Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival

Abstract Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. Disclosures No relevant conflicts of interest to declare.

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Leukemia-Initiating Activity in T-ALL Requires Active Hif1a and Wnt Signaling

Blood ◽

10.1182/blood.v124.21.57.57 ◽

2014 ◽

Vol 124 (21) ◽

pp. 57-57

Author(s):

Vincenzo Giambra ◽

Catherine E Jenkins ◽

Sonya H Lam ◽

Catherine Hoofd ◽

Miriam Belmonte ◽

...

Keyword(s):

Stem Cells ◽

Wnt Signaling ◽

Cell Biology ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Negative Regulator ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells ◽

Hematopoietic Stem

Abstract Prior work has shown that NOTCH1 is a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL) with activating NOTCH1 mutations occurring in over 50% of cases (Weng et al, Science 2004) and loss-of-function mutations in its negative regulator FBXW7 occurring in 8-15% of cases (O’Neil et al, J Exp Med 2007; Thompson et al, J Exp Med 2007). Subsequent work has shown that continued Notch signaling is required for maintenance of T-ALL leukemia stem cells (Armstrong et al, Blood 2009; Tatarek et al, Blood 2011; Giambra et al, Nat Med 2012). Several lines of evidence have substantiated genetic interactions between the Notch and Wnt signaling pathways in various contexts, and Wnt signaling has been shown to play important roles in hematopoietic stem cell biology and also in hematopoietic cancers such as acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Luis et al, Leukemia 2012). To address what role if any Wnt signaling may play in T-ALL, we generated primary murine leukemias by viral transduction of bone marrow progenitors with activated NOTCH1, then delivered a fluorescent Wnt reporter construct (7TGP; Fuerer & Nusse, PLoS ONE 2010) by lentiviral transduction, and retransplanted the leukemias to interrogate Wnt signaling activity in vivo. We report here that active Wnt signaling is restricted to minor subpopulations within bulk T-ALL tumors, and that these Wnt-active subsets are highly enriched for leukemia-initiating cell (LIC) activity. Moreover, using Ctnnb1loxP/loxP animals we show that inducible Cre-mediated deletion of β-catenin or enforced expression of a dominant-negative TCF construct severely compromises LIC activity. We also show that β-catenin levels are upregulated by hypoxia through Hif1a stabilization, and that deletion of Hif1a also severely compromises LIC activity. Interestingly, Wnt-active subsets are distributed diffusely throughout the marrow interstitial space suggesting that tumor infiltration induces formation of local hypoxic niches as opposed to taking up residence in pre-existing anatomic compartments with low oxygen tensions. Taken together, these results suggest a model in which hypoxic niches in vivo facilitate Hif1a-dependent accumulation of β-catenin which drives Wnt signaling and self-renewal of leukemia stem cells. Finally, we show using patient-derived xenografts that antagonism of Hif1a or Wnt signaling also compromises human LIC activity, suggesting that pharmacologic targeting of these pathways could have therapeutic application in patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

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Inhibition of EZH2 Depletes MLL Fusion Leukemia Stem Cells Through Restoration of p16 Expression

Blood ◽

10.1182/blood.v120.21.3510.3510 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3510-3510

Author(s):

Koki Ueda ◽

Akihide Yoshimi ◽

Masahiro Nakagawa ◽

Satoshi Nishikawa ◽

Victor E Marquez ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

High Specificity ◽

Prolonged Survival ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Colony Forming Capacity

