Mir-126 Governs Human Leukemia Stem Cell Quiescence and Chemotherapy Resistance

Abstract miRNA expression is deregulated in human acute myeloid leukemia (AML), however the impact of altered post-transcriptional programs on the genesis and maintenance of leukemia stem cells (LSC) remains undefined. In order to elucidate the functional role of miRNA in LSC and identify relevant miRNA candidates, we performed global miRNA profiling on sorted cell subpopulations from 16 AML patient and 3 umbilical cord blood samples (Eppert et al, Nature Medicine 2011). Supervised analysis guided by the ability of each sub-population to initiate leukemic engraftment after xenotransplantation into immune-deficient mice generated a unique miRNA signature. miR-126, a miRNA that we previously demonstrated to have a conserved role in maintaining hematopoietic stem cell (HSC) quiescence (Lechman et al. Cell Stem Cell, 2012), was more highly expressed in LSC-enriched fractions and chosen for further validation. To confirm that miR-126 is a bona fide LSC determinant, we utilized a bidirectional lentiviral reporter vector specific for miR-126 (Gentner et al. Science Translational Medicine, 2010) to sort cells from AML patient samples based on miR-126 bioactivity, and demonstrated that all in vivo leukemia-initiating capacity was confined to cells with elevated miR-126 bioactivity. Lentiviral enforced expression of miR-126 in primary AML patient samples significantly increased LSC frequency (3.5-52.3 fold) as assessed by limiting dilution transplantation assays, while diminishing cell cycle entry, differentiation marker expression (CD14,CD15) and colony forming potential. Sponge-mediated knockdown of miR-126 expression resulted in the opposite effects. These findings suggest that high levels of miR-126 bioactivity support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Moreover, by preserving LSC quiescence miR-126 promoted chemotherapy resistance, in part through suppression of CDK3, a gatekeeper of G0 to G1 cell cycle transit. Enforced expression of CDK3 partially rescued the functional consequences of supra-physiological levels of miR-126 bioactivity, rendering previously resistant LSC susceptible to killing by AraC/Daunorubicin combination chemotherapy. Our human LSC miRNA signature, optimized by regression analysis on a cytogenetically normal AML patient cohort, was prognostic for survival in a large independent AML patient cohort (Ley et. al N Engl. J Med, 2013) further validating the clinical significance of miRNA as stem cell determinants. Furthermore, miRNA-126 alone was prognostic for survival in two independent cohorts of AML patients with normal cytogenetics. These data demonstrate a mechanistic role for miR-126 in governing intrinsic LSC properties and establish miR-126 as a critical biomarker for clinical outcome. Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

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Hematopoietic Stem Cells Have an Intrinsic Expansion Limit

Blood ◽

10.1182/blood.v120.21.4749.4749 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4749-4749

Author(s):

Shanti Rojas-Sutterlin ◽

André Haman ◽

Trang Hoang

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Chemotherapy Regimen ◽

Gene Dosage ◽

Hematopoietic Stem ◽

Cell Functions ◽

Cell Pool ◽

Stem Cell Pool

Abstract Abstract 4749 Hematopoietic stem cell (HSC) transplantation is the first successful cellular therapy and remains the only treatment providing long-term cure in acute myeloblastic leukemia. At the apex of the hematopoietic system, quiescent HSCs are spared by chemotherapeutic treatments that target proliferating cells and therefore can regenerate the entire blood system of a patient after drug exposure. Nevertheless, the consequence of repeated chemotherapy regimen on HSC function remains to be clarified. We previously showed that Scl/Tal1 gene dosage regulates HSC quiescence and functions when transplanted at limiting dilutions (Lacombe et al., 2010). In the present study, we investigate how massive expansion in vivo influences stem cell functions. To address this question, we optimized a protocol based on 5-fluorouracil (5-FU), an antimetabolite that has been used to treat colon, rectum, and head and neck cancers. In addition, we used Scl+/− mice to address the role of Scl in controlling HSCs expansion post-5-FU. We show that within 7 days following 5-FU treatment, HSCs exit quiescence and enter the cell cycle. To deplete cycling HSCs, we injected a second dose of 5-FU and showed that the stem cell pool was disseminated. Nonetheless, the remaining HSCs proliferated extensively to re-establish the HSC pool, which was twice larger than that of untreated mice. At this point, most HSCs have exited the cell cycle and were back to quiescence. Despite a near normal stem cell pool size and a quiescent status, HSCs from these 5-FU treated mice could not compete against untreated cells to regenerate the host in transplantation assays. Furthermore, we show that this extensive proliferation in vivo severely impaired the clonal expansion of individual HSC as measured by the mean activity of stem cell (MAS). Our results demonstrate that HSCs lose their competitive potential after two 5-FU treatments, suggesting that HSCs have an intrinsic expansion limit beyond which their regenerative potential is impaired. In addition, Scl is haplodeficient for cell cycle entry and cell division but Scl gene dosage does not affect this expansion limit. Therefore, our data dissociate the control of HSC expansion under extensive proliferative stress from cell cycle control during steady state. We surmise that chemotherapy regimen based on repeated administration of 5-FU or other antimetabolites are likely to severely impair long-term stem cell functions. Disclosures: No relevant conflicts of interest to declare.

