Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj <0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR<0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

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Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽

10.1182/blood.v116.21.405.405 ◽

2010 ◽

Vol 116 (21) ◽

pp. 405-405

Author(s):

Kenichi Miharada ◽

Göran Karlsson ◽

Jonas Larsson ◽

Emma Larsson ◽

Kavitha Siva ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Distinct Population ◽

Self Renewal ◽

Hematopoietic Stem ◽

Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

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Conditional Loss of Dnmt3a Results in Myeloproliferation and Liver-Specific Myeloid Expansion

Blood ◽

10.1182/blood.v124.21.364.364 ◽

2014 ◽

Vol 124 (21) ◽

pp. 364-364

Author(s):

Olga A Guryanova ◽

Yen Lieu ◽

Kaitlyn R Shank ◽

Sharon Rivera ◽

Francine E Garrett-Bakelman ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Extramedullary Hematopoiesis ◽

Disease Phenotype ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic System ◽

Flow Cytometric

Abstract Mutations in the DNA methyltransferase 3A (DNMT3A) gene are frequent in normal karyotype de novo acute myeloid leukemia (AML) (20-35%), chronic myelomonocytic leukemia (CMML) (10-20%) and myelodysplastic syndrome (MDS) (8%). Hematopoietic-specific loss of Dnmt3a in a mouse model leads to acquisition of aberrant self-renewal by the HSCs and expansion of the stem/progenitor compartment in bone marrow transplantation studies. Despite these important insights, the impact of hematopoietic deletion of Dnmt3a on disease phenotype in primary, non-transplanted mice has not been described. Mx1-Cre-mediated Dnmt3a ablation in the hematopoietic system in primary mice led to the development of a myeloproliferative neoplasm (MPN) with a 100% penetrance (n=14) and a median age of onset at 47.7 weeks (survival difference between Dnmt3a KO and control animals p<0.0001, Figure 1A). Loss of Dnmt3a in the hematopoietic compartment resulted in thrombocytopenia (platelet counts 250±251.8 K/μl in Dnmt3a KO vs 1260±292.8 K/μl in controls, p<0.002) and overall anemia (hematocrit 25.25±7.48% vs 44.8±5.83%, p<0.006). Marked expansion of the mature Mac1+Gr1+ myeloid cell population in the peripheral blood was evident by flow cytometric analysis (52.3±18.03% in Dnmt3a knock-outs). Myeloproliferation induced by Dnmt3a loss was characterized by marked, progressive hepatomegaly (liver weights 7.25±1.195 g in Dnmt3a-deleted animals vs 1.61±0.266 g in wild-type controls, p<1.75×10^-8, Figure 1B) with moderate splenomegaly (spleen weights 457.5±379.6 mg vs 79.43±21.19 mg, p<0.033). Histopathological analysis revealed massive myeloid infiltration in spleens and livers leading to complete effacement of organ architecture, left shifted myeloid cells, and occasional blasts. In addition, the presence of megakaryocytes in spleens and livers of Dnmt3a-deleted mice was indicative of extramedullary hematopoiesis. The significant myeloid infiltration of liver parenchyma was confirmed by flow cytometric analysis of liver tissue, with Mac1+Gr1+ myeloid cells making up 66.15±11.93% of all viable cells. In line with previous reports, we observed an increased number of immunophenotypically defined stem (Lin-Sca1+cKit+, LSK, 2.013±1.200% in Dnmt3a-ablated mice vs 0.423±0.052% in controls, a 4.76-fold increase, p<0.014) and granulomonocytic progenitor (GMP, Lin-Sca1-cKit+CD34+FcγR+, 2.713±1.593% vs 1.278±0.451%, a 2.12-fold increase, p<0.024) cells in the bone marrow. Consistent with extramedullary hematopoiesis, we were able to detect expanded LSK cell populations in livers and spleens of Dnmt3a-deleted mice. Notably, the myeloid disease phenotype induced by Dnmt3a loss was fully transplantable, including the marked hepatomegaly; these data demonstrate that the liver-specific expansion reflects a cell-autonomous mechanism. To assess relative tropism for different target organs, we next performed homing studies where Dnmt3a-deleted bone marrow cells were competed against wild-type counterparts in lethally irradiated hosts. 48 hours after transplantation, we observed increased tropism of the Dnmt3aΔ/Δ BM cells to the liver and spleen, whereas control cells preferentially localized to the bone marrow (difference between homing to bone marrow and spleen/liver p<0.0115, Figure 1C). These data demonstrate that altered homing and tissue tropism of Dnmt3a KO hematopoietic cells promote extramedullary hematopoiesis and liver involvement. ERRBS and gene expression profiles by RNA-seq in stem and progenitor cell populations demonstrated differential regulation of key biologic pathways, including self-renewal, hematopoietic lineage commitment and differentiation, and heterotypic cell-cell interactions. In conclusion, our studies show that ablation of Dnmt3a in the hematopoietic system leads to myeloid transformation in vivo, with cell autonomous liver tropism and marked extramedullary hematopoiesis. These data demonstrate, in addition to its established role in controlling self-renewal, Dnmt3a serves as an important regulator of the myeloid compartment that limits expansion of myeloid progenitors in vivo. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The CXCR4 Antagonist AMD 3100 Induces Rapid Mobilization of Facilitating Cells and Hematopoietic Stem Cells in Mice

