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

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 Role of Kdm6a in Normal Hematopoiesis

Blood ◽

10.1182/blood.v128.22.1467.1467 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1467-1467

Author(s):

Ling Tian ◽

Lukas D. Wartman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Young Female ◽

Conditional Knockout ◽

Transplant Experiment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Aged Mice

Abstract The role of the X-linked H3K27 demethylase KDM6A in normal hematopoiesis remains unclear. We generated Kdm6a conditional knockout mice (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre mice to inactivate Kdm6a in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) Kdm6a conditional KO mice. We included female and male animals. The inactivation of Kdm6a alone, both in male and female mice, results in a myeloid replating phenotype indicative of aberrant self-renewal. This is present in both young and aged mice with an increased number of colonies at week 2 in the female KO mice as compared to the hemizygous male KO mice. Interestingly, heterozygous female KO mice only acquire this abnormal replating phenotype with age. Next, we found that the genetic inactivation of Kdm6a has age- and gender-dependent effects on primitive hematopoietic cell populations and progenitors (Figure 1). The phenotype outlined in Figure 1 is specific to female Kdm6a-null mice and is not present in male hemizygous or female heterozygous mice at any time point. We went on to show that the decrease in the KLS compartment seen in the young female Kdm6a-null mice is associated with an increase in the fraction of these cells in G0/G1 along with increased apoptosis. The deletion of Kdm6a in young female homozygous mice causes mild thrombocytopenia but in aged mice is associated with anemia and thrombocytopenia. There is also a myeloid skewing with an increased number of neutrophils and a B-cell lymphopenia in the bone marrow, spleen and peripheral blood that becomes more pronounced with age in these mice. The female homozygous KO mice also have mild splenomegaly and an increased number of red blood cell precursors in the spleen. We went on to perform a competitive transplant experiment mixing donor marrow from all cohorts of young Kdm6a conditional KO x Vav1-Cre mice (CD 45.2) with WT competitor marrow (CD 45.1 x 45.2) in a 1:1 ratio, which was then transplanted into lethally-irradiated CD45.1 recipients so that we could easily follow donor versus recipient chimerism. It has been shown that hematopoietic stem cells (HSCs) from female Kdm6a-null mice had a cell migration defect (Thieme S et al., Blood, 2013). We have now shown a decreased repopulation potential for all three cohorts of Kdm6a KO mice (homozygous and heterozygous females and hemizygous males). The finding is most pronounced in the female homozygous Kdm6a KO mice. Strikingly, and in keeping with results noted in the non-transplanted aged mice, there is a selective preservation of the donor SLAM compartment from all cohorts of the Kdm6a conditional KO mice, which is most pronounced in the female Kdm6a-null mice. The competitive repopulation disadvantage was observed when the bone marrow from primary recipients was transplanted into secondary mice. Again, however, relative preservation of the SLAM compartment was sustained. To circumvent the effects of a migration/engraftment defect of female Kdm6a-null stem cells, we repeated a competitive transplant experiment using young, female homozygous Kdm6a conditional KO x ERT2-Cre mice (and appropriate controls). After engraftment (6-weeks post-transplant), these mice were given tamoxifen to activate the Cre locus and to inactivate Kdm6a. In the bone marrow, we achieved approximately 50% floxing of the Kdm6a conditional allele 2-weeks after the last dose of drug. Again, we observed decreased repopulation potential in all lineages with the exception of the relative preservation of the SLAM compartment. We also repeated serial replating assays and showed that the floxed Kdm6a allele increased with serial replating as expected. Finally, we performed gene expression profiling via exon arrays on flow-sorted SLAM cells from aged female Kdm6a-null mice and aged controls. Hierarchical clustering revealed a clear distinction between cohorts. In sum, our data shows that female Kdm6a KO mice have a gender-specific phenotype that emerges with aging and is similar to human myelodysplastic syndrome (MDS). The female KO aged mice have an expansion of their HSCs with aberrant self-renewal, but these HSCs do not differentiate into downstream progeny as in normal hematopoiesis. As such, these mice become anemic and thrombocytopenic-but do not develop overt leukemia or die of these abnormalities. Disclosures No relevant conflicts of interest to declare.