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The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Blood ◽

10.1182/blood.v122.21.1175.1175 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1175-1175

Author(s):

Stefanie Kreutmair ◽

Anna Lena Illert ◽

Rouzanna Istvanffy ◽

Christina Eckl ◽

Christian Peschel ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Spleen Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mitotic Entry ◽

Stem Cell Maintenance ◽

F Box Protein

Abstract Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells. Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

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Ibrutinib for Bridging to Allogeneic Hematopoietic Stem Cell Transplantation (alloHCT) in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL) Is Safe and Effective: First Results of a Survey By the Chronic Malignancy and the Lymphoma Working Parties of the EBMT

Blood ◽

10.1182/blood.v128.22.4657.4657 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4657-4657 ◽

Cited By ~ 5

Author(s):

Peter Dreger ◽

Mauricette Michallet ◽

Jennifer Hoek ◽

Ariane Boumendil ◽

Mohamad Sobh ◽

...

Keyword(s):

Stem Cell ◽

Research Funding ◽

Kinase Inhibitor ◽

Chronic Gvhd ◽

Disease Status ◽

Observation Time ◽

Lymphocytic Leukemia ◽

Hematopoietic Stem ◽

The Impact

Abstract BACKGROUND: The advent of the Bruton's tyrosine kinase inhibitor ibrutinib has improved the outlook of patients with CLL and MCL failing chemoimmunotherapy (CIT). However, the impact of ibrutinib on the feasibility and safety of a subsequent alloHCT is unknown. Here we present results of the ibrutinib cohort of an ongoing EBMT survey on the outcome of alloHCT following prior exposure to pathway inhibitors (PI) in patients with CLL or lymphoma (EBMT study code LWP 2013-N-03/CMWP 44204425). DESIGN: Eligible were patients aged >18 years registered with the EBMT data office for a planned alloHCT for CLL or lymphoma after pre-exposure to ibrutinib at any time before transplant. Baseline patient, disease, and transplant data were collected from MED-A forms. Centers were requested to provide additional treatment and follow-up information. Statistical analysis used Gray's test to assess the impact of baseline characteristics on the cumulative incidence of relapse (REL) in a competing risk framework. RESULTS: As of July 4, 2016, 38 patients (84% male) were evaluable in the ibrutinib cohort. Diagnosis was CLL in 28 patients, MCL in 9 patients, and follicular lymphoma (FL) in 1 patient. The median age was 51 (33-68) years and the median number of treatment lines prior to ibrutinib 2 (1-9). Eight of the 9 patients with MCL but none of the other patients had a prior autoHCT. Patients had been on ibrutinib for a median of 190 (39-432) days. In 2 patients, ibrutinib had been stopped because of disease progression >100d before transplant, whereas the interval between ibrutinib withdrawal and alloHCT was 15-100d in 30%, 4-14d in 51%, and 0-1d in 14% of the patients. Of the CLL patients, 43% had a TP53 lesion, and 87% and 79% met at least one of the 2007 and 2014 EBMT criteria for high-risk CLL, respectively, including PI failure in 29%. Disease status at alloHCT was sensitive in 78% of the CLL patients, and in 60% of the patients with lymphoma. Conditioning was reduced-intensity in 60% of the transplants and included in-vivo T cell depletion with ATG (71%) or alemtuzumab (11%) in the majority of cases. Donors were identical siblings in 26%, matched unrelated in 66%, and partially matched unrelated in 8%, with PBSC (89%) being the predominant stem cell source (bone marrow 8%, cord blood 3%). The median time to reach neutrophils of >0.5/nl and platelets of >20/nl was 17 (10-26) and 15 (10-46) d post transplant, respectively. Acute GVHD grade 2-4 (3-4) was observed in 37% (10%) of 30 evaluable patients, and limited and extensive chronic GVHD occurred in 24% and 16% of 25 patients at risk. With a median observation time of survivors of 8 (1-24) months, there were only 2 non-relapse deaths, translating into a 1-year non-relapse mortality (NRM) of 6% (95%CI 0-15%). 1-year REL, progression-free survival, and overall survival was 36%, 61%, and 73% for CLL, and 14%, 75%, and 75% for lymphoma. In the 25 evaluable patients with CLL, PI-sensitive compared to refractory disease status at alloHCT tended to be associated with a lower 1-y REL (29% vs 60%; p 0.071), whereas prior PI failure, TP53 status, duration of ibrutinib exposure, interval between ibrutinib withdrawal and alloHCT, and conditioning intensity had no significant impact on REL. CONCLUSIONS: Ibrutinib for bridging to alloHCT for CLL and MCL does not appear to adversely affect engraftment, GVHD risk, and NRM. Patients with CLL still responding to ibrutinib at the time of alloHCT might benefit from ibrutinib bridging as our preliminary results indicate that also after PI exposure sensitive disease translates into a lower risk of relapse. Therefore, ibrutinib may improve the perspective of CIT-refractory patients scheduled for alloHCT. The optimum timing of ibrutinib administration in the interrelation to alloHCT in CLL and MCL needs to be defined by additional studies. Disclosures Dreger: Gilead: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Gilead: Speakers Bureau; Novartis: Consultancy; Roche: Consultancy. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Berg:Celgene: Other: Travel Funding; Astellas: Other: Travel Funding; Alexion: Other: Travel Funding. Niederwieser:Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Montoto:Gilead: Research Funding; Roche: Honoraria. Schetelig:Sanofi: Honoraria.

