Term Hematopoietic Stem Cells Research Articles

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors.

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.

Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet.To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis.The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs.We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Fbxw11 Variants Control the Quiescence of HSCs

Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. Fbxw11, a constituent of the SCF (Skp1-Cul1-F-box) ubiquitin ligase complex, targets for degradation of several important transcription factors, including NF-κB, β-cantenin and ATF4. Fbxw11 may play pivotal roles in many aspects of hematopoiesis through regulating various signal transduction pathways. However, the role of Fbxw11 on HSCs quiescence remains largely unknown. In this study, we cloned three transcript variants (Fbxw11a, Fbxw11c and Fbxw11d) to study the biological function of Fbxw11 in hematopoiesis.To elucidate Fbxw11 distribution in hematopoietic system, hematopoietic cell populations at different stages of differentiation were isolated from bone marrow of 8 week-old mice and Fbxw11 expression was studied by real-time PCR. Expression of Fbxw11 were lower in purified long term hematopoietic stem cells (LT-HSC, LSK CD34- Flt3-), but higher in short term hematopoietic stem cells (ST-HSC, LSK CD34+ Flt3-), hematopoietic stem and progenitor cells (LSK), and various hematopoietic progenitor cells. The results reveal that Fbxw11 is preferentially expressed in more mature progenitor cells. The expression of Fbxw11 in mature blood cells was also studied showing that Fbxw11 was expressed at lower level in neutrophils, higher level in B and T lymphocytes, and moderate level in monocytes.To assess the impact of Fbxw11 on reconstitution capacity of LT-HSCs, we cloned Fbxw11a, Fbxw11c and Fbxw11d into retrovirus system, respectively. LSK cells were infected with MSCV-Fbxw11a/Fbxw11c/Fbxw11d-IRES-GFP or the blank control vector MSCV-GFP. Competitive repopulation assays we performed 48h later after infection, and reconstitution in peripheral blood (PB) was analyzed every 4 weeks. Repopulation of donor cells expressing high level of Fbxw11 variants was significantly lower than those infected with control vector at 1 and 4 months in PB and at 4 months in BM after transplantation. These data indicate that Fbxw11 is negative for the long-term repopulating capacity of HSCs.To further confirm the effects of Fbxw11 variants in hematopoiesis, the effect of Fbxw11 variants on the growth and enumeration of hematopoietic progenitor cells was detected by colony-forming cell assay (CFC). The number of CFU-G, CFU-GM, CFU-GEMM and the total number of CFU were lower in LSK over-expressing Fbxw11 variants when compared with LSK control. To determine the cell-cycle distribution of HSC cells, Hoechst 33342 and Ki67 staining were performed showing that G0 phase LSK cells were decreased when they over-expressing Fbxw11 variants.In conclusion, our data reveal unrecognized roles for Fbxw11 in the regulation of HSPCs. Our findings suggest that Fbxw11 variants have negative effect on reconstitution capacity of LT-HSCs. Fbxw11 variants decrease the reconstitution capacity through promoting cell proliferation, which results in loss of hematopoietic stem cell quiescence. We anticipate that our experiments will facilitate the understanding of hematopoiesis through which Fbxw11-mediated signals control HSC quiescence and functions.The work was supported by the Grants 81300376, 81370634, 81570153 from the National Natural Science Foundation of China (NSFC); 14JCQNJC10600 from the Tianjin Science and Technology Programs; DisclosuresNo relevant conflicts of interest to declare.

Inhibition of CML Development Following Conditional SIRT1 Deletion in Transgenic BCR-ABL Mice

▪Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interferon-Gamma Impairs Expansion and Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells

Maintenance of hematopoietic stem cells (HSCs) and regulation of their quiescence and self-renewal is critical for maintaining a lifelong supply of blood cells. The ability of HSCs to stay quiescent is thought to depend on their specific niche in the bone marrow (BM). Mesenchymal stromal cells (MSC) in the BM are multipotent stem cells that form part of the vascular HSC niche and provide micro-environmental support to HSCs both in vivo and upon expansion ex vivo. Culture-expanded MSCs also exhibit immunomodulatory properties that can be enhanced by pre-treatment with interferon-gamma (IFN-γ). BM MSC are thus attractive candidates for cellular therapy after hematopoietic stem cell transplantation, for promoting rapid hematopoietic recovery and reducing the incidence or severity of graft versus host disease. Although IFN-γ pre-treatment can improve the immunomodulatory properties of MSCs, elevated IFN-γ levels have also been associated with anemia and BM failure in multiple chronic inflammatory diseases. While the impact of IFN-γ on HSC has been elucidated in recent years, it remains largely unknown whether IFN-γ can also influence hematopoietic support by BM stromal cells.In this study, we aim to elucidate the impact of IFN-γ on hematopoietic support of BM MSC.We show that in vitro expansion of primary BM MSC cultures from healthy donors was significantly reduced in the presence of IFN-γ, and this effect could be reproduced in the BM stromal cell line MS-5. Concurrently, IFN-γ diminished the clonal capacity of BM MSC, as measured by CFU-F assays. In addition, BM MSC that were pre-stimulated with IFN-γ produced significantly lower levels of CXCL12, suggesting a loss of hematopoietic support potential. Indeed, support of CD34+ hematopoietic stem and progenitor cells (HSPC) in a co-culture assay was greatly reduced in when MSC were pre-treated with IFN-γ.To determine the impact of IFN-γ on BM MSC in vivo, we investigated the BM stromal compartment of IFN-γ AU-rich element deleted (ARE-Del) mice, which constitutively express IFN-γ in steady state conditions. FACS analysis revealed a remodeling of the BM stromal compartment in ARE-Del mice compared to littermate controls, with significantly fewer MSCs, identified as CD45-Ter119-CD31-CD51+PDGFRa+ cells. Numbers of other stromal cell subsets, such as osteoblasts and fibroblasts, were not altered.The reduction of BM MSC in ARE-Del mice coincided with a loss of quiescence in HSCs; only 35% of long term HSC (LT-HSC) in ARE-Del mice were quiescent, compared to 70% in WT mice, as determined by Ki-67 staining. Loss of quiescence in LT-HSC did not lead to increased self-renewal, but rather induced increased differentiation towards short-term HSC and multi-potent progenitors.We then sorted LT-HSC from WT and ARE-Del mice and performed in vitro HSC culture assays in the absence of IFN-γ. Absolute numbers of LT-HSC were rapidly decreased in ARE-Del compared to WT cultures after 3 and 7 days of HSC culture, while numbers of more differentiated progenitors were increased. These data indicate that an IFN-γ-mediated loss of BM MSC in ARE-Del mice leads to loss of quiescent LT-HSCs and induces a tendency towards HSC differentiation over self-renewal.In conclusion, we have shown that IFN-γ has a negative impact on expansion and hematopoietic support of BM MSC in vitro and in vivo across species. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, it is obvious from our data that IFN-γ impairs their HSC supporting function. These data also provide more insight in the underlying mechanism by which IFN-γ contributes to the pathogenesis of anemia and BM failure. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for TCR and CAR based Adoptive Immunotherapy of Cancer