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Mutant p53 Drives the Development of Pre-Leukemic Hematopoietic Stem Cells through Modulating Epigenetic Regulators

Blood ◽

10.1182/blood.v126.23.307.307 ◽

2015 ◽

Vol 126 (23) ◽

pp. 307-307

Author(s):

Sarah C Nabinger ◽

Michihiro Kobayashi ◽

Rui Gao ◽

Sisi Chen ◽

Chonghua Yao ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Leukemia Stem Cells ◽

Mutant P53 ◽

Self Renewal ◽

Hematopoietic Stem ◽

Tp53 Mutations ◽

Marrow Cells

Abstract AML is thought to arise from leukemia stem cells (LSCs); however, recent evidence suggests that the transforming events may initially give rise to pre-leukemic hematopoietic stem cells (pre-leukemic HSCs), preceding the formation of fully transformed LSCs. Pre-leukemic HSCs have been shown to contribute to normal blood development and harbor a selective growth advantage compared to normal HSCs. Pre-leukemic HSCs can acquire subsequent mutations, and once differentiation capacity is impaired, leukemia emerges. Recently, acquired somatic TP53 mutations, including p53R248W and p53R273H, were identified in healthy individuals as well as AML patients, suggesting that TP53 mutations may be early events in the pathogenesis of AML. We found that p53R248W HSCs showed a multi-lineage repopulation advantage over WT HSCs in transplantation experiments, demonstrating that mutant p53 confers a pre-leukemic phenotype in murine HSCs. Although TP53 mutations are limited in AML, TP53 mutations do co-exist with mutations of epigenetic regulator, ASXL-1, or receptor tyrosine kinase, FLT3, in AML. Mutations in Asxl-1 are present in ~10-30% of patients with myeloid malignancies and confer poor prognosis. Loss of Asxl-1 in the hematopoietic compartment leads to a myelodysplastic-like syndrome in mice and reduced stem cell self-renewal. Internal tandem duplications in Flt3 (Flt3-ITD) occur in ~30% of AML patients and are associated with adverse clinical outcome. Flt3-ITD-positive mice develop a myeloproliferative neoplasm (MPN) and HSCs expressing Flt3-ITD have decreased self-renewal capabilities. We hypothesize that mutant p53 drives the development of pre-leukemic HSCs with enhanced self-renewal capability, allowing clonal expansion and subsequent acquisition of Asxl-1 or Flt3 mutations leading to the formation of fully transformed leukemia stem cells. To define the role of mutant p53 in Asxl-1+/- HSCs, we generated p53R248W/+ Asxl-1+/- mice and performed in vitro serial replating assays as well as in vivo competitivebone marrow transplantation experiments. We found that p53R248W significantly enhanced the serial replating ability of Asxl-1-deficient bone marrow cells. Interestingly, while bone marrow from Asxl-1+/- mice had very poor engraftment compared to wild type bone marrow cells 16 weeks post-transplantation, the expression of p53R248W in Asxl-1+/- bone marrow rescued the defect. To examine the role of mutant p53 in Flt3-ITD-positive HSCs, we generated p53R248W/+ Flt3ITD/+ mice. We found that p53R248W enhanced the replating ability of Flt3ITD/+ bone marrow cells. Despite the fact that Flt3ITD/+ bone marrow cells displayed decreased repopulating ability compared to wild type cells 16 weeks post-transplant, expression of p53R248W in Flt3ITD/+ cells rescued the defect. We are monitoring leukemia development in primary and secondary transplant recipients as well as in de novo p53R248W/+ Asxl-1+/- and p53R248W/+ Flt3ITD/+ animals and predict that mutant p53 may cooperate with Asxl-1 deficiency or Flt3-ITD in the formation of LSCs to accelerate leukemia development in Asxl-1 deficient or Flt-ITD-positive neoplasms. Mechanistically, dysregulated epigenetic control underlies the pathogenesis of AML and we discovered that mutant p53 regulates epigenetic regulators, including Ezh1, Ezh2, Kdm2a, and Setd2, in HSCs. H3K27me3 is catalyzed by EZH1 or EZH2 of the Polycomb repressing complex 2 (PRC2). Both Ezh1 and Ezh2 are important for HSC self-renewal. SETD2 is a histone H3K36 methyltransferase and mutations in SETD2 have been identified in 6% of patients with AML. SETD2 deficiency resulted in a global loss of H3K36me3 and increased self-renewal capability of leukemia stem cells. We found that there were increased levels of H3K27me3 and decreased levels of H3K36me3 in p53R248W/+ HSCs compared to that of the WT HSCs. In ChIP experiments, we found that p53R248W, but not WT p53, was associated with the promoter region of Ezh2 in mouse myeloid progenitor cells, suggesting that p53R248W may directly activate Ezh2 expression in hematopoietic cells. Given that Asxl-1 has been shown to regulate H3K27me3 in HSCs, the synergy between mutant p53 and Asxl-1 deficiency on LSC self-renewal could be due to changes in histone modifications. Overall, we demonstrate that mutant p53 promotes the development of pre-leukemic HSCs by a novel mechanism involving dysregulation of the epigenetic pathways. Disclosures No relevant conflicts of interest to declare.