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Leukemic Stress Targets the mTOR Pathway to Suppress Residual HSC in the BM Microenvironment

Blood ◽

10.1182/blood-2019-125682 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3730-3730

Author(s):

John T Butler ◽

Sherif Abdelhamed ◽

Lina Gao ◽

Jeong Lim ◽

Terzah M Horton ◽

...

Keyword(s):

Protein Synthesis ◽

Conflicts Of Interest ◽

Chemotherapy Resistance ◽

Hematopoietic Cells ◽

Tumor Burden ◽

Mtor Pathway ◽

Hematopoietic Stem ◽

The Impact

The remodeling of the bone marrow (BM) microenvironment that occurs along with the progressive spread of acute myeloid leukemia (AML) cells can be considered a constitutive aspect of leukemogenesis. To date most studies have focused on the functional and in part inflammatory adaptation of stroma, and its potential role in extrinsic chemotherapy resistance. Much less is known about the impact of leukemic stress on residual hematopoietic cells. We previously identified the trafficking of select microRNAs (miRs-) in extracellular vesicles (EVs) between AML cells and hematopoietic progenitor cells (HPC). These studies revealed the mechanism underlying the suppression of HPC function in the AML niche (Hornick, Doron et. al., Science Signaling 2016). Several groups, including ours also noted the relative resistance of residual hematopoietic stem cells (HSC) to elimination from the BM of xenografted animals. In the current study we set out to understand how leukemic stress in the AML xenograft niche shapes HSC fate and function. Using AML cell lines (Molm14, U937, HL60) we established NSG xenografts, systematically tracking peripheral blood AML chimerism to recover murine hematopoietic cells at low or negative tumor burden, and replicating key assays using purified EV for intrafemoral injections. In immunofluorescent studies we initially confirmed the uptake of GFP labeled xenograft-derived EVs across the spectrum of HPC and HSC (KSL/CD150+/CD48-), as well as the successive loss and peripheral displacement of HPCs, and gains in HSC frequency in the leukemic niche. These HSC were found to be enriched for G0 cell cycle status with an increase in phospho- p53, but showed no evidence of apoptosis or senescence. To understand the mechanism underlying their apparent quiescence, we performed in vitro proteomics studies of AML EV exposed HPSC identified downregulation of ribosomal biogenesis pathways. We then confirmed in vivo that residual HSC from AML xenografts experienced a loss of protein synthesis (OPP assay). We next reasoned that deficits in ribosome dysfunction and protein synthesis may reflect deregulation by specific miRNAs highly abundant in AML EV. Here, we had an opportunity to profile EV miRNA from the plasma of 12 unselected AML patients at diagnosis versus 12 control samples, and we confirmed a significant enrichment for specific miRNAs, including miR-1246. Raptor is a component of the mTOR pathway and an annotated target of miR-1246. We demonstrated in a series of experiments that miR-1246 translationally suppresses Raptor and downregulates protein synthesis in residual HSC from AML xenografts. The transfection of synthetic anti-miR1246 sequences on the other hand reversed the effects of AML EV in murine HSC. In aggregate we show that direct crosstalk between AML and hematopoietic cells adds to the adaptive changes that occur in the AML niche. Our experiments suggest a functional significance for EV miRNA that can be detected in AML patient plasma in the regulation of residual BM HSC. More broadly, the mechanisms by which leukemic stress alters hematopoietic function remain underexplored, but our observations suggest that leukemia derived EV contribute to changes in competitive fitness of residual HSC. Disclosures No relevant conflicts of interest to declare.

