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

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

Blood ◽

10.1182/blood.v128.22.5257.5257 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5257-5257

Author(s):

Wenbin Zhong ◽

Vesa Olkkonen ◽

Xu Bing ◽

Biying Zhu ◽

Guoping Li ◽

...

Keyword(s):

Stem Cells ◽

Oxidative Phosphorylation ◽

Mitochondrial Function ◽

Conflicts Of Interest ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells ◽

Ip3 Receptor ◽

Hematopoietic Stem

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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BCR/ABL Can Promote CD19+ Cell Growth but Not Render Them Long-Term Stemness

Blood ◽

10.1182/blood.v128.22.2709.2709 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2709-2709

Author(s):

Donghe Li ◽

Xuemei Zhao ◽

Bo Jiao ◽

Ping Liu ◽

Ruibao Ren

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells ◽

T Cell Therapy ◽

Hematopoietic Stem

Abstract Cancer stem cells are a subpopulation of malignant cells that have the capacity of both self-renewal and reconstitution of the cancer. Eradication of cancer stem cells is crucial for curing the malignant disease. Previous studies in hematopoietic malignancies showed that leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) chronic phase are originated from a hematopoietic stem cell (HSC), while LSCs in acute myeloid leukemia (AML) can either be derived from HSCs or be transformed from myeloid progenitors. But in acute B-lymphoblastic leukemia (B-ALL), the origin of leukemia stem cells is not clear. In this study, we tested whether BCR/ABL could transform B-lineage committed CD19+ cells to LSCs. We found that transducing BCR/ABL in CD19+ cells can promote their colony formation in vitro and induce B-ALL like disease in vivo. However, only BCR/ABL transduced whole bone marrow cells, but not CD19+ cells, can be transplanted multiple times in recipient mice, and the frequency of long-term LSCs from the latter ranges from 1/135 to 1/629. These studies suggest that LSCs in BCR/ABL+ B-ALL may originate from CD19- hematopoietic stem/progenitor cells and that CD19 chimeric antigen receptor (CAR) modified T cell therapy may not be effective in eradicating LSCs in BCR/ABL+ B-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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41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽

10.1182/blood.v124.21.2293.2293 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2293-2293

Author(s):

Ekta Kapadia ◽

Elad Jacoby ◽

Mark Kohler ◽

Waleed Haso ◽

Christopher Daniel Chien ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

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Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo

Blood ◽

10.1182/blood.v120.21.sci-41.sci-41 ◽

2012 ◽

Vol 120 (21) ◽

pp. SCI-41-SCI-41

Author(s):

Andreas Trumpp ◽

Marieke Essers

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Strong Increase ◽

Interferon Α ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract SCI-41 Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs. Disclosures: No relevant conflicts of interest to declare.

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Leukemia Stem Cells Are Resistant to In Vivo, Cell Non-Autonomous Wnt Inhibition.

Blood ◽

10.1182/blood.v114.22.1025.1025 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1025-1025

Author(s):

Steven W. Lane ◽

Cristina Lo Celso ◽

Stephen M Sykes ◽

Sebastian Shterental ◽

Mahnaz Paktinat ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Wnt Signaling ◽

Bone Marrow Microenvironment ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Dkk1 Expression ◽