Blood ◽

10.1182/blood.v118.21.4694.4694 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4694-4694

Author(s):

Hong Xu ◽

Ziqiang Zhu ◽

Yiming Huang ◽

Suzanne T. Ildstad

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Dose Titration ◽

Great Promise ◽

Hematopoietic Stem ◽

Flow Cytometric

Abstract Abstract 4694 Bone marrow transplantation (BMT) offers great promise for treating red blood cell disorders, inherited disorders of metabolism, autoimmune diseases, and inducing donor-specific tolerance to organ transplants. However, the widespread application of this approach is dependent upon the development of less toxic strategies for BMT and avoidance of graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FC) facilitate engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex barriers without causing GVHD. We previously reported that Flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) synergistically mobilize FC and HSC into the peripheral blood (PB). Recently, AMD 3100 has been found to be a rapid mobilizing agent whose effect occurs within hours after injection. It is a macrocyclic compound and potential fusion inhibitor that antagonizes CXCR4 alpha-chemokine receptor for its effect on HSC mobilization. CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), are important in HSC homing and maintenance in the bone marrow microenvironment. Here, we investigated the effects of AMD 3100 on the mobilization of FC and HSC into PB in combination with FL and G-CSF. A dose titration of AMD 3100 was first performed. B6 mice were injected subcutaneously with AMD 3100 with the doses ranging from 1.25 mg/kg to 10 mg/kg. PB was obtained 0.5, 1, 3, and 6 hours post-injection. After individual count of peripheral blood mononuclear cells (PBMC), cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−). The numbers of PBMC significantly increased even 0.5 hour after AMD 3100 treatment and peaked at 1 h. The maximal mobilization of PBMC was noted at 1 h with 5.0 mg/kg AMD 3100. Treatment with 5.0 mg/kg AMD 3100 caused a 3.1-fold increase of WBC at 1h compared with saline treated controls. An increase of FC was detectable with all doses of AMD 3100. The numbers of FC peaked between 1 and 3 h, and declined rapidly to resemble saline-treated controls at 6 h after. A 5.9-fold increase of FC was observed at 1 h with 5.0 mg/kg AMD 3100 (P = 0.012). These data suggest that AMD 3100 is a potent cell mobilizer from bone marrow to PB. We next investigated the effect of AMD 3100 in combination with FL and G-CSF on the mobilization of FC and HSC into PB. B6 mice were injected with FL (day 1 to 10), G-CSF (day 4 to 10), and AMD 3100 (day 10). PB was obtained 1 h after injection on day 10. After performing a count of peripheral WBC, cells were stained for flow cytometric analysis to enumerate FC (CD8+/TCR−) and HSC (Lin−/Sca-1+/c-kit+) mobilization. The maximal mobilization of PBMC was observed when animals were treated with AMD 3100/FL/G-CSF. The numbers of PBMC with AMD3100/FL/G-CSF treatment increased with 17.2-fold and 6.4-fold when compared with controls treated with saline or AMD 3100 alone (P < 0.00001), respectively. A maximal elevation of both FC and HSC was detected when AMD 3100 was added to FL/G-CSF treatment and reached 1.91 ± 0.42 × 103/μl (Figure 1A) and 1.89 ± 0.35 × 103/μl (Figure 1B), respectively. The increase of FC and HSC was significant. There was a 10.1-fold increase in FC and 230.8-fold increase in HSC when compared with recipients treated with AMD 3100 alone (P < 0.00001). AMD 3100/FL/G-CSF treatment resulted in a 1.7-fold of FC and 2.2-fold increase of HSC when compared with recipients treated with FL/G-CSF (P < 0.05). In summary, AMD 3100, FL, and G-CSF show a highly significant synergy on the mobilization of FC and HSC. This study may be clinically relevant in efforts to mobilize immunomodulatory FC and HSC to PB for transplantation, especially to induce tolerance for organ transplant recipients. Disclosures: Ildstad: Regenerex, LLC: Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.3061.3061 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3061-3061