Abstract Long-term survival and anti-tumor immunity critically depends on their metabolic fitness but approaches to select metabolically robust T cells for T cell based immunotherapy remains unknown. We utilized a lipophilic cationic dye (tetramethylrhodamine methyl ester) to identify and isolate metabolically robust T cells based on mitochondrial membrane potential (ΔΨm). Cells with lower membrane-potential (low ΔΨm) had a molecular profile of memory precursors and displayed an enhanced ability to enter the memory pool as compared to cells displaying higher mitochondrial potential (high ΔΨm) characteristic of short lived effectors. Interestingly, we also found that multiple distinct negative inhibitory receptors such as PD1, CTLA4, BTLA, LAG3 and TIM3 were enriched in the high ΔΨm subset compared to the low-ΔΨm subset. Comprehensive metabolomics profiling demonstrated global features of metabolic fitness (increased lipid oxidation) in low ΔΨm sorted CD8+ T cells. Transfer of low ΔΨm T cells was associated with superior long-term in vivo persistence as evidenced by 100 fold increase in the frequency of T cells 300 days after adoptive transfer, augmented autoimmunity and an enhanced capacity to eradicate established cancer compared with high ΔΨm cells. Mechanistically, we found that the IL2 driven mTOR activation caused increased mitochondrial oxygen consumption and accumulation of higher level of ROS. Surprisingly, use of Ψm to enrich for cells with superior metabolic features was observed within central memory, effector (Tc17, Th1, and Th17) T cells and long term hematopoietic stem cells (LT HSC). This novel metabolism based approach may be applicable to therapies involving transfer of lymphocytes for treatment of advanced cancer.

Aryl Hydrocarbon Receptor Deficiency in an Exon 3 Deletion Mouse Model Promotes Hematopoietic Stem Cell Proliferation and Impacts Endosteal Niche Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor belonging to the Per-Arnt-Sim (PAS) family of proteins. The AHR is involved in hematopoietic stem cell (HSC) functions including self-renewal, proliferation, quiescence, and differentiation. We hypothesize that AHR impacts HSC functions by influencing genes that have roles in HSC maintenance and function and that this may occur through regulation of bone marrow (BM) niche cells. We examined BM and niche cells harvested from 8-week-old AHR null-allele (KO) mice in which exon 3 was deleted in the Ahr gene and compared these data to cells from B6 control mice; young and old (10 months) animals were also compared. We report changes in HSCs and peripheral blood cells in mice lacking AHR. Serial transplantation assays revealed a significant increase in long term HSCs. There was a significant increase in mesenchymal stem cells constituting the endosteal BM niche. Gene expression analyses of HSCs revealed an increase in expression of genes involved in proliferation and maintenance of quiescence. Our studies infer that loss of AHR results in increased proliferation and self-renewal of long term HSCs, in part, by influencing the microenvironment in the niche regulating the balance between quiescence and proliferation in HSCs.

Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models.In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Dicer Ablations in Bone Marrow Niche Impair Hematopoietic Progenitor/Stem Cells and Induce Myelodysplasia in Young Mice but Are Dispensable for Adult Hematopoiesis

MicroRNAs (miRNAs) regulate hematopoietic cell fate and their global down-regulation by Dicer1 deletion promotes tumorigenesis in a cancer-cell-autonomous manner (Kumar M.S. et al, 2007). Raajimakers MH et al. (2010) using neonatal Osterix specific dicer deletion showed altered hematopoiesis and developed myelodysplasia. However, there is no study illustrating the role of the ablation of bone marrow (BM) niche specific miRNA processing machinery in the adult mice. Since expression and functions of different mesenchymal and osteoprogenitors vary from embryonic development to adulthood, studying the dicer ablation in adult mice may provide more insight about the role of miRNA processing in adult mice niche.Here we investigate whether adult Osterix expressing cells play a similar role in the HSC niche compared to fetal Osterix expressing cells. We crossed Osx-GFP-tTA-Cre recombinase mice with mice with floxed Dicer1 allele. Crossing generated Osx- GFP-tTA-Cre+Dicerfl/+ (OCDfl/+control) and Osx-GFP- tTA-Cre+ Dicerfl/fl (OCDfl/fl mutant) mice. Osx-GFP-tTA-Cre expression was either activated during embryonic development (young dicer KO) or suppressed using tetracycline until mice were 6 weeks of age (adult dicer KO). We found young dicer KO mice had reduced weight (p=0.0031), leukopenia, anemia, reduced mature CD19+B220- B lymphocytes (p=0.0034) and increased CD11b+Gr- monocytes and CD11b+Gr+ neutrophils (p=0.02 and p=0.04 respectively) in peripheral blood compared to OCDfl/+ control aged littler mates. The leucocytes and platelets showed dysplastic changes suggestive of myelodysplasia and had extra-medullary hematopoiesis. Adult dice KO, on the other hand, show no leukemia development 6 months after Cre activation. The number of BM hematopoietic progenitors (Lin-Sca1+ c-Kit+ cells, LSK) and long term hematopoietic stem cells (LT-HSCs, LSK CD150+CD48+ cells) in young dicer KO mice were significantly reduced compared to age matched control (OCDfl/+ control) mice. We observed increased Annexin V positive LSK, LT-HSCs and megakaryocytes erythroid progenitors (MEP) in the young dicer KO mice indicating increased apoptosis. Adult dicer KO mice didn't have significant changes in apoptosis in different hematopoietic progenitors. In young dicer KO mice, BM derived LSK and LT-HSCs showed increased cycling (SG2M phase, p=0.0133) and less quiescenece (Go phase, p=0.013). However LT-HSC from adult dicer KO didn't show any difference in cell cycling (p=0.18 and 0.09 respectively). Together these results indicate that while Osterix expressing cells in fetal and young mice give rise to a variety of HSC niche supporting cells the adult expression is limited to more mature osteoblast that are not absolutely essential for HSC maintenance. Our study provides the rationale for further exploration of the complexity in hierarchy of activity within niche constituting mesenchymal stroma progenitors and their role in different developmental stages to maintain hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Osteocyte-Mediated Parathyroid Hormone (PTH) Signaling Regulates Hematopoietic Stem Cells Under Physiologic and Continuous PTH Exposure