Hsc Niche

Abstract Abstract 1025 Poster Board I-47 Acute myeloid leukemia (AML) initiating cells reside within and utilize the bone marrow microenvironment, as a sanctuary to evade chemotherapy and to maintain self-renewal. Following treatment, these leukemia stem cells (LSC) re-emerge and reconstitute disease, leading to relapse. The canonical Wnt signaling pathway is frequently dysregulated in LSC and recent data indicates that Dkk1 (a potent endogenous Wnt inhibitor) may have a therapeutic role in treating AML. Microenvironment specific Dkk1 expression inhibits hematopoietic stem cell (HSC) Wnt and extinguishes HSC self-renewal in vivo, identifying the Wnt pathway as essential in normal HSC-niche homeostasis. We investigated the importance of bone marrow microenvironment Wnt signaling in LSC survival. AML was generated using retroviral transduction of murine bone marrow with the MLL-AF9 fusion oncogene. We then assessed the potential for niche-directed Wnt inhibition of LSC using 2.3kbColl1alpha-Dkk1 transgenic mice in which Dkk1 expression is restricted to osteoblasts. AML was observed in the Dkk1 or wild type mice with similar disease latency and phenotype. AML was also observed in secondary transplant recipients, although there was a reduction of LSC (linlowcKithighSca-1-FcGRII/III+CD34+) derived from Dkk1 mice (LSC frequency 2.8% WT vs 1.6% Dkk1, p<0.05), correlating with a subtle prolongation in disease latency (n=15, 20 days WT vs. 24 days Dkk1, p<0.001). To determine the status of Wnt signaling in MLL-AF9 AML, we generated AML in bone marrow derived from TOPGal reporter mice that harbor a Tcf/Lef responsive promoter with a LacZ reporter, and quantified LacZ expression or galactosidase protein levels. Wnt activation was increased following transformation of bone marrow with MLL-AF9 (relative TOPGal expression 1.35 empty vector vs 2.58 MLLAF9, p=0.03). To assess the effects of osteoblast-restricted Dkk1 expression in vivo, Wnt signaling was measured in LSC purified by high-speed multiparameter flow cytometry. Reporter activity (fluorescein di-β-D-galactopyranoside (FDG), Invitrogen) was unchanged in LSC from WT or Dkk1 recipients (Median fluorescent intensity 552 vs 542, p=0.85), indicating that, in contrast with normal HSC, Wnt signaling in LSC is relatively resistant to Dkk1 expression in the niche. To better understand the mechanism of LSC resistance to Dkk1, we examined the homing and micro-localization of LSC in vivo using live, 3 dimensional two photon-confocal hybrid imaging of the bone marrow microenvironment. LSC proliferate with similar kinetics in Dkk1 or WT recipients (proliferating fraction 57.7% WT vs 50.3% Dkk1 LSC p=0.48). However, when compared to HSC, LSC home with less affinity to osteoblasts and may escape the effects of osteoblast specified Dkk1 expression through residence in a niche that is physically distant from endosteum (Median distance to osteoblast 18um WT vs 20.6um Dkk1 LSC, p=0.13). Taken together, these data indicate that MLL-AF9 LSC can escape the normal HSC-niche homeostatic constraints regulated by Wnt, an observation that may have important therapeutic implications. Disclosures: Scadden: Fate Therapeutics: Consultancy. Gilliland:Merck: Employment.

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Leukemia stem cells in a genetically defined murine model of blast-crisis CML

Blood ◽

10.1182/blood-2007-02-073031 ◽

2007 ◽

Vol 110 (7) ◽

pp. 2578-2585 ◽

Cited By ~ 102

Author(s):

Sarah J. Neering ◽

Timothy Bushnell ◽

Selcuk Sozer ◽

John Ashton ◽

Randall M. Rossi ◽

...

Keyword(s):

Stem Cells ◽

Kinase Inhibitor ◽

Blast Crisis ◽

Myelogenous Leukemia ◽

In Vivo Studies ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Treatment Regimens ◽

Resistant Disease

Myeloid leukemia arises from leukemia stem cells (LSCs), which are resistant to standard chemotherapy agents and likely to be a major cause of drug-resistant disease and relapse. To investigate the in vivo properties of LSCs, we developed a mouse model in which the biologic features of human LSCs are closely mimicked. Primitive normal hematopoietic cells were modified to express the BCR/ABL and Nup98/HoxA9 translocation products, and a distinct LSC population, with the aberrant immunophenotype of lineage−, Kit+/−, Flt3+, Sca+, CD34+, and CD150−, was identified. In vivo studies were then performed to assess the response of LSCs to therapeutic insult. Treatment of animals with the ABL kinase inhibitor imatinib mesylate induced specific modulation of blasts and progenitor cells but not stem- cell populations, thereby recapitulating events inferred to occur in human chronic myelogenous leukemia (CML) patients. In addition, challenge of leukemic mice with total body irradiation was selectively toxic to normal hematopoietic stem cells (HSCs), suggesting that LSCs are resistant to apoptosis and/or senescence in vivo. Taken together, the system provides a powerful means by which the in vivo behavior of LSCs versus HSCs can be characterized and candidate treatment regimens can be optimized for maximal specificity toward primitive leukemia cells.

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Targeting Pre-Leukemic Stem Cells in T-Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.527.527 ◽

2016 ◽

Vol 128 (22) ◽

pp. 527-527

Author(s):

Bastien Gerby ◽

Diogo F.T Veiga ◽

Jana Krosl ◽

Julianne Ouellette ◽

André Haman ◽

...