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PDK1 Controls the Differentiation of Hematopoietic Stem Cells Via Modulating ROS Levels

Blood ◽

10.1182/blood.v126.23.896.896 ◽

2015 ◽

Vol 126 (23) ◽

pp. 896-896

Author(s):

Tianyuan Hu ◽

Cong Li ◽

Le Wang ◽

Yingchi Zhang ◽

Luyun Peng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Conditional Knockout ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Counts

Abstract Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Evidence That a Bioactive Lipid, Ceramide-1 Phosphate (C1P), Is Upregulated In Bone Marrow Microenvironment After Myeloablative Therapy and Is a Potential Novel Homing Factor for Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v116.21.401.401 ◽

2010 ◽

Vol 116 (21) ◽

pp. 401-401

Author(s):

Chihwa Kim ◽

Wu Wan ◽

Ahmed Abdel-Latif ◽

Marcin Wysoczynski ◽

Magdalena Kucia ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Membrane Lipids ◽

Proteolytic Enzymes ◽

Heat Inactivation ◽

Bioactive Lipids ◽

Hematopoietic Stem ◽

Ceramide 1

Abstract Abstract 401 Stromal derived factor-1 (SDF-1), when employed at supra-physiological concentrations, is a potent in vitro chemoattractant for hematopoietic stem progenitor cells (HSPCs). However, because this chemokine is extremely sensitive to degradation by proteolytic enzymes (e.g., MMP-2) and as we have observed myeloablative conditioning for hematopoietic transplantation induces a highly proteolytic microenvironment in bone marrow (BM), SDF-1 secreted by stromal cells and osteoblasts must be rapidly degraded under these conditions. While a role for the SDF-1–CXCR4 axis in retention of HSPCs in BM is undisputed, the role of SDF-1 in the homing of HSPCs in a highly proteolytic microenvironment is somewhat less certain and some redundant homing mechanisms may exist. This latter notion is supported by several observations, such as that i) CXCR4-/- fetal liver HSPCs may home to BM in an SDF-1- independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced, and iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy are able to engraft even in lethally irradiated recipients. To reappraise the role of SDF-1 and other new potential factors in homing of HSPCs, we employed several complementary strategies. First we measured expression of SDF-1 mRNA in BM at 24 and 48 hours after lethal irradiation and observed a ∼3-fold increase. By contrast, the SDF-1 protein level in BM, evaluated by ELISA, surprisingly decreased as compared to non-irradiated mice. Next, we found that after blocking SDF-1 with AMD3100 treatment, conditioned media (CM) from irradiated BM cells still chemoattracted HSPCs. This SDF-1- independent chemotactic activity was resistant to heat inactivation, but was eliminated after stripping by activated charcoal, suggesting the possible involvement of bioactive lipids. Therefore, we began a search for unknown chemoattractants that could direct trafficking of HSPCs, with bioactive lipids as strong candidates, because, as small molecules, they are resistant to proteases. We focused especially on ceramide-1 phosphate (C1P) and sphingosine-1 phosphate (S1P), which are products of membrane-lipids metabolism. It is known that C1P, in contrast to S1P, is retained intracellularly and can be released mostly from damaged cells. Mass spectrometry (MS) analysis revealed that the major isoforms of C1P were detected at higher concentration in supernatant from irradiated BM when compared to supernatant from non-irradiated BM, which suggests that this bioactive lipid and chemoattractant is released from “leaky” BM cells damaged by myeloablative irradiation. We report here for the first time that C1P i) is a strong chemoatttractant for murine and human HSPCs, ii) activates phosphorylation of MAPKp42/44 and AKT in these cells, iii) induces expression of matrix metallopeptidases (MMPs), and iv) modulates adhesion of HSPCs to stroma and endothelium. Furthermore, in direct