The bone marrow microenvironment, including osteolineage cells, regulates hematopoietic stem cell (HSC) fate choices. Intermittent pharmacologic treatment of mice with parathyroid hormone, PTH (1-34), indirectly increases HSCs through their niche, as HSCs do not express the PTH receptor (PTH1R). Osteocytes, the most abundant osteolineage cells in bone, are a critical target of the skeletal actions of PTH and coordinate multiple cell types that are components of the HSC niche including osteoblasts, osteoclasts and resident macrophages. While osteocytes express the PTH1R, the role of osteocytes in HSC regulation is unclear. Therefore, we studied the role of osteocyte-mediated PTH regulation of HSCs, using cre recombinase driven by the 8kb-DMP1 promoter to conditionally delete PTH1R in osteocytes (OCyPTHRko mice).OCyPTHRko mice were viable, fertile, did not exhibit any significant skeletal defect as juveniles or at 6 months of age, had no significant difference in serum PTH levels, and had no significant difference in osteoblastic or mesenchymal stem cell numbers compared to WT mice. In juvenile OCyPTH1Rko mice there was a decrease in long-term HSCs as measured by flow cytometric analysis (0.0029 ± 0.00028 vs. 0.0021 ± 0.00021 % of cells, WT vs. OCyPTH1Rko p≤0.05 N≥19 mice/group). OCyPTH1Rko mice had 4 fold lower long-term engraftment capacity as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥10 mice/group) that was evident in all hematopoietic lineages. Short-term engraftment however was increased in OCyPTH1Rko mice as measured by primary competitive transplantation (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.01, N≥9 mice/group). These data demonstrate that physiologic PTH signaling in osteocytes regulates the balance of long-term and short-term HSC potential in juvenile, growing mice. Adult OCyPTH1Rko mice also had 5 fold lower long-term engraftment as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥15 mice/group). These findings demonstrate a previously unrecognized physiologic role of PTH signaling in HSC regulation. Having demonstrated a role for PTH signaling in HSC homeostasis, we investigated if sustained PTH elevations (as are found in vitamin D deficiency and in hyperparathyroidism) alter HSC function. Therefore, we utilized a murine model of secondary hyperparathyroidism caused by a low calcium (LCa) diet. In juvenile mice placed on the LCa diet immediately upon weaning, serum PTH levels were significantly elevated. Fourteen days on the LCa diet caused a significant reduction in long-term engraftment potential as measured by secondary competitive transplants over 22 weeks (Normal vs. LCa diet donors, 2-way ANOVA p≤0.001, N≥20 mice/group), while there was no decrease in HSCs when adult mice were placed on the LCa diet. These data suggest that sustained PTH signaling decreases microenvironmental support for HSCs in juvenile mice. We utilized the OCyPTHRko mice to study the role of osteocytes in hyperparathyroidism-induced loss of functional HSCs. In juvenile mice the lack of PTH signaling in osteocytes rescued the long-term engraftment defects, suggesting that PTH signaling in osteocytes mediates the loss of long-term HSC support caused by the LCa diet. In further support of a deleterious effect mediated by the PTH1R in osteocytes in the setting of continuous PTH, adult OCyPTH1Rko mice placed on LCa diet had superior long term HSC function.Our findings demonstrate a physiologic role for PTH in HSC regulation and identify osteocytes as a critical constituent of the HSC niche that, either directly or indirectly, contribute to maintenance of the long-term repopulating HSC pool. In addition, we show that continuous exposure to elevated levels of PTH in a model of secondary hyperparathyroidism leads to osteocyte-mediated loss of long-term engraftment potential of HSCs in juvenile mice. We speculate that removing the effect of continuous PTH from osteocytes uncovers additional HSC-supportive effects of continuous PTH, mediated by non-osteocyte HSC niche cellular populations. Together these data establish PTH as a critical regulatory signal in the HSC niche, and show that the relative contributions of niche populations to HSC regulation are modulated by age. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

S6K1 Regulates Self-Renewal of Leukemia Initiating Cells and Normal Hematopoietic Stem Cells

