C-MYC and NF-E2 Genes Regulate Proplatelet Formation in Cultured Megakaryocytes with Different Levels of Polyploidization.

Abstract Abstract 2289 Introduction: One of the goals of stem cell medicine is the production of platelets from stem cells for transfusion therapy. The process of platelet production from megakaryocytes is complex and depends on a wide spectrum of internal and external stimuli. The degree of cell polyploidization, amount of demarcation membrane system (DMS), and the cells capacity to form proplatelets are major determines of the quantity of platelets released by each megakaryocyte. Both polyploidization and proplatelet formation depend on the synchronous function of microtubules and actin/myosin. This complex machinery is regulated in part by the actions of the C-Myc and NF-E2 promoters. These promoters play a crucial role during polyploidization and proplatelet formation. We have examined the potential role of these promoters in the efficacy of various chemical and cell culture-based methods of driving hematopoietic stem cells to megakaryocyte differentiation and platelet production. Methods: Human cord blood derived CD34+ cells were isolated and 5×104cells were cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 12 days. Megakaryocytes were cultured along with reagents that inhibit distinct mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Combinations of reagents were used in order to determine their interactions and to maximize megakaryocyte ploidy. The DMS was analyzed and quantified with Di-8 ANEPPS in flow cytometry and morphology was studied with Electron Microscopy (EM). On day 12 proplatelets were analyzed with an inverted microscope and platelets were counted with an Advia 120 cell counter. Total RNA was extracted and analyzed for C-Myc and NF-E2 mRNA by QRT-PCR. Results: All treatments increased megakaryocyte ploidy, except MLCKI. RRI reached the highest ploidy (p=0.0007), followed by NIC (p=0.003), SI (p=0.026) and ABI (p=0.018). Combinations all significantly increased polyploidization; however the only combination that equaled RRI alone was the combination of all of the other inhibitors (p<0.0001). EM showed normal megakaryocyte structure. DMS quantification showed that higher ploidy megakaryocytes had more extensive DMS (p<0.02). Higher ploidy megakaryocytes released more proplatelets and platelets than control and low ploidy cells (p=0.01). Treatments that had the lowest increase proplatelet formation were ABI and the combination of NIC-SI-ABI-MLCKI. RRI-NIC resulted in the highest release of proplatelet formation. C-Myc gene expression was down-regulated in most of the treatments. NF-E2 expression was up-regulated in megakaryocytes treated with ABI and in the combination of NIC-SI-ABI-MLCKI, but not in other treatments. Conclusion: RRI proved to be the most effective agent in driving megakaryocyte polyploidization. The summation of effects of all of the other cytokinesis inhibitors increased polyploidization only to the same extent as RRI. Light and EM imaging showed that the cultured megakaryocytes were morphologically normal. Higher ploidy megakaryocytes with an extended DMS were able to extend more proplatelets and release more platelets in culture, especially after treatment with RRI-NIC. Gene expression analysis showed that down-regulation of C-Myc in late stages of development was correlated with increased proplatelet formation and platelet release. NF-E2 up-regulation on day 12 was associated with ABI treatment and decreased proplatelet formation in cultured megakaryocytes. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v116.21.1209.1209 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1209-1209

Author(s):

Chun Shik Park ◽

Koramit Suppipat ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

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

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Efficiency of Leukemic Stem Cell Separation From Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.4997.4997 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4997-4997

Author(s):

Lu Zhang ◽

Susanne Hofmann ◽

Lars Bullinger ◽

Marlies Goetz ◽

Markus Wiesneth ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Cell Sorting ◽