Author(s):

Jennifer Lynch ◽

Hangyu Yi ◽

Brendon Martinez ◽

Florida Voli ◽

Jenny Yingzi Wang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Death ◽

Blood Cells ◽

Mitochondrial Membrane ◽

Leukemia Stem Cells ◽

Ros Production ◽

Self Renewal ◽

Jnk Signaling

Abstract 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. Disclosures No relevant conflicts of interest to declare.

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Expression of a HoxC4 Retroviral Vector Mediates Expansion of Murine Hematopoietic Stem Cells with Normal Hematopoiesis in Transplanted Mice.

Blood ◽

10.1182/blood.v104.11.2788.2788 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2788-2788

Author(s):

Lilia Stepanova ◽

Brian P. Sorrentino

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Retroviral Vector ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Murine Bone Marrow

Abstract Homeobox (Hox) transcription factors are important regulators of hematopoietic cell proliferation and differentiation. Of them, HoxB4 is of particular interest because overexpression promotes rapid expansion of mouse hematopoietic stem cells (HSCs) without causing neoplastic transformation. Despite the effects of HoxB4 overexpression on HSCs, mice that are homozygous for HoxB4 gene deletion have only subtle defects in HSCs and progenitor cells. We hypothesized that other paralogs of HoxB4 may also be capable of inducing HSC expansion could thereby compensate for loss of HoxB4 function. To test this hypothesis, we have studied the effects of retroviral overexpression of a HoxC4 gene in murine progenitors and HSCs. The murine HoxC4 cDNA was cloned and inserted into an MSCV vector that co-expresses an IRES-YFP reporter gene. We transduced murine bone marrow cells with a MSCV-HoxC4-YFP vector and compared the secondary replating efficiency of myeloid colonies (CFU-Cs) to that seen using either a MSCV-HoxB4-GFP or an MSCV-GFP vector. This assay tests for progenitor cell self-renewal which is increased using HoxB4-expressing vectors. Cells transduced with the MSCV-HoxC4-YFP vector formed 20–40 times more secondary CFU-Cs than with cells transduced with the MSCV-GFP control vector. This increase in CFU-C replating efficiency was equivalent to that seen with the MSCV-HoxB4-IRES-GFP vector. To test the in vivo effects of the MSCV-HoxC4-YFP vector on self-renewal of HSCs, we transplanted lethally irradiated mice with a mixture of cells; 20% transduced with the MSCV-HoxC4-YFP vector and 80 % mock-transduced. Peripheral blood analysis of the transplanted recipients up to 28 weeks post-transplantation showed that the percentage of cells transduced with the MSCV-HoxC4-YFP vector was 70–85% in both lymphoid and myeloid cells in the peripheral blood. A similar degree of chimerism was noted in concurrent controls using the MSCV-HoxB4-GFP vector. In contrast, the percentages of peripheral blood cells transduced with the MSCV-GFP vector was only 15–25%, paralleling the input ratios of transplanted cells. Secondary transplantation experiments showed stable levels of chimerism in both HoxC4 and HoxB4 groups, indicating that the expansion seen with the MSCV-HoxC4-YFP vector occurred at the HSC level. These results indicate that retroviral-mediated expression of HoxC4, like HoxB4, can cause significant expansion of HSCs in vivo. Because several other Hox genes can cause hematopoietic abnormalities and leukemia when expressed from a retroviral vector, we transplanted lethally irradiated mice with 4x106 cells that were transduced with the MSCV-HoxC4-YFP vector and monitored the animals for survival and complete blood counts. Now, at 33 weeks post transplantation, no tumor formation was observed in mice expressing either the HoxB4 or the HoxC4 vector, and peripheral blood counts have remained normal. Our results show that retroviral overexpression of HoxC4 can induce a significant expansion of the HSCs in vivo, and suggest that expression of HoxC4 may compensate for the loss of HoxB4 in knockout mice. We are currently analyzing the effects of HoxA4 and HoxD4 to determine if they share the same functional characteristics, and are also determining whether HoxB4 and HoxC4 are modulating the same downstream genes using microarray analysis of transduced murine bone marrow cells.