The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult hematopoietic stem cells (HSCs) and regulation of progenitor population. Translocations in MLL have been detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. Various genes, including AF4, AF5, AF9, ELL and ENL have been identified as partners for translocation in MLL-rearranged leukemia. In hematopoietic cells, expression of MLL fusion proteins result in a block of differentiation. AML patients who successfully undergo treatment but relapse suggest the importance of targeting leukemia initiating cells (LICs) within the MLL leukemia. LICs remain in a quiescent state and are capable of survival despite treatment with chemotherapeutic agents or targeted molecular inhibitors. In mouse models, LICs are defined by the capability of successfully propagating disease upon serial transplantation. In order to prevent disease relapse, identification of molecules, which regulate the self-renewal of LICs, is essential.The phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway is a key regulator of self-renewal of both HSCs and LICs. Deletion of Raptor, a subunit of mTORC1, does not affect initiation and progression of acute myeloid leukemia (AML), but Raptor deficiency results in delayed propagation of AML. Upon its activation, mTORC1 phosphorylates and activates p70 ribosomal protein S6 kinase (S6K1) and inhibits the activity of eukaryote translation initiation factor 4E binding protein 1 (4E-BP1). S6K1 has been shown to be hyperactivated in hematopoietic cells expressing oncogenic MLL-AF9 fusion protein.In our study, we have assessed the role of S6K1 in the initiation, progression and propagation of AML using a genetic model of S6K1 knockout mice (S6K1-/-). We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and transplanted them into lethally irradiated recipients. Recipients of both WT and S6K1-/- HSC/Ps bearing MLL-AF9 displayed high white blood cell (WBC) count, splenomegaly and developed AML. There was no difference in survival between the WT and S6K1-/- recipients. In order to determine whether S6K1 regulates the self-renewal of LICs, we transplanted lethally irradiated mice with cells from WT and S6K1-/- primary recipients who developed AML. Recipients of S6K1 deficient AML cells survived significantly longer compared to controls (n=17/group, p<0.001). S6K1 deficient HSC/Ps expressing MLL-AF9 showed reduced activation of Akt as well as decreased mTORC1 activity, suggesting that deletion of S6K1 results in reduced activation of PI-3K-Akt-mTORC1 pathway both upstream and downstream of mTORC1 which indicates that S6K1 might be involve in a feedback loop within this pathway.To determine the role of S6K1 in normal HSC development and maintenance, we analyzed bone marrow derived HSCs in WT and S6K1-/- mice. S6K1 deficiency did not alter the frequency of long term HSCs (LT-HSCs) as defined by CD150+ CD48- Lin- Sca1+ c-Kit+ surface markers, but the absolute number of LT-HSCs were significantly reduced in S6K1 deficient mice (p<0.02). The absolute number of multipotent progenitors (MPPs) (p<0.001), common myeloid progenitors (CMPs) (p<0.01) and megakaryocyte-erythroid progenitors (MEPs) (p<0.01) were also significantly reduced in S6K1 deficient mice. Deficiency of S6K1 resulted in reduced quiescence of LT-HSCs (p<0.05). Expression level of p21, an inhibitor of cell cycle progression, was significantly decreased in LT-HSCs derived from S6K1-/- mice compared to LT-HSCs from control group. To study the role of S6K1 in HSCs' function, we performed competitive repopulation assay. Sorted LT-HSCs from bone marrow cells derived from either WT or S6K1-/- mice were transplanted with competitor cells into lethally irradiated recipients. S6K1 deficient LT-HSCs displayed reduced repopulating ability in secondary recipients (n=9-10/group, p<0.001). Expression level of p21 was downregulated in donor-derived HSCs isolated from secondary recipients of S6K1 deficient HSCs compared to control. Overall, our study establishes S6K1 as a critical regulator of self-renewal of both LICs and HSCs. Deficiency of S6K1 in AML cells results in delayed propagation of disease and deficiency of S6K1 in HSCs results in decreased self-renewal potential. DisclosuresNo relevant conflicts of interest to declare.

Selective Deletion of ATG5 in Bone Marrow Leptin-Receptor-Expressing Perivascular Stromal Cells Causes Leukopenia

Bone marrow niche cells, specifically the Leptin-receptor-expressing perivascular stromal cells (LepR+), CXCL12-abundant reticular cells (CAR), and endothelial cells play an important role in the maintenance and self-renewal functions of hematopoietic stem cells (HSCs). This maintenance function provided by the niche cells is mediated by a number of secretory-related molecules such as stem cell factor (SCF), CXCL12 and Angiopoietin. In addition, functional autophagy within HSCs is known to play a critical role for maintaining HSC homeostasis, as the loss of ATG7 in HSCs leads to loss of normal HSC functions and severe myeloproliferation. However, it remains unclear as to how hematopoiesis will be affected if autophagy is selectively blocked in bone marrow niche cells. To investigate the effect of autophagy deficiency within bone marrow niche cells on hematopoiesis, we generated a knockout mouse model allowing for a selective ablation of an autophagy-essential protein, ATG5, in LepR+ perivascular stromal cells. We have confirmed that the deletion of Atg5 is indeed restricted to LepR+ cells, and that the loss of ATG5 is sufficient to block autophagic function, as evident through the accumulation of p62 protein. We found that the white blood cell count (WBC) is significantly reduced in 16-week old male ATG5 KO mice compare to wild type (wt) littermate control mice (p<0.05). Further, the myeloid population (CD11b+ Ly6G+) in both bone marrow and peripheral blood is significantly decreased in ATG5 KO mice (p value < 0.05, ATG KO vs. wt control). WBC count continues to decline in 24-week old ATG5 KO male mice (p=0.02) but not in female mice (p=0.58). Both LSK (Lin- Sca-1+ c-Kit+) and long term HSC (LT-HSCs, CD150+ CD48- LSK) populations are decreased in both 16-week and 24-week old male mice; however, this trend did not reach statistical significance (p>0.05). Although the absolute cell count of lymphocytes, including B and T cells (CD19+ and CD3+, respectively) in peripheral blood, is significantly dropped in ATG5 KO mice compared to wt controls, there is no significant change of mature B and T cells in bone marrow. These preliminary results suggest that deletion of a key autophagy regulator in LepR+ bone marrow niche cells will cause leukopenia. The underlying mechanism is currently under investigation and it will help us to better understand the role of autophagy in hematopoiesis within the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Loss of IKKβ but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells.

NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.

Microenvironmental Contribution to Dysfunctional Hematopoiesis in a Murine Model of Myelodysplastic Syndrome

In vitro data provide evidence of an altered bone marrow microenvironment (BMME) in the myelodysplastic syndromes (MDS). To assess the role of the BMME in MDS in vivo, we used a well-established transgenic murine model with expression of the translocation product Nup98-HOXD13 (NHD13) in hematopoietic cells that leads to development of an MDS phenotype, fully penetrant by 5 months of age. In order to assess whether the BMME contributes to diminished hematopoiesis as a feature of MDS, we transplanted marrow from 5-month-old NHD13 mice and normal competitor marrow into irradiated NHD13 mice and their wild type (WT) littermates. Serial analysis of peripheral blood (PB) indicated engraftment of NHD13 marrow was improved in WT recipients relative to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.0001). Flow cytometric analysis of marrow harvested at 16 weeks post-transplant revealed increased NHD13 donor contribution to the hematopoietic stem and progenitor cell (HSPC) pool in WT relative to NHD13 recipients (28.2 ± 4.3 vs. 2.4 ± 0.5 % of total Lineage-, cKit+, Sca1+ (LSK) cells, p<0.01). Surprisingly, leukopoiesis was improved after transplantation of NHD13 marrow into WT as compared to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.01). These data establish that the MDS BMME interferes with the ability of MDS HSPCs to function similarly to normal HSPCs. After the identification of a microenvironmental defect in adult NHD13 mice, we further investigated the NHD13 BMME support for hematopoietic progenitors. By flow cytometric analysis, there were no differences in marrow multipotent progenitors (MPPs) and long term hematopoietic stem cells (LT-HSCs) from NHD13 mice vs. WT littermates at 3 weeks of age. However, in adults there was a progressively severe decline in the NHD13 HSPC pool. HSPCs were not diminished in the spleens of NHD13 mice, suggesting a specific BMME defect. The decrease in phenotypic HSPCs in NHD13 mice was confirmed functionally by competitive repopulation assays using NHD13 or WT donor marrow transplanted into irradiated WT recipients. NHD13-derived PB cells demonstrated marked myeloid skewing relative to WT-derived cells, indicative of a differentiation defect in NHD13-associated hematopoiesis. At 16 weeks post-transplant, recipient marrow was assayed for relative NHD13 and WT donor contributions to the HSPC pool. Consistent with the decreased NHD13 donor contribution to PB counts, NHD13 donor contribution to the HSPC pool in the marrow was diminished (59.4 ± 8.7 vs. 15.5 ± 5.6, % WT donor vs. NHD13 donor contribution to total LSK cells, p<0.001). Despite robust engraftment of WT competitor marrow, cytopenias and macrocytosis were observed in the recipients of NHD13 marrow, suggesting a bystander effect by the NHD13 clone on the function of the normal competitor marrow. To determine NHD13 long-term engraftment function, secondary transplantation of marrow harvested from the primary recipients of NHD13 and WT donors was performed using WT recipients. Serial PB flow cytometric data demonstrated improved overall engraftment of the NHD13 relative to WT donor marrow with persistent and even more marked myeloid skewing of NHD13 donor derived blood cells than was seen in the primary transplant. Consistent with PB data, at 16 weeks post-transplant, the contribution of NHD13 and WT donors to the HSPC pool was similar. Improved NHD13 HSPC number and function in the secondary recipients may be related to BMME rejuvenation through serial passage into a WT BMME. Our data indicate that in this model (1) MDS hematopoietic function is improved in a normal compared to MDS microenvironment (2) the HSPC pool is defective and (3) there is suppression of non-clonal hematopoiesis via a bystander effect, possibly mediated by the MDS BMME. In aggregate our data demonstrate a contributory role of the BMME to ineffective hematopoiesis in MDS, and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Role of Enhanced Microenvironmental Interleukin-1 (IL-1) Expression and Increased IL-1 Responsiveness in Persistence of Leukemia Stem Cells in TKI Treated CML Patients