Peripheral Blood ◽

Recovery Rate ◽

Mononuclear Cells ◽

Separation Efficiency ◽

Conflicts Of Interest ◽

Cell Number ◽

Hematopoietic Stem

Abstract Abstract 4997 Leukemic stem cells (LSC) are the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSCs probably represent a critical target for therapeutic options. Xenograft models confirmed repeatedly that LSCs from AML patients reside mainly in CD34+CD38- compartment of leukemic blasts which makes the pure and efficient separation of this population mandatory to identify new therapeutic drugs to target LSC in different AML subtypes. We separated this subpopulation out of primary AML peripheral blood mononuclear cells (PBMC) samples with fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) and compared the efficiency of both methods. In order to profile gene expression of LSCs and hematopoietic stem cells (HSC) MicroArrays were performed using GeneChip Human Genome U133 Plus 2.0 from Affymetrix. The CD34+CD38- subpopulation was separated from PBMCs of 12 AML patients and 5 healthy volunteers using FACS. Concerning the 12 primary AML samples, the ratio of CD34+CD38- cells ranges between 0.79% and 86.2% using 1–5×107 PBMC for separation. After sorting, the purity of those AML samples increased to 88.4–98.4% while 2×104-3.6×106 cells were obtained. MACS was used to separate 2 representative samples, in which the CD34+CD38- subpopulation was rather small (sample1: 0.78%) or large (sample2: 86.1%). Those sorted subpopulations were compared to the samples sorted via FACS. In order to evaluate separation efficiency in a standardized manner, we defined the recovery rate: (CD34+CD38- cell number obtained /total CD34+CD38- cell number) × 100%. The total CD34+CD38- cell number was calculated through a pre-sorting FACS analysis. For sample 1, MACS resulted in a recovery rate of 4.2–6.4% with a purity of 86.6–90.3%, which is inferior to the recovery rate of 17% and the purity of 92.1% using FACS. For Sample 2, MACS resulted in a recovery rate of 0.4% with a purity of 98.8%, compared to the recovery rate of 11.6% with a purity of 98.1% by FACS. Comparing both methods it is obvious that the purity doesn't differ a lot, but the yield is much higher using FACS. This could represent a powerful tool, when managing rare samples. Finally, by comparing purity and yield, we showed that FACS is the adequate separation method. At the moment MicroArrays are being performed in order to investigate the gene expression profile for 12–15 AML patients and 5 HVs. Taken together, we showed a widely efficient method to routinely separate LSCs from patients with different subtypes of AML. Microarrays, that have been performed, represent a method that allows the comparison of the characteristics of LSCs in different AML subtypes and also of LSCs from bone-marrow with LSCs from peripheral blood and with HVs. These array data analyses are ongoing and will be presented. Disclosures: No relevant conflicts of interest to declare.

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Lgr4-Mediated Potentiation Of Wnt/β-Catenin Signaling Promotes MLL Leukemogenesis Via An Rspo3/Wnt3a-Gnaq Pathway In Leukemic Stem Cells

Blood ◽

10.1182/blood.v122.21.887.887 ◽

2013 ◽

Vol 122 (21) ◽

pp. 887-887 ◽

Cited By ~ 3

Author(s):