Abstract Abstract 3510 Leukemia stem cells (LSCs) are resistant to conventional chemotherapy and persistent LSCs after chemotherapy are supposed to be a major cause of disease relapse or refractoriness. However, information on genetic or epigenetic regulation of stem cell properties is still limited and LSC-targeted drugs have scarcely been identified or used in clinical settings so far. Epigenetic regulators are associated with many cellular processes such as cell cycle, proliferation, and apoptosis. Of note are polycomb group proteins, because they potentially control stemness including activity of cancer stem cells, and can be pharmacologically targeted by a selective inhibitor of H3K27, 3-deazaneplanocin A (DZNep). We first administrated DZNep to MLL-related leukemia mouse model in order to test whether DZNep has potential to eradicate LSCs of the leukemic mice. Remarkably, the leukemic granulocyte-macrophage progenitors (LGMPs) in MLL/AF9 positive cells were significantly decreased in number by administration of DZNep while AraC did not affect the number of LGMPs, which implied that LSCs were targeted by DZNep. These data were reproduced by transplantation assays using short hairpin RNA (shRNA)-mediated knockdown of EZH2, a major component of polycomb repressive complex 2 (PRC2) which is responsible for H3K27 tri-methylation. Significantly, DZNep administration to wild-type mice led to only mild suppression of hematopoiesis, suggesting that this agent spares normal hematopoietic stem cells while eliminating LSCs, which is consistent with a previous report that genetic depletion of EZH2 did not compromise adult hematopoiesis in mice. Serial replating assay of MLL/AF9-induced leukemia cells showed that DZNep treatment in vivo diminished their colony forming capacity. Limiting dilution transplantation assays revealed that frequency of LSCs was markedly reduced by DZNep administration. DZNep treatment or EZH2 knockdown significantly prolonged survival of MLL/AF9 and MLL/ENL leukemic mice. To elucidate a molecular mechanism underlying the effects of DZNep on LSCs, we investigated transcriptional or epigenetic changes during DZNep treatment and EZH2 knockdown. Gene expression profiling revealed that p16 was significantly upregulated by EZH2 knockdown or DZNep administration. Knockdown of p16 completely canceled the survival advantage of the leukemia mice which received DZNep in vivo and restored the colony forming capacity of leukemia cells transduced with shRNA for EZH2 in vitro. These results supported the idea that p16 upregulation derived from EZH2 attenuation is central to the LSC reduction. Next, we investigated epigenetic status around p16 promoter and transcription start site (TSS) by chromatin immunoprecipitation (ChIP) assays. In MLL/ENL leukemia cells, both H3K4 and H3K27 methylation marks were highly enriched around the TSS of p16, together with EZH2 and Bmi1, a component of PRC1. Therefore removal of EZH2 is supposed to convert the promoter of p16 from a bivalent to an active state. The results of ChIP assays also indicated that MLL/ENL fusion protein binds to p16 coding region. In order to clarify whether dependency on EZH2 is specific for MLL fusion leukemia or can be applied for other types of leukemia, we evaluated the consequence of EZH2 inhibition in several types of leukemia. DZNep or shRNA for EZH2 strongly suppressed the proliferation of leukemia cell lines and immortalized cells harboring MLL fusion genes with high specificity. Administration of DZNep or transduction of shRNA targeting EZH2 significantly prolonged survival of MLL/AF9 and MLL/ENL-induced leukemia mice while TEL/PDGFRA-AML1/ETO-induced leukemia was not sensitive to DZNep, although bone marrow (BM) cells from either mice became globally hypo-methylated on H3K27 by exposure to this drug. Serial replating assay with DZNep or EZH2-shRNA demonstrated high sensitivity to EZH2 inhibition of MLL/AF9-transduced BM cells but not of AML1/ETO-transduced BM cells, E2A/HLF-transduced BM cells, or normal c-kit+ BM cells. Thus, the anti-leukemia effect of EZH2 inhibition is thought to be specific for MLL fusion leukemia. Collectively, our findings indicate that EZH2 is a potential therapeutic target of LSCs of MLL fusion leukemia to overcome the poor prognosis, encouraging the development of inhibitors against EZH2 with high specificity. Disclosures: No relevant conflicts of interest to declare.

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Inhibition Of Microenvironmental Interleukin-1 Signaling Enhances TKI-Mediated Targeting Of Chronic Myelogenous Leukemia Stem Cells

Blood ◽

10.1182/blood.v122.21.512.512 ◽

2013 ◽

Vol 122 (21) ◽

pp. 512-512 ◽

Cited By ~ 1

Author(s):

Bin Zhang ◽

Yin Wei Ho ◽

Tessa L. Holyoake ◽

Ravi Bhatia

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Chronic Myelogenous Leukemia ◽