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in the treatment of chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. In previous studies we have shown that altered expression of inflammatory cytokines in the CML bone marrow (BM) microenvironment provides a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) (Cancer Cell 2012, 21:577). Our studies suggest an important role for the pivotal pro-inflammatory cytokine Interleukin-1α/β (IL-1α/β) in selectively promoting growth of CML LTHSC. Using a transgenic BCR-ABL mouse model of CML and human CML and normal CD34+CD38- cells, we showed that inhibition of IL-1 signaling using recombinant IL-1 receptor antagonist (IL-1RA) in combination with nilotinib (NIL) resulted in significantly greater inhibition of CML LSC, compared with NIL alone (Blood 2013, abstract 512). To further investigate the mechanisms underlying increased IL-1 sensitivity of CML stem cells, we evaluated expression of the IL-1 receptor components, IL-1 receptor-associated protein (IL-1RAP) and IL-1R1, on CML and normal stem cells using flow cytometry. Expression of both IL-1RAP and IL-1R1 were increased on primary CML CD34+CD38-CD90+ cells compared to their normal counterparts (n=5, p<0.05). Exposure to IL-1α (10ng/ml) resulted in increased expression of p-NF-kB (p65), p-p38 MAPK and p-JNK in CML compared to normal CD34+CD38-CD90+ cells as evaluated by flow cytometry, indicating enhanced sensitivity to IL-1 induced signaling (n=5, p<0.05). The expression of p-NF-kB(p65), p-p38 MAPK and p-JNK in CML CD34+CD38-CD90+ cells cultured in CML BM conditioned medium (CM) was reduced after treatment with NIL or IL-1RA, and further reduced by the combination of NIL and IL-1RA (n=4, p<0.05). Immunohistochemistry (IHC) analysis showed that nuclear NF-κB p65 protein was reduced in NIL and IL-1RA treated CML CD34+CD38-CD90+ cells compared with controls. Treatment with NIL and IL-1RA also significantly reduced expression of the NF-κB target genes NFκB1A, BCL2L1, BIRC3 and CD83 (n=6, p<0.001), and of the inflammatory cytokines IL6, CXCL1, CXCL2, CCL2, CCL3, CCL4 and TNF-α (n=6, p<0.05), as assessed by Q-RT-PCR. We evaluated IL-1 expression in BM samples from CML patients with undetectable minimal residual disease (UMRD) using Q-RT-PCR. Interestingly IL-1α, but not IL-1β, expression was increased in BM samples from CML patients with UMRD compared to normal BM samples (n=12, p<0.05). To evaluate the source of increased IL-1α expression we analyzed selected monocyte (CD45+CD14+), non-monocytic myeloid cell (CD45+CD14-CD33+), T cell (CD45+CD14-CD33-CD3+), B cell (CD45+CD14-CD33-CD19+), endothelial cell (CD45-GPA-CD31+) and mesenchymal cell (CD45-GPA-CD31-) populations from BM samples obtained from CML patients with UMRD and from normal healthy controls. These studies revealed significantly elevated IL-1α expression in BM CD14+ monocytic and CD31+ endothelial cells from CML patients with UMRD compared to normal controls (n=12, p<0.05). Our studies indicate that CML LSC demonstrate increased IL-1 receptor expression and IL-1 induced NF-kB, p38 MAPK and JNK signaling. We also observe enhanced IL-1α expression in BM endothelial and monocytic cells from CML patients achieving UMRD, indicating persistence of an inflammatory microenvironment that may contribute to persistence of residual LSC. Our studies provide a strong rationale for the application of anti-IL-1 directed strategies to inhibit inflammatory signaling and enhance LSC elimination in TKI treated CML patients. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Clonal Mapping of Hematopoiesis in Vivo in Humans By Retroviral Vector Insertional Barcoding

▪Hematopoietic stem cells (HSC) are endowed with the unique role of generating an adequate and efficient pool of blood cells throughout human life. Data derived from clonal tracking of HSC activity and hematopoietic dynamics directly in vivo in humans would be of paramount importance for the design of therapies for hematological disorders and cancers. Our gene therapy (GT) clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS) based on the infusion of genetically engineered HSC, constitute unique clinical settings where each vector-marked progenitors and its blood cell progeny is traceable being univocally barcoded by a vector integration site (IS). To study early dynamics of hematopoietic reconstitution in humans, we collected by LAM-PCR + Illumina-Miseq sequencing 14.807.407 sequence reads corresponding to 71.981 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 36 months after GT. We firstly identified and quantified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as marker of the real-time clonal output of individual vector-marked HSC clones in vivo. We unraveled the timing of short, intermediate and long term HSC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. By unsupervised clustering of IS similarities among lineages we unveiled diverse input of HSPC clonal differentiation towards lymphoid, myeloid and megakaryo-erythroid cells and found that NK cells have a distinct relationship with HSPC as compared to T and B cells. We also profiled the level of HSPC output overtime showing that early reconstitution is markedly skewed towards myeloid production. Importantly, clonogenic progenitors generated in vitro from ex vivo purified CD34+ patients’ cells, showed a IS profile coherent with that of freshly purified BM and PB cell types from the same time-point.We also studied population clonal entropy through 7 different diversity indexes and uncovered that progenitor output occurs in distinct waves during the first 6-9 months after transplantation reaching a “homeostatic equilibrium” only by 12 months after GT. At steady state we estimated by mark-recapture mathematical approaches that 1900-7000 transduced HSC clones were stably contributing to the progenitors repertoire for up to 3 years after infusion of gene corrected CD34+ cells. To evaluate the long-term preservation of activity by transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC over a period of 5 years without the manifestation of clonal quiescence phases. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy, can still, once infused, retain in vivo long-term activity in humans. We exploited IS similarities among the lineages for both WAS and ADA-SCID datasets to reconstruct the hematopoietic hierarchy by combining conditional probability distributions and static/dynamic graphical models of dependencies. Notably, preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of hematopoiesis based on a delayed branching of myeloid and lymphoid lineages. Further mathematical models are being applied to specifically study population dynamics and single HSPC contribution to hematopoiesis including stochastic models of neutral clonal drift. More detailed analysis are also being performed on IS collected from 7 distinct CD34+ subtypes isolated from GT patients and FACS sorted according to the most recent markers of HSPC differentiation. Overall our work constitute the first molecular tracking of individual hematopoietic clones in humans providing an unprecedented detailed analysis of HSC activity and dynamics in vivo. The information gathered will be crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors. DisclosuresNeduva:GSK: Employment. Dow:GSK: Employment.

Oncogenic transcription factors, from hematopoietic stem cells to thymocyte reprogramming