Hangyu Yi ◽

Jianlong Wang ◽

Maria Kavallaris ◽

Jenny Yingzi Wang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

G Protein ◽

Colony Formation ◽

Conflicts Of Interest ◽

Active Form ◽

Leukemic Stem Cells ◽

Western Blots ◽

Hematopoietic Stem

Abstract Although the clinical importance of aberrant Wnt/β-catenin signaling has been recognized in various cancers, including MLL-rearranged acute myeloid leukemia (MLL AML), its key tractable pathway components have not yet been discovered in leukemic stem cells (LSC). Our studies have identified an Rspo3/Wnt3a-Lgr4-Gnaq pathway, which significantly potentiates β-catenin signaling in MLL LSC. Genetic and pharmacological targeting of this pathway impairs LSC self-renewal and survival, inhibiting MLL-AF9-induced leukemia progression in vivo. Gene expression analysis of AML patient samples (Nucleic Acids Res, 41:D1034-9, 2013) revealed an approximately 3-fold increase (p=0.00002) in expression of leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) in leukemic cells from patients with MLL AML compared to normal human hematopoietic stem cells (HSC). As recent studies have highlighted a critical link between R-spondin (Rspo)/Lgr4 and Wnt/β-catenin signaling pathways, we hypothesized that up-regulation of Lgr4 is associated with aberrant activation of β-catenin signaling in MLL LSC. We have previously demonstrated that β-catenin is highly expressed in HSC transformed by MLL-AF9 and is lower in HSC transduced with leukemic oncogenes such as Hoxa9/Meis1, while increased β-catenin expression is correlated with a poor survival rate in mice. In this study, western blots confirmed high levels of Lgr4 expression in HSC expressing MLL-AF9 compared to Hoxa9/Meis1. ShRNA-mediated stable knockdown of Lgr4 markedly reduced colony formation of HSC expressing MLL-AF9 by 55-65% (p=0.0001) and significantly prolonged mouse survival (p=0.0019) through its inhibition of endogenous β-catenin expression. This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Furthermore, ectopic expression of Lgr4 alone was not sufficient for triggering the leukemic transformation of HSC but conferred a growth advantage in vivo to HSC expressing Hoxa9/Meis1 and significantly accelerated the onset of Hoxa9/Meis1-induced AML in mice (p=0.0011). These data support an oncogenic role of Lgr4 in promoting tumor formation through activation of β-catenin signaling. As Lgr4 has recently been identified as a receptor for the Rspo family of secreted proteins (Rspo1–Rspo4), we sought to determine if Rspo is a positive regulator of β-catenin signaling in MLL AML. We found that only the combination of Rspo3 and Wnt3a potently enhanced β-catenin signaling in HSC expressing MLL-AF9 whereas Rspo and Wnt3a alone or the combination of Wnt3a with other Rspo had no effects on β-catenin activity. Depletion of Lgr4 completely abolished Rspo3/Wnt3a-induced β-catenin signaling, suggesting Rspo3/Wnt3a potentiating β-catenin signaling through Lgr4. Next, we assessed if Lgr4 signals through G protein pathways. By testing G protein alpha inhibitors in MLL LSC, we demonstrated that G protein alpha-q (Gnaq) was required for maintenance of stem cell properties by chemical suppression of the Gnaq-activated β-catenin pathway with a Gnaq selective inhibitor, which exhibited a 3-fold decrease in colony formation (p=0.0001) and a 4-fold reduction in cell number (p=0.0009), and was sufficient to induce substantial cell differentiation and apoptosis. Treatment with Gnaq inhibitor abolished the effect of Lgr4 on β-catenin transactivation, implicating an Lgr4-Gnaq-β-catenin signaling pathway in MLL LSC. Microarray analysis of gene expression confirmed enrichment of genes related to cancer cell proliferation, migration and growth, as well as enrichment of Wnt target genes in LSC expressing Lgr4. Taken together, we report here an Rspo3/Wnt3a-Lgr4-Gnaq-β-catenin signaling circuit in MLL leukemogenesis. Interference with components of the circuit can block β-catenin signaling and perturb leukemia development. Thus, our findings provide potential therapeutic targets in treating LSC-based hematological malignancy driven by Wnt/β-catenin signaling. Disclosures: No relevant conflicts of interest to declare.

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A New Flowcytometry-Based Method to Discriminate Malignant From Normal Stem Cells in CML.

Blood ◽

10.1182/blood.v114.22.3245.3245 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3245-3245

Author(s):

Jeroen J.W.M. Janssen ◽

Wendy Deenik ◽

Karlijn G.M. Smolders ◽

Monique Terwijn ◽

Angele Kelder ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Risk Scores ◽

Fish Analysis ◽

Leukemic Stem Cells ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Culture Techniques

Abstract Abstract 3245 Poster Board III-182 Tyrosine kinase inhibitor (TKI) insensitivity of CML hematopoietic stem cells prevents eradication of the disease by these drugs and is presumably implicated in development of TKI resistance. Probably, improvement of treatment results will involve leukemic stem cell directed therapy. Therefore, more knowledge of stem cell specific targets would be instrumental. Previously, leukemic stem cells could only be identified indirectly by using culture techniques. We developed a new flowcytometric approach that enables to directly distinguish CML stem cells from their normal counterparts within single patient samples. In 24 newly diagnosed CML patients CML CD34+CD38- stem cells could be discriminated from normal stem cells by higher CD34 and CD45 expression and different forward/sideward light scatter properties, reflecting differences in size and granularity. In addition, aberrant expression of CD7, CD11b and CD56 was demonstrated on malignant stem cells, allowing clear discrimination from benign stem cells, that were always negative for these markers. Above all, in all tested CML patients we were able to demonstrate that high CD90 expression is a specific feature of CML stem cells, while CD90 expression is low on their normal counterparts. FISH analysis on FACS sorted cells proved that populations were BCR-ABL positive (in case of high CD34 and CD45 expression and high CD90 expression) or negative (in case of low CD34 and CD45 expression and low CD90 expression), while long term liquid culture assays with subsequent CFU assays and FISH analysis proved their malignant/normal stem cell character. Patients with a large proportion of non-leukemic stem cells had significantly lower clinical risk scores (Sokal, Euro) than patients with few remaining normal stem cells. This new technique will expand our possibilities to identify new CML stem cell specific targets and may improve efficacy assessment of CML treatment as well. Disclosures No relevant conflicts of interest to declare.