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Identification and Characterization of Human Leukemia Stem Cell Functional Regulators

Blood ◽

10.1182/blood.v118.21.2955.2955 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2955-2955

Author(s):

Kolja Eppert ◽

Karin G Hermans ◽

John E. Dick

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cord Blood ◽

Hematopoietic Stem Cell ◽

Prognostic Significance ◽

Cell Populations ◽

Human Leukemia ◽

Transcriptional Program ◽

Hematopoietic Stem

Abstract Abstract 2955 Emerging evidence shows that many cancers are organized as cellular hierarchies sustained by a subpopulation of cancer stem cells (CSC). It is thought that CSC properties influence therapy response, disease relapse, and overall survival. However, little is currently known about the molecular pathways that control stem cell behaviour. Our goal is to identify and characterize the molecular regulatory networks that govern the self-renewal and developmental program of human leukemia stem cells (LSC). To expand our understanding of LSC function, we selected genes from our previously established LSC transcriptional program as novel candidate regulators of stem cell function. The LSC transcriptional program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations that were subjected to sensitive in vivo LSC assays. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from functionally defined LSC fractions. Using similar methodology, a hematopoietic stem cell (HSC) enriched gene signature was established from human cord blood. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, revealing the molecular machinery that underlies stemness properties. The LSC and HSC signatures have prognostic significance independent of other factors when validated on a large cohort of cytogenetically normal AML patients. We have begun experiments to determine the role of 17 candidate stem cell regulators using functional in vivo and in vitro assays. We first examined the effect of candidate gene overexpression in normal HSC using xenograft assays. Lineage negative cord blood cells were transduced with lentiviral overexpression vectors, injected into immune-deficient mice and engraftment of human cells was measured after 12 weeks. In the preliminary round of in vivo analysis, over-expression of GPR56 increased the engraftment capability of HSCs in immune-deficient recipients compared to control (p=0.0019). GPR56 is involved in cell adhesion and differentiation and evidence is accumulating for a role in various cancers such as melanoma, esophageal cancer, and glioma. By qPCR analysis we observed that GPR56 mRNA is more highly expressed in both LSC-enriched and primitive normal hematopoietic cell populations, including highly purified HSC, compared to more mature cell populations such as AML blasts and normal lineage committed progenitor cells. In conclusion, our LSC and HSC data established that determinants of stemness influence clinical outcome of AML patients. We have identified multiple novel candidate stem cell-related genes and provided evidence for a role for GPR56 in hematopoietic stem cell regulation. Disclosures: No relevant conflicts of interest to declare.

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A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency

Blood ◽

10.1182/blood-2018-10-881441 ◽

2019 ◽

Vol 133 (20) ◽

pp. 2198-2211 ◽

Cited By ~ 3

Author(s):

Kerstin B. Kaufmann ◽

Laura Garcia-Prat ◽

Qiang Liu ◽

Stanley W. K. Ng ◽

Shin-Ichiro Takayanagi ◽

...