Normal hematopoietic stem cells (HSCs) are uniquely endowed with long term self-renewal capacities to sustain blood cell production throughout life whereas progenitors and mature blood cells have finite life spans. HSCs normally reside in the protective microenvironment of the bone marrow where they remain dormant. These cells divide infrequently to produce mature hematopoietic cells without compromising the pool of HSCs. Our results indicate that the SCL/TAL1 transcription factor which is activated in 25% of T-cell acute lymphoblastic leukemia (T-ALL), preserves long term HSC self-renewal activity by maintaining these cells in a quiescent state. SCL occupies a large number of loci in hematopoietic cells, among which Kit is well documented to sustain long term HSC self-renewal, survival and quiescence in adult mice, and to retain HSCs in their niches via interaction with membrane-linked Kit ligand. We now show that Scl and Kit are centrally involved in a feed forward loop that controls hematopoietic cell fate. In acute leukemias, the self-renewal capacity of leukemia initiating cells can be conferred by the cell of origin of leukemia, i.e. primitive HSCs. Alternatively oncogenes acting on committed progenitors can induce stemness genes, transforming non-self-renewing progenitors into pre-leukemic stem cells (pre-LSCs). We capitalized on a transgenic mouse model of T-ALL induced by thymic expression of two oncogenic transcription factors, SCL and LMO1 to show that SCL-LMO1 reprogram a subset of non-self-renewing double-negative thymocytes into self-renewing pre-LSCs. Surprisingly, the NOTCH1 oncogene which is activated in more than 55% of T-ALLs does not reprogram thymocytes on its own. Rather, NOTCH1 enhances the oncogenic reprogramming activity of SCL-LMO1 in a dosage-dependent manner. We will discuss how chromosomal translocations result in the inappropriate activation of HSC self-renewal genes in immature thymocytes and how oncogenic reprogramming differs from cellular reprogramming. Normal hematopoietic stem cells (HSCs) are uniquely endowed with long term self-renewal capacities to sustain blood cell production throughout life whereas progenitors and mature blood cells have finite life spans. HSCs normally reside in the protective microenvironment of the bone marrow where they remain dormant. These cells divide infrequently to produce mature hematopoietic cells without compromising the pool of HSCs. Our results indicate that the SCL/TAL1 transcription factor which is activated in 25% of T-cell acute lymphoblastic leukemia (T-ALL), preserves long term HSC self-renewal activity by maintaining these cells in a quiescent state. SCL occupies a large number of loci in hematopoietic cells, among which Kit is well documented to sustain long term HSC self-renewal, survival and quiescence in adult mice, and to retain HSCs in their niches via interaction with membrane-linked Kit ligand. We now show that Scl and Kit are centrally involved in a feed forward loop that controls hematopoietic cell fate. In acute leukemias, the self-renewal capacity of leukemia initiating cells can be conferred by the cell of origin of leukemia, i.e. primitive HSCs. Alternatively oncogenes acting on committed progenitors can induce stemness genes, transforming non-self-renewing progenitors into pre-leukemic stem cells (pre-LSCs). We capitalized on a transgenic mouse model of T-ALL induced by thymic expression of two oncogenic transcription factors, SCL and LMO1 to show that SCL-LMO1 reprogram a subset of non-self-renewing double-negative thymocytes into self-renewing pre-LSCs. Surprisingly, the NOTCH1 oncogene which is activated in more than 55% of T-ALLs does not reprogram thymocytes on its own. Rather, NOTCH1 enhances the oncogenic reprogramming activity of SCL-LMO1 in a dosage-dependent manner. We will discuss how chromosomal translocations result in the inappropriate activation of HSC self-renewal genes in immature thymocytes and how oncogenic reprogramming differs from cellular reprogramming.

Inhibition Of Microenvironmental Interleukin-1 Signaling Enhances TKI-Mediated Targeting Of Chronic Myelogenous Leukemia Stem Cells

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. Most CML patients need continued TKI treatment to prevent disease relapse, and new strategies to eliminate residual leukemia stem cells are required to enhance possibility of achieving treatment-free remission. In previous studies we have shown that increased several cytokines expressed by leukemia cells may provide a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) within the CML BM microenvironment. Studies evaluating the effects of individual factors indicated that exposure to Interleukin-1α/β (IL-1α/β) at concentrations similar to those observed in CML BM resulted in significantly increased growth of CML compared with normal LTHSC (Cancer Cell 2012, 21:577). Consistent with previous reports (PNAS 2010, 107:16280), we observed that expression of the IL-1 receptor-associated protein (IL-1RAP), an important IL-1 signaling component, was increased in primitive CML cells, potentially explaining enhanced IL-1 sensitivity. To further evaluate the role of microenvironmental IL-1 in maintenance of CML LTHSC, we used recombinant IL-1 receptor antagonist (IL-1RA) to block IL-1 receptor signaling. IL-1RA is clinically approved for the treatment of rheumatoid arthritis. Purified LTHSC (Lin-Sca-1+Kit+Flt3-CD150+CD48- cells) from the SCL-tTA/BCR-ABL inducible mouse model of CML (CD45.1) and from congenic FVBN mice (CD45.2) were mixed in a 1:1 ratio and cultured with CML BM plasma, with and without IL-1RA. Culture with CML BM plasma for 7 days results in significantly increased growth of CML compared to normal LTHSC. The ratio of CML to normal cells was significantly reduced in the presence of IL-1RA (2.5μg/ml) (3.6:1 without IL-1RA, 1.7:1 with IL-1RA, p=0.0002), indicating that inhibition of IL-1 signaling reduced the growth advantage of CML LTHSC cultured in CML BM plasma. We next investigated the effect of IL-1RA on CML hematopoiesis in vivo. BM cells from CML mice (CD45.1) were transplanted into congenic FVBN mice (CD45.2) to generate CML-like disease in recipient mice. Four weeks after transplantation mice were treated with Nilotinib (NIL, 50mg/kg/d, gavage), IL-1RA (150mg/kg/d s.c.), the combination of NIL and IL-1RA, or vehicle (control) for 3 weeks. Treatment with NIL plus IL-1RA resulted in significantly greater reduction in CD45.1+ CML cells in blood, and in CML LTHSC, MPP, CMP and GMP in BM, compared with NIL alone (CML LTHSC/2 femurs: control 738±122, NIL 486±94, IL-1RA 525±49, combination 360±33, P=0.01 combination vs. Nilotinib). Mice treated with NIL plus IL-1RA also showed significantly prolonged survival after completion of treatment compared to mice treated with NIL alone (median survival 6 days for NIL alone versus 45 days for combination, p=0.02). Following transplantation of BM cells from treated mice into 2nd recipients (CD45.2), significantly lower CML cell engraftment in BM and reduced development of leukemia was seen after transplantation of cells from mice treated with the combination compared with NIL or untreated controls (8 out of 8 mice developed leukemia for control, 6 out of 8 for NIL, 5 out of 8 for IL-1RA, 3 out of 8 for the combination). We also studied the effect of treatment with NIL (5μm), IL-1RA (5μg/ml), NIL+IL-1RA, or vehicle for 72 hours on human CML and normal CD34+CD38+ and CD34+CD38- cells cultured with CML BM conditioned medium (CM). The combination of NIL and IL-1RA significantly reduced CML CD34+CD38+ and CD34+CD38- cell growth compared to Nilotinib alone (CD38- cells: NIL 23.7±10.1%, combination 13.1±8.9% of control, p<0.05), cell division (measured by CFSE labeling) (CD38- proliferation index: NIL 3.3±1.0, combination 2.4±0.6, p=0.06) and CFC frequency in methylcellulose progenitor assays (CD38- cells: NIL 67±22 per 1000 cells, combination 39±26, p<0.05); and moderately increased apoptosis of CML CD34+CD38- cells. We conclude that inhibition of microenvironmental IL-1 signaling using IL-1RA significantly increases inhibition of self-renewing murine and human CML stem cells in combination with NIL. Our results support further evaluation of IL-1 inhibition as a strategy to enhance elimination of CML LSC in TKI-treated patients. Disclosures:Holyoake:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees.