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Platelet Production System Using An Immortalized Megakaryocyte Cell Line Derived From Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.2.2 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2-2 ◽

Cited By ~ 2

Author(s):

Sou Nakamura ◽

Naoya Takayama ◽

Hiromitsu Nakauchi ◽

Koji Eto

Keyword(s):

Stem Cells ◽

Cell Line ◽

Pluripotent Stem Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Negative Regulator ◽

Vector System ◽

Transfusion Therapy ◽

Platelet Production ◽

Promising Source

Abstract Abstract 2 Human induced pluripotent stem cells (hiPSCs) are a promising source of blood cells, including platelets, for transfusion. However, there remains a need for: 1) a method to obtain large numbers of cells, 2) a system to provide cells of a predefined quality, and 3) a method to overcome the storage limitations of platelets caused by their short shelf life. To address these issues, we attempted to establish an immortalized megakaryocyte progenitor cell line derived from hiPSCs. We recently showed that the temporal profile of c-MYC activation during megakaryopoiesis is critical for normal platelet production from hiPSCs; that is, peak activation of c-MYC in megakaryocyte progenitors must be followed by a reduction of c-MYC expression for further maturation (Takayama et al. J Exp Med, 2010). Mechanistic analysis revealed that overexpression (O/E) of c-MYC increased megakaryocyte numbers but also induced apoptosis and senescence. Here we demonstrate that this phenomenon is primarily regulated by induction of the INK4A and ARF genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/E and BCL-XL (negative regulator of caspase family) O/E, and c) c-MYC O/E and BMI1 (negative regulator for both INK4A and ARF genes) O/E in hematopoietic progenitors derived from human embryonic stem cells (hESCs), we found that only c-MYC and BMI1 O/E (protocol b) increased numbers of CD41a+/CD42b+ non-polyploid megakaryocytes in an exponential manner for over 3 months. Neither c-MYC O/E and p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficient to maintain an increase in the megakaryocyte population, which suggests that down-regulation of INK4A and ARF contributes mainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thus appears that we were able to establish an immortalized megakaryocyte cell line (MKCL). As mentioned, a decline in c-MYC activation during hiPSC-derived megakaryocyte maturation is required for generation of functional CD41a+/CD42b+ platelets in vitro. Excessively sustained c-MYC expression in megakaryocytes was accompanied by increased ARF and INK4A expression and decreased GATA1 and NF-E2 expression, eventually leading to megakaryocyte senescence and apoptosis, and CD41a+/CD42blow/- platelet generation. By using an inducible expression vector system with c-MYC and BMI1 in this context, the MKCL was capable of generating polyploid megakaryocytes (>8N; 40%). The MKCL also subsequently showed proplatelet formation leading to the release of “functional” CD41a+/CD42b+ platelets. Furthermore, following transfusion of 6×108 platelets originally derived from the our immortalized MKCL into immunodeficient NOG mice, the platelets appeared to exhibit normal circulation a high degree of chimerism (human CD41a/ human CD41a + mouse CD41 was ∼67% at 2 hrs and 26% at 24 hrs post transfusion). We therefore propose that establishment of immortalized MKPCs through gene manipulation could potentially provide a stable supply of platelets at a predefined quality and quantity for transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

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Platelet Production System Using An Immortalized Megakaryocyte Cell Line Derived From Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.mrg-1.mrg-1 ◽

2011 ◽

Vol 118 (21) ◽

pp. MRG-1-MRG-1

Author(s):

Sou Nakamura ◽

Naoya Takayama ◽

Hiromitsu Nakauchi ◽

Koji Eto

Keyword(s):

Stem Cells ◽

Cell Line ◽

Pluripotent Stem Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Negative Regulator ◽