Keyword(s):

Stem Cell ◽

Molecular Mechanisms ◽

Data Sets ◽

Human Leukemia ◽

Human Cord Blood ◽

Orphan Gene ◽

Hematopoietic Stem ◽

Concomitant Reduction ◽

Stem And Progenitor Cells

Abstract There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

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Inhibition of osteoclast function reduces hematopoietic stem cell numbers in vivo

Blood ◽

10.1182/blood-2010-05-282855 ◽

2011 ◽

Vol 117 (5) ◽

pp. 1540-1549 ◽

Cited By ~ 89

Author(s):

Stefania Lymperi ◽

Adel Ersek ◽

Francesca Ferraro ◽

Francesco Dazzi ◽

Nicole J. Horwood

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Absolute Number ◽

Hematopoietic Stem ◽

Hsc Niche ◽

M Phase ◽

Unit Cells ◽

Osteoclast Function

Abstract Osteoblasts play a crucial role in the hematopoietic stem cell (HSC) niche; however, an overall increase in their number does not necessarily promote hematopoiesis. Because the activity of osteoblasts and osteoclasts is coordinately regulated, we hypothesized that active bone-resorbing osteoclasts would participate in HSC niche maintenance. Mice treated with bisphosphonates exhibited a decrease in proportion and absolute number of Lin−cKit+Sca1+ Flk2− (LKS Flk2−) and long-term culture–initiating cells in bone marrow (BM). In competitive transplantation assays, the engraftment of treated BM cells was inferior to that of controls, confirming a decrease in HSC numbers. Accordingly, bisphosphonates abolished the HSC increment produced by parathyroid hormone. In contrast, the number of colony-forming-unit cells in BM was increased. Because a larger fraction of LKS in the BM of treated mice was found in the S/M phase of the cell cycle, osteoclast impairment makes a proportion of HSCs enter the cell cycle and differentiate. To prove that HSC impairment was a consequence of niche manipulation, a group of mice was treated with bisphosphonates and then subjected to BM transplantation from untreated donors. Treated recipient mice experienced a delayed hematopoietic recovery compared with untreated controls. Our findings demonstrate that osteoclast function is fundamental in the HSC niche.

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Nucleotide Receptor P2Y14 Modulates Hematopoietic Stem Cell Response to Tissue Injury Altering Stem Cell Preservation and Tissue Recovery.

Blood ◽

10.1182/blood.v108.11.679.679 ◽

2006 ◽

Vol 108 (11) ◽

pp. 679-679 ◽

Cited By ~ 1

Author(s):

Yoriko Saito ◽

Eyal Attar ◽

Samyukta Jana ◽

David Dombkowski ◽

Viktor Janzen ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Chemical Injury ◽

Cell Cycle Entry ◽

Induced Apoptosis ◽

Lethal Irradiation

Abstract P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.

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Osteoclast Function Is Essential in the Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v112.11.547.547 ◽

2008 ◽

Vol 112 (11) ◽

pp. 547-547

Author(s):

Stefania Lymperi ◽

Nikki Horwood ◽

Francesco Dazzi

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Absolute Number ◽

Hematopoietic Stem ◽

Micro Pump ◽

Hsc Niche ◽

Osteoclast Function

Abstract Hematopoietic stem cell (HSC) function and number are tightly regulated in a specific microenvironment - the HSC niche - constituted of several cell types. Amongst them, osteoblasts have a prominent role since their increment is associated with an expansion of HSC. However, we demonstrated that an overall increase in osteoblasts does not necessarily promote hematopoiesis (Lymperi et al, Blood 2008 111:1173-81). We hypothesised that osteoclast mediated bone resorption, a component of the bone remodelling process which regulates osteoblast formation, takes active part in the HSC niche. To address this question we inhibited osteoclast function with the bone catabolic agents bisphosphonates. In vivo administration of bisphosphonates to C57BL/6 mice altered the architecture of the trabecular bone as defined by micro-CT and histomorphometric analysis. Treated mice exhibited increased bone volume and trabecular number and reduced trabecular separation and trabecular pattern factor compared to the untreated controls. This was accompanied to a decrease in the proportion and absolute number of HSC in the bone marrow (BM) as assessed by both the immunophenotypic enumeration of Lin-Sca1+c-kit+Flk2-HSC and the long-term culture- initiating cell (LTC-IC) assay. The number of the colony forming unit-cells (CFU-C) in the BM of bisphosphonate treated mice was increased, while more Lin-Sca1+c-kit+ enriched cells were found in the S/M phase of the cell cycle. These results indicate that, in osteoclast-impaired mice a proportion of HSC enters the cell cycle changing the balance in favour of hematopoietic progenitors. The ability of BM cells from bisphosphonate treated mice to engraft and reconstitute the hematopoietic system was tested in a competitive transplantation assay, whereby BM cells from treated and untreated mice were transplanted into syngeneic irradiated recipients. The long-term engraftment of treated BM cells was inferior to the one of controls, indicating a decrease in HSC numbers. Furthermore, recipient mice treated with bisphosphonates prior to conditioning and BM transplantation experienced a delayed hematopoietic recovery as compared to untreated recipients. Since it has been shown that parathyroid hormone (PTH) administration increases the number of HSC by stimulating osteoblast numbers, mice receiving micro-pump delivered PTH were also injected with bisphosphonates. We observed that the administration of bisphosphonates abolished the ability of PTH to increase the primitive HSC pool size as measured by FACS analysis of the Lin-Sca1+c-kit+Flk2- BM cells and their capacity to engraft and reconstitute irradiated recipients. Our findings demonstrate that the osteoclast function is fundamental for the HSC niche, possibly by promoting the recruitment of osteoblasts with HSC supporting capacity.