clonogenic studies, we did not observe any toxic effect of C1P on proliferation of murine and human clonogenic progenitors. We therefore propose a novel paradigm in which C1P is a chemoattractant for HSPCs that, in contrast to SDF-1, is highly resistant to proteolysis. In the proteolytic microenvironment induced in BM after myeloablative radio/chemotherapy, it could play along with SDF-1 an important and, until now, unrecognized role in the homing of HSPCs after transplantation. Furthermore, C1P secreted by damaged cells in other organs (e.g., infarcted myocardium) may in these highly proteolytic or necrotic microenvironments play a similar role in the homing of circulating stem cells involved in regeneration. Disclosures: No relevant conflicts of interest to declare.

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Id2 Is Required for the Self-Renewal and Proliferation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v126.23.1173.1173 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1173-1173 ◽

Cited By ~ 1

Author(s):

Lei Sun

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Conditional Knockout ◽

The Self ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The production of mammalian blood cells is sustained throughout life by the self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation in this system leads to different pathologies including anemia, bone marrow failure and hematopoietic malignancies. The Helix-Loop-Helix transcriptional regulator Id2 plays essential roles in regulating proliferation and cell fate of hematopoietic progenitors; however, its role in regulating HSC development remains largely unknown. To assess the function of Id2 in HSCs, we developed two mouse models, including an Id2 conditional knockout model and an Id2-EYFP model, in which EYFP expression is driven by endogenous Id2 promoter. When we examined HSC function by serial transplantation, we found that mice transplanted with Id2F/F Mx1-Cre+ conditionally deleted bone marrow cells became moribund more rapidly after primary and secondary transplantation, compared to those transplanted with Id2+/F Mx1-Cre+ bone marrow, suggesting that HSC self-renewal is impaired when Id2 is deleted. To further determine if self-renewal and maintenance of HSCs depends on the expression level of Id2, we purified HSCs with different levels of Id2 expression using Id2-EYFP mice to specifically address the role of Id2 in HSCs. First, we confirmed Id2 is highly expressed in HSCs in this model. Second, when HSCs with either low or high levels of Id2-EYFP were transplanted into irradiated mice, cells with high levels of Id2 reconstituted transplanted recipients faster than those with low levels of Id2 at 3 weeks and longer, suggesting that Id2 expression is associated with repopulation advantage. Furthermore, Ki-67 staining showed that HSCs with high levels of Id2 have 15-fold more cells in G2/M phase, and fewer cells in G0. BrdU staining also suggested that there are 5-fold more BrdU+ cells in HSCs with high levels of Id2, indicating that Id2 expression correlates with cell cycle progression in HSCs. In addition, p57 has been reported to be required for quiescence of HSCs. Our preliminary data showed that p57 is downregulated in HSCs with high levels of Id2, and p57 is correspondingly upregulated in Id2-null HSCs. Altogether, our data demonstrate that Id2 is required for the self-renewal and proliferation of HSCs, and suggest a link between Id2 and the transcriptional regulatory networks that regulate the functional hematopoietic system. Since Id2 is also expressed in other adult stem cells including muscle and neuronal stem cells, as well as cancer cells, we believe our results can improve our understanding of stem cell biology and cancer development, and contribute to the identification of novel molecules that may be targeted to eliminate cancer stem cells. Disclosures No relevant conflicts of interest to declare.