Interleukin 1 ◽

Myelogenous Leukemia ◽

Proliferation Index ◽

Leukemia Stem Cells ◽

Advisory Committees ◽

Hematopoietic Stem

Abstract BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. Most CML patients need continued TKI treatment to prevent disease relapse, and new strategies to eliminate residual leukemia stem cells are required to enhance possibility of achieving treatment-free remission. In previous studies we have shown that increased several cytokines expressed by leukemia cells may provide a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) within the CML BM microenvironment. Studies evaluating the effects of individual factors indicated that exposure to Interleukin-1α/β (IL-1α/β) at concentrations similar to those observed in CML BM resulted in significantly increased growth of CML compared with normal LTHSC (Cancer Cell 2012, 21:577). Consistent with previous reports (PNAS 2010, 107:16280), we observed that expression of the IL-1 receptor-associated protein (IL-1RAP), an important IL-1 signaling component, was increased in primitive CML cells, potentially explaining enhanced IL-1 sensitivity. To further evaluate the role of microenvironmental IL-1 in maintenance of CML LTHSC, we used recombinant IL-1 receptor antagonist (IL-1RA) to block IL-1 receptor signaling. IL-1RA is clinically approved for the treatment of rheumatoid arthritis. Purified LTHSC (Lin-Sca-1+Kit+Flt3-CD150+CD48- cells) from the SCL-tTA/BCR-ABL inducible mouse model of CML (CD45.1) and from congenic FVBN mice (CD45.2) were mixed in a 1:1 ratio and cultured with CML BM plasma, with and without IL-1RA. Culture with CML BM plasma for 7 days results in significantly increased growth of CML compared to normal LTHSC. The ratio of CML to normal cells was significantly reduced in the presence of IL-1RA (2.5μg/ml) (3.6:1 without IL-1RA, 1.7:1 with IL-1RA, p=0.0002), indicating that inhibition of IL-1 signaling reduced the growth advantage of CML LTHSC cultured in CML BM plasma. We next investigated the effect of IL-1RA on CML hematopoiesis in vivo. BM cells from CML mice (CD45.1) were transplanted into congenic FVBN mice (CD45.2) to generate CML-like disease in recipient mice. Four weeks after transplantation mice were treated with Nilotinib (NIL, 50mg/kg/d, gavage), IL-1RA (150mg/kg/d s.c.), the combination of NIL and IL-1RA, or vehicle (control) for 3 weeks. Treatment with NIL plus IL-1RA resulted in significantly greater reduction in CD45.1+ CML cells in blood, and in CML LTHSC, MPP, CMP and GMP in BM, compared with NIL alone (CML LTHSC/2 femurs: control 738±122, NIL 486±94, IL-1RA 525±49, combination 360±33, P=0.01 combination vs. Nilotinib). Mice treated with NIL plus IL-1RA also showed significantly prolonged survival after completion of treatment compared to mice treated with NIL alone (median survival 6 days for NIL alone versus 45 days for combination, p=0.02). Following transplantation of BM cells from treated mice into 2nd recipients (CD45.2), significantly lower CML cell engraftment in BM and reduced development of leukemia was seen after transplantation of cells from mice treated with the combination compared with NIL or untreated controls (8 out of 8 mice developed leukemia for control, 6 out of 8 for NIL, 5 out of 8 for IL-1RA, 3 out of 8 for the combination). We also studied the effect of treatment with NIL (5μm), IL-1RA (5μg/ml), NIL+IL-1RA, or vehicle for 72 hours on human CML and normal CD34+CD38+ and CD34+CD38- cells cultured with CML BM conditioned medium (CM). The combination of NIL and IL-1RA significantly reduced CML CD34+CD38+ and CD34+CD38- cell growth compared to Nilotinib alone (CD38- cells: NIL 23.7±10.1%, combination 13.1±8.9% of control, p<0.05), cell division (measured by CFSE labeling) (CD38- proliferation index: NIL 3.3±1.0, combination 2.4±0.6, p=0.06) and CFC frequency in methylcellulose progenitor assays (CD38- cells: NIL 67±22 per 1000 cells, combination 39±26, p<0.05); and moderately increased apoptosis of CML CD34+CD38- cells. We conclude that inhibition of microenvironmental IL-1 signaling using IL-1RA significantly increases inhibition of self-renewing murine and human CML stem cells in combination with NIL. Our results support further evaluation of IL-1 inhibition as a strategy to enhance elimination of CML LSC in TKI-treated patients. Disclosures: Holyoake: Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees.

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Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.

Blood ◽

10.1182/blood.v114.22.1475.1475 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1475-1475

Author(s):

Isabelle Andre-Schmutz ◽

Andrea Ditadi ◽

Amine Boudil ◽

Sophie Ezine ◽

Marina Cavazzana-Calvo

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Conflicts Of Interest ◽

Calf Serum ◽

Embryonic Stem ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Serum Free

Abstract Abstract 1475 Poster Board I-498 We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells. Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression. Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications. 1. Ditadi 2009 Disclosures: No relevant conflicts of interest to declare.