Vector System ◽

Transfusion Therapy ◽

Platelet Production ◽

Promising Source

Abstract Abstract MRG-1 Human induced pluripotent stem cells (hiPSCs) are a promising source of blood cells, including platelets, for transfusion. However, there remains a need for: 1) a method to obtain large numbers of cells, 2) a system to provide cells of a predefined quality, and 3) a method to overcome the storage limitations of platelets caused by their short shelf life. To address these issues, we attempted to establish an immortalized megakaryocyte progenitor cell line derived from hiPSCs. We recently showed that the temporal profile of c-MYC activation during megakaryopoiesis is critical for normal platelet production from hiPSCs; that is, peak activation of c-MYC in megakaryocyte progenitors must be followed by a reduction of c-MYC expression for further maturation (Takayama et al. J Exp Med, 2010). Mechanistic analysis revealed that overexpression (O/E) of c-MYC increased megakaryocyte numbers but also induced apoptosis and senescence. Here we demonstrate that this phenomenon is primarily regulated by induction of the INK4A and ARF genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/E and BCL-XL (negative regulator of caspase family) O/E, and c) c-MYC O/E and BMI1 (negative regulator for both INK4A and ARF genes) O/E in hematopoietic progenitors derived from human embryonic stem cells (hESCs), we found that only c-MYC and BMI1 O/E (protocol b) increased numbers of CD41a+/CD42b+ non-polyploid megakaryocytes in an exponential manner for over 3 months. Neither c-MYC O/E and p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficient to maintain an increase in the megakaryocyte population, which suggests that down-regulation of INK4A and ARF contributes mainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thus appears that we were able to establish an immortalized megakaryocyte cell line (MKCL). As mentioned, a decline in c-MYC activation during hiPSC-derived megakaryocyte maturation is required for generation of functional CD41a+/CD42b+ platelets in vitro. Excessively sustained c-MYC expression in megakaryocytes was accompanied by increased ARF and INK4A expression and decreased GATA1 and NF-E2 expression, eventually leading to megakaryocyte senescence and apoptosis, and CD41a+/CD42blow/− platelet generation. By using an inducible expression vector system with c-MYC and BMI1 in this context, the MKCL was capable of generating polyploid megakaryocytes (>8N; 40%). The MKCL also subsequently showed proplatelet formation leading to the release of “functional” CD41a+/CD42b+ platelets. Furthermore, following transfusion of 6×108 platelets originally derived from the our immortalized MKCL into immunodeficient NOG mice, the platelets appeared to exhibit normal circulation a high degree of chimerism (human CD41a/ human CD41a + mouse CD41 was ∼67% at 2 hrs and 26% at 24 hrs post transfusion). We therefore propose that establishment of immortalized MKPCs through gene manipulation could potentially provide a stable supply of platelets at a predefined quality and quantity for transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

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Fractionated Osteoblastic Niche Cells Enhances the Homing Activity of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.560.560 ◽

2009 ◽

Vol 114 (22) ◽

pp. 560-560

Author(s):

Fumio Arai ◽

Yuka Nakamura ◽

Kentaro Hosokawa ◽

Isao Kobayashi ◽

Hiroko Iwasaki ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

The Other ◽

Quiescent State ◽

Mesenchymal Progenitor Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Other Hand

Abstract Abstract 560 Hematopoietic stem cells (HSCs) retain quiescent state in the adult bone marrow (BM), interacting with specialized niches along the endosteum (osteoblastic niche) and in perivascular sites adjacent to endothelial and reticular cells (perivascular niche). We have previously reported that the regulations of HSCs in the osteoblastic niche through the receptor-cytokine, cell-to-cell, and cell-to-extracellular matrix (ECM) interactions are critical for the maintenance of cell cycle quiescence. However, osteoblast population is heterogeneous in terms of the degree of differentiation and function of each cell. In this study, we characterized the cellular components of osteoblastic niche and examined the ability for the maintenance of HSCs. We found that CD45–CD31–Ter119– endosteal population can be subdivided into three fractions, ALCAM+Sca-1–, ALCAM–Sca-1+, and ALCAM–Sca-1– cells. Expression of osteoblast markers and differentiation potential of these three fractions revealed that osteoblasts were enriched in the Sca-1– populations. On the other hand, ALCAM–Sca-1+ cells had characteristics of mesenchymal progenitor cells (MPCs). Microarray analysis showed that ALCAM+Sca-1– fraction tend to highly express the genes related to cell-to-cell or cell-to-ECM adhesions, such as N-cadherin, Osteopontin, and Alcam, compared to other fractions, indicating that this population might regulate HSCs through the physiological interactions. On the other hand, ALCAM–Sca-1+ fraction highly expressed the genes related to growth factor and cytokine that are involved in the regulation of both quiescence and proliferation of long-term-HSCs, such as Angiopoietin-1, Flt3l, Cxcl12, Thrombopoietin, and Kitl. These data rise the possibility that multiple cell populations in endosteum collaborate as a complex to regulate the balance between HSC proliferation and quiescence in the endosteum in adult BM. Next we examined whether these fractionated cells could maintain the long-term repopulation (LTR) activity of HSCs in vitro. BM Lin–Sca-1+Kit+ (LSK) cells were cocultured with each fractionated endosteal population without growth factors. After 2 days of coculture, Ly5.1+ cells were sorted and transplanted into lethally irradiated mice. We found that all three fractions maintained LTR-activity of LSK cells. In particular, LSK cells cocultured with ALCAM+Sca-1– cells showed significantly higher LTR-activity compared to that cocultured with other fractions. Then we analyzed the gene expression implicated in the homing and lodgment of HSCs. Q-PCR array analysis revealed that upregulation of Cxcr4, integrins, Cd44, N-cadherin and Alcam was induced in LSK cells cocultured with Sca-1– populations. In particular, ALCAM+Sca-1– cells significantly upregulated N-cadherin expression in HSCs. These data suggest that osteoblasts increased homing activity of HSCs in culture or enriched cell population that had high homing activity. Furthermore, the cell-to-cell adhesion between HSCs and ALCAM+Sca-1– cells enhanced the interaction of HSCs with niche complex and maintained self-renewal activity of HSCs. Disclosures: No relevant conflicts of interest to declare.