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Angiopoietin-Like 3 Affects Homing Ability of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2632.2632 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2632-2632

Author(s):

Masato Umikawa ◽

Junke Zheng ◽

HoangDinh Huynh ◽

Chengcheng Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Physiological Role ◽

Wild Type ◽

Hematopoietic Stem ◽

Intrinsic Effect

Abstract Abstract 2632 Angiopoietin-like proteins (Angptls) are a seven-member family of secreted glycoproteins that share sequence homology with angiopoietins. It is known that several members of the Angptl family including Angptl3 support ex vivo expansion of hematopoietic stem cells (HSCs). However, the physiological role of Angptls in the hematopoietic system is not well known. Here we show that Angptl3 is expressed by both bone marrow stromal cells and HSCs. To study the intrinsic effect of Angptl3 in mouse HSCs, we isolated the same number of HSCs from wild-type and Angptl3-null mice and performed reconstitution analysis. Adult bone marrow Angptl3-null HSCs showed decreased repopulation compared to wild-type HSCs, suggesting that Angptl3 has cell-autonomous effect on HSC activity. By contrast, HSCs isolated from liver of the null mice had enhanced HSC repopulation activity than their wild-type counterparts. To study whether this effect is caused by difference in homing, we injected CFSE labeled wild-type HSCs and Angptl3 null HSCs into lethally irradiated mice, and checked the homing to bone marrow, spleen, and liver. While homing of these two types of cells to bone marrow or spleen was not significantly different, Angptl3 null HSCs homed better to the liver than the wild-type HSCs. Our result suggests that Angptl3 is important for the retention of HSCs in the bone marrow, and the absence of Angptl3 leads HSCs to move to extramedullary organs such as liver. Disclosures: No relevant conflicts of interest to declare.

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HIF1α Is Required for Survival Maintenance of Chronic Myeloid Leukemia Stem Cells

Blood ◽

10.1182/blood.v118.21.449.449 ◽

2011 ◽

Vol 118 (21) ◽

pp. 449-449

Author(s):

Haojian Zhang ◽

Huawei Li ◽

Shaoguang Li

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Bone Marrow Cells ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Cycle Arrest