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TET Family Oncogene Fus Is Essential for the Maintenance of Self-Renewing Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2529.2529 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2529-2529

Author(s):

Takeaki Sugawara ◽

Hideyuki Oguro ◽

Atsushi Iwama

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Transcriptional Repression ◽

Cell Activation ◽

B Lymphocyte ◽

Rna Binding ◽

Conflicts Of Interest ◽

Hematopoietic Stem

Abstract Abstract 2529 Poster Board II-506 A Proto-oncogene FUS (fusion derived from malignant liposarcoma), also known as TLS (translocated in liposarcoma), was originally identified in chromosomal translocation of human soft tissue sarcoma. FUS is also known to be fused with an ETS family transcription factor ERG in human myeloid leukemia with t(16;21) which is associated with poor prognosis. Based on its protein structure, DNA- and RNA-binding activity and involvement in many human cancers as the fusion with various transcription factors, FUS is now grouped with EWS and TAFII68 into TET (FET) oncogene family. Multiple functions have been postulated for FUS, including non-coding-RNA-mediated transcriptional repression, posttranscriptional RNA processing and the maintenance of genomic integrity. Fus-deficient (Fus−/−) mice showed a non-cell-autonomous defect in B lymphocyte development, defective B cell activation and increased sensitivity to radiation in previous studies. However, its physiological function in hematopoiesis remains unknown. In this study we performed detailed analyses of Fus−/− hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 exhibited a mild reduction in numbers of hematopoietic stem and progenitor cells compared with the wild type. Disruption of Fus, however, did not grossly affect proliferation or differentiation of hematopoietic progenitors. Of note, Fus−/− HSCs had significantly reduced repopulating activity of hematopoiesis in competitive repopulation assays, and did not repopulate hematopoiesis at all in tertiary recipients. Moreover, Fus−/− HSCs were highly sensitive to radiation both in vitro and in vivo and showed a drastic reduction in numbers in recipient mice after sublethal irradiation. All these findings implicate Fus in the maintenance and radioprotection of HSCs. Studies of chromosome stability, telomere length, apoptosis and levels of reactive oxigen species (ROS) appeared not accountable for the apparent defect of Fus−/− HSCs. However, gene expression profiling identified changes in expression of several genes in Fus−/− HSCs, and dysregulated expression of some of these genes might be responsible for the defective function of Fus−/− HSCs. Disclosures: No relevant conflicts of interest to declare.

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Stromal Cell Regulation of Murine Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1566.1566 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1566-1566

Author(s):

Stefan Wohrer ◽

Keegan Rowe ◽

Heidi Mader ◽

Claudia Benz ◽

Michael R Copley ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Stromal Cell ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Self Renewal ◽

Hematopoietic Stem ◽

Daughter Cells

Abstract Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (>20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (>1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. Disclosures: No relevant conflicts of interest to declare.

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A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors

Blood ◽

10.1182/blood.v118.21.1290.1290 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1290-1290

Author(s):

Julian Pulecio ◽

Leopoldo Laricchia-Robbio ◽

Juan Carlos Izpisua ◽

Montserrat Barragan ◽

Marianna Vitaloni ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

In Vitro Assays ◽

Main Role ◽

In Vitro Differentiation ◽

Related Factors ◽

Hematopoietic Stem

Abstract Abstract 1290 After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors. Disclosures: No relevant conflicts of interest to declare.

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GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis

Blood ◽

10.1182/blood.v122.21.3781.3781 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3781-3781

Author(s):