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The Trithorax Group Protein Ash1l Is An Essential Epigenetic Regulator of Adult Hematopoietic Stem Cell Maintenance

Blood ◽

10.1182/blood.v118.21.387.387 ◽

2011 ◽

Vol 118 (21) ◽

pp. 387-387

Author(s):

Morgan Jones ◽

Michelle L Brinkmeier ◽

Julien Schira ◽

Ann Friedman ◽

Sami Malek ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Stem Cell ◽

Fetal Liver ◽

Set Domain ◽

Hematopoietic Stem ◽

Trithorax Group ◽

Adult Bone

Abstract Abstract 387 The Trithorax family of epigenetic regulators is intimately linked to normal and malignant hematopoiesis. While substantial work indicates that Mixed lineage leukemia (Mll) is required for hematopoietic stem cell (HSC) homeostasis, the functions of many other Trithorax family members have not been evaluated. We have discovered that the mammalian Trithorax group gene absent, small, or homeotic 1-like (ash1l) is required for the maintenance of adult, but not fetal HSCs. Mice homozygous for a gene trap insertion into the first intron of ash1l (GT/GT) had a ca. 90% reduction in ash1l transcripts. These animals had normal numbers of phenotypically defined fetal liver HSCs (CD150+CD48−Lin−Sca-1hic-Kithi cells), but a 10-fold reduction in adult bone marrow HSCs already apparent by 6 weeks after birth. GT/GT bone marrow HSC depletion began in the first three weeks of life, the period during which HSCs turn off their fetal homeostasis program and enter a state of increased quiescence in the bone marrow. Cell cycle analysis revealed that GT/GT HSCs had an increased cycling fraction with a profound reduction in quiescent HSCs in the G0 phase of the cell cycle. This suggested that ash1l is essential to establish and/or maintain a quiescent population of adult-type HSCs. Furthermore, competitive and non-competitive transplantation assays showed that both fetal liver and adult bone marrow were incapable of providing long-term reconstitution in lethally irradiated recipients, indicating that neither compartment could sustain long-term HSC activity after reaching the recipient's bone marrow. To understand this profound HSC defect, we next sought to analyze the in vivo function of Ash1l. Like MLL, Ash1l is a SET domain-containing histone methyltransferase. Although MLL functions as an H3K4 methyltransferase, the in vivo specificity of the Ash1l SET domain has not been described. Transcriptional analysis of GT/GT HSCs indicated that expression of Hoxa9, a known target of MLL, was reduced by 50%. This suggested that Hoxa9 could be a useful locus at which to evaluate the biochemical activity of Ash1l by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). ChIP-qPCR revealed that GT/GT bone marrow cells had reduced H3K36 dimethylation but preserved H3K4 trimethylation at the Hoxa9 locus. This was consistent with previous in vitro analysis of Ash1l SET domain activity and computer predictions showing a high degree of conservation between Ash1l and SET2, an H3K36 methyltransferase. Thus, Ash1l and MLL are both required for Hoxa9 expression although their enzymatic activities differ, suggesting that Ash1l and MLL may act cooperatively at Hoxa9 and other target loci. To evaluate the functional consequences of this interaction, we studied mice deficient for ash1l and lacking Menin, a factor required for proper MLL targeting to target loci. Strikingly, these animals rapidly progressed to hematopoietic failure with a complete obliteration of the hematopoietic stem and progenitor compartment, a phenotype not observed in either genetic background independently. These data indicate that Ash1l and MLL/menin work cooperatively in hematopoiesis. Together, our findings reveal an essential function of ash1l in the maintenance of adult HSCs. Furthermore, they indicate that Ash1l functions in vivo as an H3K36 methyltransferase and suggest that different Trithorax family members can coregulate target genes by providing distinct activating histone marks. Future work will reveal the full extent and mechanisms of these cooperative effects in normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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