Abstract Abstract 449 Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder induced by the BCR-ABL oncogene, and available BCR-ABL kinase inhibitors fail to completely eradicate leukemia stem cells (LSCs) to cure the disease. The challenge lies in the identification of genes that play a critical role in survival regulation of LSCs. Hypoxia-inducible factor-1α (HIF1α), a master transcriptional regulator of the cellular and systemic hypoxia response, is essential for the maintenance of self-renewal capacity of normal hematopoietic stem cells (HSCs). It is still unknown about the role of HIF1α in survival regulation of LSCs in CML. Using a mouse model of CML, here we report that HIF1α plays a crucial role in survival maintenance of LSCs. We conducted a DNA microarray analysis to compare the gene expression profiles between LSCs and normal HSCs in our bone marrow transplantation (BMT) mouse model of CML. We retrovirally transduced bone marrow cells from C57BL/6J (B6) mice with BCR-ABL-GFP or GFP alone (as a normal HSC control) and transplanted the transduced cells into lethally irradiate B6 recipient mice to induce CML. Two weeks after BMT, we sorted GFP+LSK (Lin−Sca-1+c-Kit+) cells from bone marrow of the mice for the Affymetrix microarray analysis. HIF1α gene was up-regulated by BCR-ABL in LSCs. We next examined expression of genes known to be specifically regulated by HIF1α, and found that expression of VEGF, GLUT1 and TGFa, except for PGK1, were significantly higher in LSCs than in HSCs. Real time RT-PCR assay confirmed the up-regulation of HIF1a and other hypoxia-responsive genes by BCR-ABL in LSCs. To determine the role of HIF1α in BCR-ABL leukemiogenesis, we crossed mice carrying a loxP-flanked HIF1a allele with Cre transgenic mice in which expression of Cre is driven by the Vav regulatory element to induce the deletion of the HIF1a gene mainly in the hematopoietic system. We transduced bone marrow cells from 5-FU-treated wild type (WT) or HIF1a−/− mice with BCR-ABL-GFP retrovirus, and then transplanted into lethally irradiated recipient mice to induce primary CML, followed by a secondary transplantation. We found that HIF1α−/− LSCs failed to induce CML in the secondary recipient mice, whereas WT LSCs efficiently induced CML. The defective CML phenotype in the absence of HIF1α was consistent with a gradual decrease of the percentages and total numbers of leukemia cells in peripheral blood and with much less severe splenomegaly. These results indicate that HIF1α is required for CML development, and suggest that HIF1α is required for survival maintenance of LSCs. To understand the underlying mechanisms, we analyzed the effect of HIF1α on cell cycle progression and apoptosis of LSCs, and found that the percentage of HIF1α−/− LSCs in the S-G2/M phase was significantly lower than that of WT LSCs, indicating that the HIF1α deficiency causes a cell cycle arrest of LSCs. Furthermore, we examined whether deletion of HIF1α induces apoptosis of LSCs by staining the cells with annexin V and 7AAD, and found that HIF1α−/− LSCs had a higher apoptotic rate than WT LSCs. We further compared expression levels of three cyclin-dependent kinase inhibitors p16Ink4a, p19Arf, and p57 between HIF1α−/− and WT LSCs, and found that the cell cycle arrest caused by the HIF1α deficiency was associated with significantly higher levels of expression of p16Ink4a, p19Arf and p57 in HIF1α−/− LSCs than in WT LSCs. In addition, we observed an increased expression of the apoptotic gene p53 in HIF1α−/− LSCs, explaining the increased apoptosis of HIF1α−/− LSCs. In summary, our results demonstrate that HIF1α represents a critical pathway in LSCs and inhibition of the HIF1α pathway provides a therapeutic strategy for eradicating LSCs in CML. Disclosures: No relevant conflicts of interest to declare.

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Role of CXCL12-Expressing Mesenchymal Stromal Cell Niches in Maintaining Treatment-Resistant Leukemia Stem Cells

Blood ◽

10.1182/blood-2018-99-117762 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1291-1291

Author(s):

Puneet Agarwal ◽

Stephan Isringhausen ◽

Hui Li ◽

Andrew J Paterson ◽

Jianbo He ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Tyrosine Kinase ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Leukemia Stem Cells ◽