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CITED2 Cooperates with Low PU.1 and DNMT3A to Maintain Self-Renewal in Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.309.309 ◽

2015 ◽

Vol 126 (23) ◽

pp. 309-309

Author(s):

Hein Schepers ◽

Patrick Korthuis ◽

Marjan Geugien ◽

Jennifer Jaques ◽

Tihomira I. Todorova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Stem Cells ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Gene Expression Pattern ◽

Dependent Manner ◽

Self Renewal ◽

Hematopoietic Stem ◽

Specific Subset

Abstract CITED2 has a conserved role in the maintenance of normal hematopoiesis. We have recently shown that ~70% of acute myeloid leukemia (AML) patients display enhanced CITED2 expression levels. Interfering with CITED2 expression is detrimental for leukemia maintenance in vitro and in vivo, demonstrating that CITED2 is critically important for the survival of leukemic stem cells (LSCs). Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) led to significantly better human engraftment in transplanted NSG mice, consistent with the maintenance of very primitive lin- CD34+ CD38- CD90+ CD45RA- HSCs within the bone marrow 28 weeks after transplantation. Although the CITED2-engrafted mice displayed enlarged spleens, blood development appeared normal, as measured through myeloid, B and T cell staining. This indicates that CITED2 as a single hit is not sufficient to transform human CD34+ cells. CITED2 expression frequently coincides with low expression of the myeloid transcription factor PU.1, suggesting that combined effects, rather than single events are important during AML development. To investigate this, we combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and studied hematopoietic development. CITED2 increased the percentage of immature CD34+ CD38- cells 5-fold, which was not further increased by the additional downregulation of PU.1. However, functional analysis through limiting dilution LTC-iC assays indicated that combining PU.1 down-, with CITED2 upregulation led to a synergistic 8.5-fold increase in LTC-iC frequency, whereas only changing PU.1 or CITED2 induced a respective 1.4 to 3-fold change in HSC frequency. To more stringently assess self-renewal, we cultured transduced cells for 4 weeks on MS5 cells under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently performed CFC assays. Whereas after 4 weeks all groups displayed similar colony numbers, secondary and tertiary replatings demonstrated that self-renewal could only be maintained for more than 10 weeks when CITED2 upregulation was combined with PU.1 downregulation. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+ CD38- HSCs, as replating of CD34+ CD38+ progenitor-derived colonies did not yield new CFCs. In order to investigate the underlying mechanisms, we performed transcriptome analysis on human HSCPs after knockdown of PU.1, overexpression of CITED2 or the combination of both. PU.1Low-CITED2High cells displayed a gene expression pattern different from the PU.1Low or CITED2High only cells, suggesting that the two events have synergistic effects. Some genes, like HLX and SF3B1 have been shown to cause or are mutated in AML, demonstrating that the synergistic changes are related to AML. When comparing the differentially regulated genes in the PU.1Low -CITED2High cells to the gene expression in the Hemaexplorer database, a similar pattern was observed, when compared between AML and normal cells. In order to investigate the effects of the PU.1low CITED2high combination on AML development, we resorted to a PU.1-dependent mouse model of AML development. CITED2 expression in BM cells from PU.1KD/KD mice (in which deletion of an Upstream Regulatory Element leads to an 80% downregulation of PU.1), led to a steady increase of GFP+ cells over time as compared to control cells and demonstrated a dramatic expansion of Gr-1+ Mac-1+ cells, a hallmark of AML in these mice. This suggests that CITED2 contributes to a faster progression towards AML upon lowering of PU.1. To identify if our model corresponds to AMLs with a specific subset of mutations, we clustered publically available AML data (TCGA), based on the gene expression changes in the PU.1Low -CITED2High cells. The majority of AMLs clustered together in 2 groups, in which FLT3, p53 and DNMT3A mutations were most prevalent. FLT3 mutations, through its activation of STAT5, are consistent with high CITED2 expression, whereas p53 mutations are consistent with our data indicating that CITED2 loss regulates HSCs in a p53-dependent manner. The presence of DNMT3A mutations suggests that DNA methylation changes collaborate with high CITED2 and low PU.1 during leukemogenesis. This is currently under investigation. In summary, our data imply that CITED2, low PU.1 and potentially changes in DNA methylation all contribute to maintenance of self-renewal and leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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SETD2, a H3K36 Lysine Methyltransferase, Is Essential for Adult Normal Hematopoiesis and Leukemia Stem Cells

