tim-3 which expresses on T cells can suppress immune responses indirectly by inducing expansion of myeloid-derived suppressor cells (MDSCs). MDSCs at the leukemia site can also differentiate into tumor-associated macrophages (TAMs). TAMs can promote proliferation and survival of LSCs by the diversion of adaptive immunity and the facilitation of extracellular matrix remodeling, angiogenesis, and lymphangiogenesis. Our previous study in AML patient bone marrow samples showed CD68(+) macrophages around AML clone. Based on the known evidence and our experimental findings, we hypothesize that Tim-3, which specifically expresses on LSCs, is beneficial for LSCs survival and AML progression by promoting expansion of MDSCs and differentiating into TAMs at the leukemia site.

G0S2–A new player in leukemia

G0S2–A new player in leukemia

Normal ABL1 Is a Tumor Suppressor and Therapeutic Target In BCR-ABL1–positive Leukemias

Abstract BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation resulting in BCR-ABL1 kinase expression, initiating chronic myeloid leukemia in chronic phase (CML-CP). At the initial stages of CML-CP both oncogenic BCR-ABL1 kinase and normal ABL1 kinase are expressed, however, loss of ABL1 kinase expression in CML-CP can result from an interstitial deletion in the normal chromosome 9 [del(9q34)] which may be combined with the transcriptional silencing of the alternative ABL1 promoter within the translocation eventually leading to disease progression and drug resistance. We found that BCR-ABL1 Abl1-/- cells generated a CML-blast phase (BP)-like disease phenotype in SCID mice compared to CML-CP-like disease from BCR-ABL1 Abl1+/+ cells. To determine the mechanisms responsible for blastic transformation of BCR-ABL1 Abl1-/- cells, we examined the role of ABL1 in proliferation, differentiation, apoptosis, genomic instability, and stemness. The presence of ABL1 inhibited proliferation in BCR-ABL1 cells as BCR-ABL1 Abl1-/- cells had higher clonogenic activity and proliferative rate compared to their wild-type counterparts. ABL1 is essential for myeloid differentiation since BCR-ABL1 Abl1-/- cells showed an immature blast phenotype when stained with Wright-Giemsa and myeloid differentiation markers Gr-1 and CD11b. ABL1 promoted apoptosis in response to genotoxic stress as revealed by reduced clonogenicity and elevated expression of p53, phosphoserine-15 p53 and activated caspase 3 in BCR-ABL1 Abl1 +/+ compared to knock-out cells. Although the absence of ABL1 did not enhance ROS and oxidative DNA damage, it appears that an impaired DNA damage response may be responsible for higher chromosome numbers and an accumulation of high numbers of chromosomal aberrations in BCR-ABL1 Abl1-/- cells. We detected an expansion of Lin-c-Kit+Sca-1+ leukemia stem cells (LSCs) in BCR-ABL1 Abl1-/- cells compared to BCR-ABL1 Abl1+/+ or non-transformed counterparts; among the LSCs, there was a higher percentage of CD34-Flt3- long-term and CD34+Flt3-short-term stem cells. These results showed that ABL1 is involved in regulating the LSC compartment in BCR-ABL1 cells. DNA microarray analysis revealed changes in mRNA levels of several genes involved in proliferation, myeloid differentiation, apoptosis, DNA damage response and stemness in BCR-ABL1 Abl1-/- cells in comparison to BCR-ABL1 Abl1+/+ cells. Together, these results demonstrated a critical role of ABL1 in BCR-ABL1-induced leukemia, prolonging survival in mice by suppressing proliferation and expansion of LSC, inducing myeloid differentiation, apoptosis and DNA damage response in BCR-ABL1 cells. Thus, it appears that ABL1 acts as a tumor suppressor in BCR-ABL1 –positive CML cells. Moreover, we hypothesized that the enhancement of the tumor suppressor function of ABL1 may have a significant impact on CML treatment. A small molecule activator of ABL1 kinase, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin (DPH), had been reported to interact with the myristoyl-binding site of ABL1 and destabilize the bent conformation of the α-1 helix, thereby preventing the auto-inhibitory conformation. DPH partially restored ABL1 activity in imatinib-treated cells. DPH-mediated stimulation of ABL1 tumor suppressor activity enhanced the effect of imatinib and ponatinib against CML CD34+ cells, Philadelphia chromosome-positive B-ALL (Ph+B-ALL) cells and relapsed Ph+B-ALL cells harboring T315I mutation without affecting normal counterparts. In summary, ABL1 is a potential tumor suppressor in BCR-ABL1-induced leukemia and stimulation of its function may play a significant role in the development of novel therapeutic strategies for CML and Ph+ALL. Disclosures: No relevant conflicts of interest to declare.

Targeting Of Leukemia Stem Cells By Interfering With Niches Using CD44 Variant 6 Chimeric Antigen Receptor Redirected Central Memory T Cells