Philipp A Dietrich ◽

Murray D Norris ◽

Jenny Yingzi Wang

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Active Form ◽

Leukemic Stem Cells ◽

Poor Survival ◽

Functional Link ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Inappropriate activation of Wnt/β-catenin signaling confers hematopoietic progenitors the property of self-renewal that promotes malignant transformation in MLL-rearranged acute myeloid leukemia (AML). However, it has been noted that activation of β-catenin is observed in tumors without clear mutations in the major components of the pathway or increase in Wnt signaling. This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of β-catenin. Recently, a number of G protein-coupled receptors (GPCRs) have been shown to activate β-catenin signaling to recruit the key downstream components of the canonical Wnt pathway in distinct cell types, including stem cells. GPCRs, the largest family of cell-surface molecules involved in signal transmission, have emerged as crucial players in tumor growth and metastasis, and represent one of the most important drug targets in pharmaceutical development. Given the close functional link with activation of β-catenin signaling, a GPCR signaling pathway may act as the upstream regulator of β-catenin signaling in the establishment of leukemic stem cells (LSC). In this study, our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified GPR84, a proinflammatory GPCR, as a potential LSC-specific candidate target. An analysis of the comprehensive patient outcome database (Oncogenomics – maintained by the National Cancer Institute) showed that high levels of GPR84 were significantly associated with poor survival in patients with leukemia (P=0.0048), implying its potential clinical relevance in predicting disease prognosis. Western blot and flow cytometric analyses confirmed the microarray results and revealed a positive correlation between GPR84 and β-catenin expression. We previously demonstrated that β-catenin was highly expressed in HSC transformed by MLL-AF9 (HSC-MLLAF9) and had lower expression in HSC transduced with leukemic oncogenes Hoxa9/Meis1 (HSC-Hoxa9/Meis1), while increased β-catenin expression was correlated with a poor survival rate in vivo. Herein, our results showed that forced expression of GPR84 induced a robust upregulation of β-catenin in HSC-Hoxa9/Meis1. Conversely, shRNA-mediated ablation of GPR84 in HSC-MLLAF9 led to highly significant downregulation of both GPR84 (P=0.0003) and β-catenin (P=0.0008). Further in vitro functional studies showed that GPR84 knockdown significantly reduced HSC-MLL-AF9 colony forming units (P=0.0006), and induced a marked reduction of cells in S-phase (P=0.0017). This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Importantly, subsequent in vivo survival studies using leukemia transplantation mouse models showed that GPR84 knockdown significantly reduced LSC frequency and severely impaired maintenance (P<0.0001; 11 mice per cohort) of HSC-MLL-AF9 induced leukemia, a highly aggressive and drug-resistant subtype of AML. The defect in disease phenotype resulted from inhibited expression of both GPR84 and β-catenin. Furthermore, forced overexpression of GPR84 alone was not sufficient for leukemic transformation of HSC but conferred a growth advantage in vivo to HSC-Hoxa9/Meis1 cells and significantly accelerated the onset of Hoxa9/Meis1-induced AML (P=0.0039), establishing a completely malignant phenotype similar to HSC-MLL-AF9 in vivo (P=0.9986). These data support an oncogenic role of GPR84 in MLL-AF9-induced leukemogenesis. In conclusion, our studies have identified a novel β-catenin regulator that contributes to leukemia maintenance by sustaining aberrant activation of a stem cell self-renewal pathway in LSC, and drugs targeting GPR84 may represent a novel and promising strategy for improving the therapy and outcome of AML patients. Disclosures: No relevant conflicts of interest to declare.

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Myc-Miz-1 signaling promotes self-renewal of leukemia stem cells by repressing Cebpα and Cebpδ

Blood ◽

10.1182/blood.2019001863 ◽

2020 ◽

Author(s):

Lei Zhang ◽

Jing Li ◽

Hui Xu ◽

Xianyu Shao ◽

Li Fu ◽

...

Keyword(s):

Stem Cells ◽

Leukemia Stem Cells ◽

Mutant Form ◽

Self Renewal ◽

Hematopoietic Stem ◽

Reduced Frequency ◽

Almost All

c-Myc (Myc hereafter) is found to be deregulated and/or amplified in most acute myeloid leukemias (AML). Almost all AML cells are dependent upon Myc for their proliferation and survival. Thus Myc has been proposed as a critical anti-AML target. Myc has Max-mediated trans-activational and Miz1-mediated trans-repressional activities. The role of Myc-Max-mediated trans-activation in the pathogenesis of AML has been well-studied; however the role of Myc-Miz1-mediated trans-repression in AML is still somewhat obscure. MycV394D is a mutant form of Myc which lacks trans-repressional activity due to a defect in its ability to interact with Miz1. We found that, compared to Myc, the oncogenic function of MycV394D is significantly impaired. The AML/myeloproliferative disorder which develops in mice receiving MycV394D-transduced hematopoietic stem/progenitor cells (HSPCs) is significantly delayed compared to mice receiving Myc-transduced HSPCs. Using a murine MLL-AF9 AML model, we found that AML cells expressing MycV394D (intrinsic Myc deleted) are partially differentiated and show reductions in both colony-forming ability in vitro and leukemogenic capacity in vivo. The reduced frequency of leukemia stem cells (LSCs) among MycV394D-AML cells and their reduced leukemogenic capacity during serial transplantation suggest that Myc-Miz1 interaction is required for the self-renewal of LSCs. In addition, we found that MycV394D-AML cells are more sensitive to chemotherapy than are Myc-AML cells. Mechanistically, we found that the Myc represses Miz1-mediated expression of Cebpα and Cebpδ, thus playing an important role in the pathogenesis of AML by maintaining the undifferentiated state and self-renewal capacity of LSCs.

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β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.2899.2899 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2899-2899

Author(s):

Jodi Murakami ◽

Baohui Xu ◽

Christopher B. Franco ◽

Xingbin Hu ◽

Stephen J. Galli ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Adhesion ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Effector Cells ◽

Cell Cycle Status

Abstract α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche. Disclosures No relevant conflicts of interest to declare.

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