Blood ◽

10.1182/blood.v128.22.1055.1055 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1055-1055

Author(s):

Yile Zhou ◽

Yunzhu Dong ◽

Jiachen Bu ◽

Xiaomei Yan ◽

Yoshihiro Hayashi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

The Other ◽

Leukemia Stem Cells ◽

Transcriptional Networks ◽

Loss Of Function ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are characterized by their capability for self-renewal and multi-potency. Hematopoiesis is dynamically controlled by the interplay between epigenetic and transcriptional networks. Dysregulation of these networks can lead to unfitness of hematopoiesis, cell transformation, and hematological diseases. The human SETD2 gene was originally isolated from HSCs and progenitors. SETD2 is a histone methyltransferase, which specifically catalyzes tri-methylation of histone 3 lysine 36 (H3K36me3). SETD2 functions as a tumor suppressor, as loss-of-function mutations have been identified in many cancers. However, the role of SETD2 in hematopoiesis has not been fully understood. To assess the function of Setd2 in hematopoiesis, we generated three Setd2 mouse alleles with Crispr/CAS9 technology; Setd2F2478/WT knock-in, Setd2Exon6-Δ/WT, and Setd2-Exon6flox/flox/Mx1-Cre conditional knockout alleles, as homozygous Setd2 mutation showed embryonic lethality. Setd2-F2478 point mutation, which is located in the SRI domain, can express SETD2 mutant protein but completely lose the interaction with RNA pol II. Setd2Exon6-Δ/WT allele results in a frame shift and nonsense mediated decay of Setd2 mRNA and protein. After induction of excision with pIpC injection, Setd2-exon6flox/flox/Mx1-Cre+ (Setd2Exon6-Δ/Δ) mice showed severe anemia, increased platelet count, and a reduction in bone marrow (BM) cellularity compared to wild-type (WT) mice, while Setd2F2478/WT and Setd2Exon6-Δ/WT mice did not show any obvious hematological changes. The Lin- Sca-1+ c-Kit+ (LSK) population in Setd2Exon6-Δ/Δ mice was 2.5-fold decreased compared to those in WT, while the LSK populations in Setd2F2478/WT and Setd2Exon6-Δ/WT mice were comparable with those in WT. Interestingly, all three of these Setd2 mutant alleles showed a higher frequency of Lin- Sca-1- c-Kit+ (LK) cells in the BM. In the LK populations, we found an increased CMP population in Setd2F2478/WT and Setd2Exon6-Δ/WT mice; of note, the CMP population in the Setd2Exon6-Δ/Δ mice had disappeared while the MEP population expanded with higher expression of CD16/32. Next, to assess the function of the HSPCs, we performed CFU assays and competitive bone marrow transplantations (CBMT). Consistent with our phenotypic findings, the number of colonies derived from Setd2F2478/WT and Setd2Exon6-Δ/WT BM cells was increased in the first two passages, while the number of colonies derived from Setd2Exon6-Δ/Δ mice was significantly decreased. In CBMT, we found that mice transplanted with Setd2Exon6-Δ/Δ BM cells showed anemia and an impaired BM reconstitution, compared to the control (p = 0.0002). On the other hand, the Setd2F2478/WT and Setd2Exon6-Δ/WT models showed comparable capabilities of BM reconstitution. Taken together, these results suggest that Setd2 has an essential role in the maintenance of adult hematopoiesis. SETD2 mutations (mainly one allele mutation) have been frequently identified in acute leukemia, especially in about 22% of MLL leukemia. To understand the role of SETD2 in leukemic stem cells, Setd2 mutant mice were bred with the Mll-AF9 knock-in mouse. The Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice showed higher frequencies of LK and LSK populations compared to Mll-AF9 mice, indicating that Setd2 mutations may increase the stemness of leukemia stem cells (LSCs). The cells derived from Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice resulted in a significantly higher yield of colonies and growth advantage in serial replating CFU assay compared to the cells derived from Mll-AF9 mice. After BMT of equal numbers of cells from Mll-Af9 or Mll-AF9/ Setd2F2478/WT mice into recipient mice, the Mll-AF9/ Setd2F2478/WTBMT mice developed leukemia with significantly shortened latencies compared with MLL-Af9 BMT mice. In conclusion, our data suggests that Setd2 plays an important role in maintaining normal HSPCs. Half the doses of Setd2 can still maintain the normal hematopoiesis while a total loss of Setd2 leads to a failure of hematopoiesis. In leukemia, heterozygous mutants of Setd2 can accelerate leukemogenesis by expanding LSCs. Whether the remaining WT allele is required for leukemia maintenance is unclear. Further reduction of Setd2 levels, or complete deletion of the other WT allele, may diminish SETD2-mutated leukemia. Such tumor vulnerability can be explored as a therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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The Methylation-Sensitive Enhancer Derare Maintains Hematopoietic Stem Cells through Regulation of Hoxb Cluster