Hematopoietic Stem ◽

Tki Treatment

Abstract Chronic myeloid leukemia (CML) results from hematopoietic stem cell (HSC) transformation by the BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKI), although effective in inducing remissions in CML, fail to eradicate leukemia stem cells (LSC) which persist as source of relapse. LSC resistance to TKI-treatment occurs through kinase-independent mechanisms, which include alterations in intrinsic cell-regulatory mechanisms as well as signals from the bone marrow (BM) microenvironment that support LSC persistence. HSC have been shown to be regulated by C-X-C motif chemokine ligand 12 (CXCL12)-expressing bone marrow niches, but the nature and regulatory role of BM niches for LSC remains poorly understood.Here, we used CXCL12-GFP mice and CXCL12f/fmice crossed with Cre lines targeting specific CXCL12-expressing cells to investigate the contribution of CXCL12-expressing populations to LSC regulation. We found that targeted deletion of CXCL12 from Prx1+ mesenchymal stromal cells (MSC) reduced normal HSC numbers. In contrast, deletion of CXCL12 from Prx1+ MSC in the setting of CML, resulted in increased leukocytosis, neutrophilia, BM cellularity and LSC numbers, and reduced survival, compared to control CML mice. CXCL12 deletion from Prx1+ MSC was found to enhance LSC cycling. Despite increased cycling, the expanded LSC from these mice maintained their in vivo repopulating capacity. To evaluate the effect of CXCL12 deletion on LSC and MSC distribution, we performed 3D imaging of BM volumes from Prx1-Cre mice crossed with tdTomato reporter mice. CML development resulted in formation of large pathological tissue niches harboring an abnormally high density of MSCs as well as c-Kit+ leukemia progenitor cells. However, these MSC and leukemic progenitor clusters were not observed in Prx1-Cre+CXCL12fl/fl mice, indicating that formation of tissue structures with colocalized leukemic progenitors is dependent on CXCL12 expression in MSC. We performed gene expression analysis of LSC from Prx1-Cre+CXCL12f/f and Cre-negative mice. Gene expression analysis revealed enrichment of cell cycling and MYC related genes in CML LSC from Prx1-Cre+CXCL12f/f mice, and downregulation of Polycomb Repressive Complex 2 (PRC2) target genes, indicating increased PRC2 activity. We confirmed that EZH2 expression and H3K27 trimethylation were increased in LSC from Prx1-Cre+CXCL12f/f mice. Treatment with the EZH2 inhibitor, GSK 343, resulted in significant reduction in WBC, neutrophils, BM cellularity and LSC in Prx1-Cre+CXCL12f/f mice, but not control CML mice. These results support a role for increased PRC2 activity in LSC expansion in mice with CXCL12 deletion from Prx1+ MSC. We evaluated the effect of CXCL12 deletion from Prx1+ MSC on LSC sensitivity to treatment with the TKI Nilotinib. Treatment of Cre-negative CML mice with Nilotinib reduced WBC counts, spleen cellularity, and splenic LSC numbers, but did not reduce BM cellularity or LSC numbers. On the other hand, Nilotinib treatment significantly reduced BM cellularity and LSC numbers, and enhanced survival, in Prx1-Cre+CXCL12f/f CMLmice. Transplantation of BM from TKI-treated Prx1-Cre+CXCL12f/f mice to irradiated normal recipients resulted in significantly reduced long-term donor engraftment and donor LSC numbers compared to vehicle-treated mice. These results indicate that CXCL12 deletion from Prx1+ MSC leads to enhanced sensitivity of CML LSC to elimination by TKI treatment. In conclusion, our studies show that deletion of CXCL12 from Prx1+ MSC niches leads to loss of MSC clustering and colocalization with leukemic progenitors, and to loss of quiescence and expansion of LSC, dependent on enhanced PRC2 activity. CXCL12 deletion from MSC also increases TKI-mediated targeting of resistant, quiescent, self-renewing CML LSC. Strategies to inhibit CXCL12-mediated niche interactions represent a promising approach for LSC depletion to enhance opportunities for cures in CML. Disclosures No relevant conflicts of interest to declare.

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The Role of the Bone Marrow Microenvironment in the Response to Infection

Frontiers in Immunology ◽

10.3389/fimmu.2020.585402 ◽

2020 ◽

Vol 11 ◽

Author(s):

Courtney B. Johnson ◽

Jizhou Zhang ◽

Daniel Lucas

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Immune Cells ◽

Critical Role ◽

Primary Source ◽

Bone Marrow Microenvironment ◽

Future Research ◽

Multipotent Stem Cells ◽

Hematopoietic Stem

Hematopoiesis in the bone marrow (BM) is the primary source of immune cells. Hematopoiesis is regulated by a diverse cellular microenvironment that supports stepwise differentiation of multipotent stem cells and progenitors into mature blood cells. Blood cell production is not static and the bone marrow has evolved to sense and respond to infection by rapidly generating immune cells that are quickly released into the circulation to replenish those that are consumed in the periphery. Unfortunately, infection also has deleterious effects injuring hematopoietic stem cells (HSC), inefficient hematopoiesis, and remodeling and destruction of the microenvironment. Despite its central role in immunity, the role of the microenvironment in the response to infection has not been systematically investigated. Here we summarize the key experimental evidence demonstrating a critical role of the bone marrow microenvironment in orchestrating the bone marrow response to infection and discuss areas of future research.

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