Leukemic stem cells (LSCs), are resistant to radio/chemotherapy and responsible for disease relapse. Therefore, prevention of leukemia recurrence relies on the eradication of LSCs, which remains challenging for the treatment of hematopoietic malignancies. CD44 and its ligand are critical components of LSC niches that contain crucial signals for LSCs self-renewal. A CD44-specific mAb (clone H90) has been shown to specifically eradicate LSCs in immunodeficient mouse models. Further, a splice variant of CD44, CD44 variant 6 (CD44v6) is over expressed on malignant hematopoietic cells including ALL, AML, CML, NHL and multiple myeloma and plays an important role for proliferation, homing, and metastasis of LSCs. With the aim of targeting LSCs, we constructed a second generation Chimeric Antigen Receptor (CAR) specific for CD44v6 containing truncated EGFR as a selection and ablation molecule (CD44v6CAR/EGFRt). Central memory CD8+ T cells (CD62L+CD45RO+CD8+; CD8+TCM) were isolated from a healthy donor and transduced with lentivirus encoding CD44v6CAR/EGFRt after CD3/CD28 beads activation. Gene modified cells were immunomagnetically purified after labeling with biotinylated Erbitux followed by anti-biotin microbeads and expanded in rapid expansion medium (REM) containing OKT3 and feeder cells in the presence of IL-2 (50U/ml) and IL-15 (1ng/ml). The purified CD44v6CAR/EGFRt CD8+ T cells were expanded 300 fold following each cycle of 2 stimulations. 40-50% of the expanded CD44v6CAR/EGFRt CD8+T cells preserved central memory T-cell phenotype markers such as CD62L, CD28 and CD27. In a 4 hour chromium release assay, CD44v6CAR/EGFRt CD8+ T cells specifically recognized and lysed AML cells (THP-1) and lymphoblastoid cell lines (LCL) cells expressing CD44v6, while sparing CD44v6-negative KG1-a target cells (Fig. 1). To study in vivo efficacy of the CD44v6CAR/EGFRt CD8+ T cells, a xenogeneic AML model was established by intravenous injection of 2x10^6 firefly luciferase expressing THP-1 cells (THP-1-ffluc) into NSG mice. Adoptive transfer of 5x10^6 CD44v6CAR/EGFRt CD8+ T cells dramatically reduced THP-1-ffluc tumors as evidenced by bioluminescent imaging 14 days post T cell infusion. To prove the specificity of targeting, CD19+LCL-ffluc cells that are 50% CD44v6 positive were systemically infused into NSG mice. As expected, CD44v6CAR/EGFRt CD8+ T exhibited less antitumor activity against LCL tumor than THP-1 tumor. Accordingly, irrelevant CE-7CAR+T cells (L1CAM specific) that were derived from the CD8+ TCM of the same donor had no antitumor activity. These results indicate that targeting CD44v6 with CAR-transduced CD8+ TCM cells has the potential to treat hematological malignancies. Moreover, CD44v6 redirected T cell therapy would apply to various hematopoietic malignancies that aberrantly over-express CD44v6. Disclosures: No relevant conflicts of interest to declare.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.

A tumor suppressor function of the Msr1 gene in leukemia stem cells of chronic myeloid leukemia

AbstractWe have shown that Alox5 is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL–induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5 pathway represents a major molecular network that regulates LSC function. Therefore, we sought to dissect this pathway by comparing the gene expression profiles of wild type and Alox5−/− LSCs. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5 expression. In particular, we noted that the expression of the Msr1 gene was upregulated in Alox5−/− LSCs, suggesting that Msr1 suppresses the proliferation of LSCs. Using CML mouse model, we show that Msr1 is downregulated by BCR-ABL and this down-regulation is partially restored by Alox5 deletion, and that Msr1 deletion causes acceleration of CML development. Moreover, Msr1 deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-Catenin. Together, these results demonstrate that Msr1 suppresses LSCs and CML development. The enhancement of the tumor suppressor function of Msr1 may be of significance in the development of novel therapeutic strategies for CML.

SALL4 and hematologic neoplasms

SALL4 gene is closely related to body malformations related diseases, embryonic stem cell development, and hematopoietic malignancies. SALL4 can activate hematopoietic stem cell through Wnt signaling pathways, and promote continued proliferation of leukemia stem cells, leading to leukemia. In-depth study of SALL4 gene and its protein function will help clarify the pathogenesis of leukemia, providing a new target for the treatment of leukemia. Key words: Genes,SALL4 ; Neoplasms ; Leukemia ; Stem cells ;

The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

AbstractAbstract 1209Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures:No relevant conflicts of interest to declare.

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter*

Bmi-1, the first functionally identified polycomb gene family member, plays critical roles in cell cycle regulation, cell immortalization, and cell senescence. Bmi-1 is involved in the development and progression of carcinomas and is a potent target for cancer therapy. One important pathway regulated by Bmi-1 is that involving two cyclin-dependent kinase inhibitors, p16(Ink4a) and p19(Arf), as Bmi-1 represses the INK4a locus on which they are encoded. A close correlation between the up-regulation of Bmi-1 and down-regulation of p16 has been demonstrated in various tumors; however, how Bmi-1 regulates p16 expression is not clear. In this study, we revealed that Bmi-1 regulates the expression of p16 by binding directly to the Bmi-1-responding element (BRE) within the p16 promoter. The BRE resided at bp -821 to -732 upstream of the p16 ATG codon. BRE alone was sufficient to allow Bmi-1-mediated regulation of the CMV promoter. Bmi-1 typically functions by forming a complex with Ring2; however, regulation of p16 was independent of Ring2. Chromatin immunoprecipitation sequencing of Bmi-1-precipitated chromatin DNA revealed that 1536 genes were targeted by Bmi-1, including genes involved in tissue-specific differentiation, cell cycle, and apoptosis. By analyzing the binding sequences of these genes, we found two highly conserved Bmi-1-binding motifs, which were required for Bmi-1-mediated p16 promoter regulation. Taken together, our results revealed the molecular mechanism of Bmi-1-mediated regulation of the p16 gene, thus providing further insights into the functions of Bmi-1 as well as a sensitive high-throughput platform with which to screen Bmi-1-targeted small molecules for cancer therapy.