Blood ◽

10.1182/blood.v128.22.725.725 ◽

2016 ◽

Vol 128 (22) ◽

pp. 725-725

Author(s):

Pengxu Qian ◽

Bony De Kumar ◽

Youngwook Ahn ◽

Christof Nolte ◽

Ariel Paulson ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Regulatory Elements ◽

Genetic Regulatory Networks ◽

Hematopoietic Stem ◽

Apoptosis Pathway ◽

Fold Reduction

Abstract The balance between self-renewal and multilineage differentiation in hematopoietic stem cells (HSCs) is orchestrated by genetic regulatory networks, which are constituted by hundreds of thousands of cis-regulatory elements, such as promoters, enhancers, insulators, etc. Aberrant mutations in these cis-acting modules and their trans-acting factors have been frequently found in hematopoietic malignancies including leukemia. Although next-generation sequencing technologies have identified comprehensive maps of these modules, much remains unknown about their physiological functions and underlying mechanisms in HSC maintenance and leukemogenesis. Our transcriptome analysis in 17 murine hematopoietic cell types, including HSCs, committed progenitors and lineage cells, showed that most of HoxB cluster genes were predominantly enriched in the permanently reconstituting long-term (LT) HSCs. Interestingly, one of the two putative enhancers within HoxB cluster, identified by H3K27ac ChIP-Seq analysis, shared the same sequence as the retinoic acid responsive element DERARE, which was recently reported to regulate multiple HoxB gene expression in the central nervous system. To test whether DERARE is required for normal hematopoiesis, we utilized the DERARE knockout mouse and found that homozygous deletion of DERARE led to 2-fold reduction in both the frequency and absolute number of LT-HSCs. Functionally, limiting dilution, competitive repopulating unit assays showed a 2.5-fold decrease in functional HSCs of DERAREΔ mice compared to wildtype control. We further performed serial transplantation and observed a 4.3-fold reduction of repopulation rate after secondary transplantation of DERAREΔ HSCs, indicating long-term reconstitution capacity was impaired. Mechanistically, RNA-Seq in LT-HSCs from DERAREΔ mice exhibited significantly enriched apoptosis pathway in DERAREΔ compared with that from Wt control. Furthermore, DNA methylation analysis showed gradually gained methylation on DERARE during HSC differentiation, which is negatively correlated with HoxB cluster gene expression, suggesting DERARE might be a methylation-sensitive enhancer to control HoxB genes. Moreover, HoxB gene expression is markedly upregulated in Dnmt3a KO HSCs, indicating that Dnmt3a is responsible for the DERARE methylation in HSCs. Finally, the analysis of clinical data from acute myeloid leukemia 200 patients in the Cancer Genome Atlas project revealed that lowly methylated DERARE was significantly correlated with overexpression of HoxB genes, high cytogenetic risk, and poor prognosis, suggesting abnormal regulation of DERARE contributes to leukemogenesis. Collectively, our study demonstrates the essential roles of the cis-regulatory element DERARE in both maintenance of LT-HSCs and contribution to leukemogenesis through regulation of HoxB cluster genes. Disclosures No relevant conflicts of interest to declare.

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