Keyword(s):

Stem Cells ◽

Acute Lymphoblastic Leukemia ◽

High Throughput ◽

High Throughput Screening ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Primary Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass with, however, undesirable long term consequences and remains ineffective in adolescent and adult T-ALL. Furthermore, relapse can be caused by pre-leukemic stem cells (pre-LSCs) that were spared by current protocols and evolved to malignancy. A distinctive characteristic of pre-LSCs is their critical dependence on interactions with the microenvironment for survival, which guided our strategy to target pre-LSCs using niche-based screening assays. Using transgenic mouse models that closely reproduce the human disease, we showed that the SCL/TAL1 and LMO1 oncogenic transcription factors establish a pre-leukemic state by reprogramming normal pro-T cells into aberrantly self-renewing pre-LSCs (Gerby et al. PloS Genetics, 2014). We now provide direct evidence that pre-LSCs are much less chemosensitive than leukemic blasts to current drugs, due to a distinctive lower proliferative state as assessed by real-time imaging in a competitive assay. We therefore designed a robust protocol for high-throughput screening (HTS) of compounds targeting primary pre-LSCs that are maintained on stromal cells engineered for optimal NOTCH1 activation to mimick the thymic microenvironement. The multiparametric readout takes into account the intrinsic complexity of primary cells to specifically monitor pre-LSCs. We screened a targeted library of 1904 compounds and identified UM0119979 that disrupts both cell autonomous and non-cell autonomous pathways: UM0119979 abrogates pre-LSC viability and self-renewal activity in vivo by specifically inhibiting the translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remain functional. Moreover, in vivo administration of UM0119979 efficiently reduced the leukemia propagating activity of primary human T-ALL samples in xenografted mice. Finally, in addition to SCL-LMO-induced T-ALL, our results reveal a novel possibility of therapeutic intervention in MYC-dependent hematologic malignancies. In summary, our screening assay, built on the genetic dependencies of pre-LSCs, revealed their vulnerabilities to compounds that inhibit both the primary oncogenes and non-cell autonomous pathways triggered by the microenvironment. The results illustrate how recapitulating tissue-like properties of primary cells in high throughput screening is a promising avenue for innovation in cancer chemotherapy. Disclosures No relevant conflicts of interest to declare.

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Δ12-prostaglandin J3, an omega-3 fatty acid–derived metabolite, selectively ablates leukemia stem cells in mice

Blood ◽

10.1182/blood-2010-11-317750 ◽

2011 ◽

Vol 118 (26) ◽

pp. 6909-6919 ◽

Cited By ~ 48

Author(s):

Shailaja Hegde ◽

Naveen Kaushal ◽

Kodihalli C. Ravindra ◽

Christopher Chiaro ◽

Kelsey T. Hafer ◽

...

Keyword(s):

Stem Cells ◽

Fatty Acid ◽

Polyunsaturated Fatty Acid ◽

Myelogenous Leukemia ◽

Leukemia Stem Cells ◽

Omega 3 ◽

Hematopoietic Stem ◽

Omega 3 Fatty Acid ◽

Dietary Fish

Abstract Targeting cancer stem cells is of paramount importance in successfully preventing cancer relapse. Recently, in silico screening of public gene-expression datasets identified cyclooxygenase-derived cyclopentenone prostaglandins (CyPGs) as likely agents to target malignant stem cells. We show here that Δ12-PGJ3, a novel and naturally produced CyPG from the dietary fish-oil ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA; 20:5) alleviates the development of leukemia in 2 well-studied murine models of leukemia. IP administration of Δ12-PGJ3 to mice infected with Friend erythroleukemia virus or those expressing the chronic myelogenous leukemia oncoprotein BCR-ABL in the hematopoietic stem cell pool completely restored normal hematologic parameters, splenic histology, and enhanced survival. More importantly, Δ12-PGJ3 selectively targeted leukemia stem cells (LSCs) for apoptosis in the spleen and BM. This treatment completely eradicated LSCs in vivo, as demonstrated by the inability of donor cells from treated mice to cause leukemia in secondary transplantations. Given the potency of ω-3 polyunsaturated fatty acid–derived CyPGs and the well-known refractoriness of LSCs to currently used clinical agents, Δ12-PGJ3 may represent a new chemotherapeutic for leukemia that targets LSCs.

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