Autophagy Is Required For Long-Term Hematopoietic Stem Cell (HSC) Function and G-CSF-Induced HSC Mobilization

Hematopoiesis originates from a rare pool of hematopoietic stem cells (HSC) that are uniquely capable of both self-renewal and terminal differentiation into lineage-committed progenitor cells. Autophagy is a process of cytoplasmic protein recycling which maintains cellular homeostasis and protects the cell during periods of metabolic stress and nutrient deprivation and has an established role in the survival and function of immunological cells. Recent publications have linked autophagy with preservation of normal long term HSCs (LT-HSCs) during aging (Nature; 2013: 494:323-7). We therefore sought to determine the role of autophagy in LT-HSC function at homeostasis and during the clinically relevant stress of G-CSF-induced HSC mobilization.Using single cell imaging flow cytometry to monitor autophagosome formation in LC3-GFP transgenic mice (LC3-GFP punctae formation is a reporter of autophagy activity) we demonstrated autophagic activity in HSC populations but not in committed myeloid progenitors. In line with this, inhibition of autophagy degradation by chloroquine administration resulted in the accumulation of autophagy related protein p62 in purified HSCs compared to myeloid progenitors. To determine the contribution of autophagy to HSC development and function, we analyzed mice deficient in Atg5, a protein essential for autophagosome formation. Autophagy was not required for fetal liver (FL) HSC development, however, Atg5-/- FL HSCs showed mildly reduced long-term repopulating function in bone marrow (BM) transplantation assays (16 weeks peripheral blood (PB) engraftment Atg5-/- 83.45% vs. WT 93.10%, n=12, p<0.001). Importantly, Atg5-/- LT-HSCs (Lin-Sca-1+c-kit+Flk2-CD150+CD48-) were markedly reduced in congenic recipients compared to WT FL LT-HSCs (Atg5-/- 0.02% vs. WT 0.04%, n=11, p<0.001). Secondary competitive transplantation was used to determine the effect of autophagy loss on LT-HSC function in vivo. Atg5-/- LT-HSCs exhibited a profound impairment in the repopulation of secondary congenic recipients (Atg5-/-4.65% vs. WT 32.3%, n=5, p<0.01).Mechanistically, microarray analysis of purified LT-HSCs from Atg5-/- vs. WT FL chimeras demonstrated clear differences in gene expression by unsupervised hierarchical clustering. Differentially expressed genes included Cxcl12, Sdc2 and Apex1, regulators of HSC fate. Gene Ontology enrichment analysis demonstrated that autophagy deficient LT-HSCs had impaired metabolism, enhanced cellular differentiation, enforced proliferation and increased apoptosis. Validating these findings, there was a loss of quiescence in the Atg5-/- compared to WT LT-HSC (quiescent Atg5-/- 30% vs. WT 39%, n=16 p<0.05) and Atg5-/- FL HSCs exhibited enhanced apoptosis after culture in cytokine enriched media (Atg5-/-21.2% vs. WT 14.2%, n=5, p<0.01).Given the requirement for autophagy in homeostasis of LT-HSC and its role in proliferation and metabolic stress, we next investigated whether autophagy participated in the HSC response to G-CSF. G-CSF is commonly used to ameliorate neutropenia in patients treated with chemotherapy and is also used to mobilize HSCs for patients undergoing HSC transplantation. Using single cell imaging flow cytometry, autophagosome formation was enhanced in HSCs after 6 days of G-CSF mobilization (10mcg/day). G-CSF treatment efficiently mobilized PB neutrophils and colony forming units (CFU) in WT chimeras, however Atg5-/- chimeras showed a striking reduction in G-CSF-induced neutrophil (Atg5-/- 4.95 x106/mL vs. WT 10.46 x106/mL, n=20, p<0.0001) and PB CFU mobilization post-G-CSF (Atg5-/- 47/100µL vs. WT 126/100µL, n=5, p<0.01). BM CFU numbers were similar in Atg5-/- and WT FL chimeras both pre- and post-G-CSF mobilization. Atg5-/-neutrophils demonstrated increased apoptosis after G-CSF treatment suggesting that autophagy limits PB neutrophil survival, but not BM neutrophil development during G-CSF mobilization.These data demonstrate that autophagy is an active process in LT-HSCs and that genetic deletion of Atg5 results in the failure of adult LT-HSC maintenance and function. Autophagic activity is augmented by G-CSF-induced HSC stress and is required for G-CSF-induced HSC mobilization. These findings are particularly relevant to HSC transplantation and hematopoietic function in the context of a rapid rise in the clinical use of agents that have modulatory effects on autophagy. Disclosures:No relevant conflicts of interest to declare.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures:No relevant conflicts of interest to declare.