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Differential Expression of c-Kit Identifies Hematopoietic Stem Cells with Variable Self-Renewal Potential.

Blood ◽

10.1182/blood.v120.21.2325.2325 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2325-2325

Author(s):

Joseph Yusup Shin ◽

Wenhuo Hu ◽

Christopher Y. Park

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Differential Expression ◽

Peripheral Blood ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2325 Hematopoietic stem cells (HSC) can be identified on the basis of differential cell surface protein expression, such that 10 out of 13 purified HSC (Lin−c-Kit+Sca-1+CD150+CD34−FLK2−) exhibit long-term reconstitution potential in single-cell transplants. HSCs express c-Kit, and interactions between c-Kit and its ligand, stem cell factor, have been shown to be critical for HSC self-renewal; however, HSCs express a log-fold variation in c-Kit levels. We hypothesized that differing levels of c-Kit expression on HSCs may identify functionally distinct classes of HSCs. Thus, we measured the function and cellular characteristics of c-Kithi HSCs and c-Kitlo HSCs (defined as the top 30% and bottom 30% of c-Kit expressors, respectively), including colony formation, cell cycle status, lineage fates, and serial engraftment potential. In methylcellulose colony assays, c-Kithi HSCs formed 5-fold more colonies than c-Kitlo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kithi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kitlo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kitlo HSCs and 300,000 competitor bone marrow cells exhibited increasing levels of donor chimerism, peaking at a mean of 80% peripheral blood CD45 chimerism by 16 weeks post-transplantation, whereas mice transplanted with c-Kithi HSCs reached a mean of 20% chimerism (p<0.00015). Evaluation of the bone marrow revealed an increase in HSC chimerism from 23% to 44% in mice injected with c-Kitlo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kithi HSC-transplanted mice (P<0.00021). Levels of myeloid chimerism in the bone marrow and peripheral blood were not significantly different during the first 4 weeks following transplantation between mice transplanted with c-Kithi or c-Kitlo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kithi and c-Kitlo HSCs, indicating that differential homing is not the reason for the observed differences in long-term engraftment. Donor HSCs purified from mice transplanted with c-Kithi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kitlo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kithi HSC exhibited a chimerism level of 40% to 3% from week 4 to 8 post-secondary transplant, whereas chimerism levels remained at 6% in mice injected with c-Kitlo HSC. These results indicate that c-Kithi HSCs exhibit reduced self-renewal capacity compared with c-Kitlo HSCs, and that the differences in c-Kithi and c-Kitlo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kithi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kitlo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kithi HSC. Consistent with this finding, the transplanted c-Kithi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kitlo HSC. To determine the potential mechanisms underlying the transition from c-Kitlo to c-Kithi HSCs, we assessed the activity of c-Cbl, an E3 ubiquitin ligase known to negatively regulate surface c-Kit expression in a Src-dependent manner. Flow cytometric analysis revealed 6-fold more activated c-Cbl in freshly purified c-Kitlo HSC compared to c-Kithi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kitlo HSCs. Mice treated for nine days with Src inhibitors, which inhibit c-Cbl activity, experienced a 1.5-fold and 2-fold increase in the absolute number of c-Kithi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kithi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kithi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kitlo HSC represent a population of cells enriched for long-term self-renewal capacity, characterization of this cell population provides an opportunity to better understand the mechanisms that regulate HSC function. 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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