ALBERT

Description: 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

Description: Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system. Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions. To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p 〈 0.05). Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors 〉50 years old compared to

Description: Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of Cdh2, the gene encoding N-cadherin, has been targeted to cells of the osteoblastic lineage. Targeted deletion of N-cadherin resulted in an age-dependent bone phenotype, ultimately characterized by decreased mineralized bone, but no difference in steady-state HSC numbers or function at any time tested, and normal recovery from myeloablative injury. Intermittent parathyroid hormone (PTH) treatment is well established as anabolic to bone and to increase marrow HSCs through microenvironmental interactions. Lack of osteoblastic N-cadherin did not block the bone anabolic or the HSC effects of PTH treatment. This report demonstrates that osteoblastic N-cadherin is not required for regulation of steady-state hematopoiesis, HSC response to myeloablation, or for rapid expansion of HSCs through intermittent treatment with PTH.

Description: The bone marrow provides an essential regulatory microenvironment for adult hematopoiesis, however the relationship between the bone marrow microenvironment and malignant hematopoiesis remains poorly understood. To investigate the interactions between leukemia and the bone marrow microenvironment we utilized a mouse model of blast-crisis chronic myelogenous leukemia (BC-CML), in which primitive normal murine hematopoietic cells are modified to leukemic cells by expressing the translocation products BCR/ABL and Nup98/HoxA9. The presence of each translocation was confirmed by their co-expression of Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP) respectively. Ten days after injection of GFP+/YFP+ leukemic cells into strain-matched immunocompetent, non-myeloablated recipient mice, 50% of the bone marrow was composed of leukemic cells as determined by flow cytometric analysis. Histologic analysis of the contralateral tibiae and femora demonstrated not only progressive replacement of the bone marrow by leukemic cells, but also a significant bone loss. Histomorphometric analysis confirmed 50% decreased trabecular bone volume in leukemic mice compared to control mice that were not injected with leukemic cells (bone volume/total volume (%): 12±2 vs 26±2 p=0.01). Interestingly, numerous multi-nucleated osteoclasts were observed in the bone marrow of leukemic mice and were localized adjacent to leukemic cells, suggesting that leukemic cells may affect osteoclastogenesis and result in massive bone loss. To test this hypothesis, we first measured the expression of known regulators of osteoclastogenesis, including RANKL, in our leukemic cells by quantitative RT-PCR analysis. Compared to GFP−/YFP− cells, GFP+/YFP+ cells have 3-fold increased expression of RANKL, a major osteoclastogenic cytokine. We then examined if leukemic cells can give rise to osteoclasts in the presence of RANKL and M-CSF in vitro and found that these cells were unable to differentiate into osteoclasts themselves. To determine if leukemic cells can induce osteoclastogenesis of normal osteoclast progenitors, we cocultured spleen-derived osteoclast precursors from wild-type mice with GFP+/YFP+ leukemic cells or GFP−/YFP− non-leukemic cells in osteoclastic differentiating media containing optimal concentrations of M-CSF and RANKL. As expected, there was abundant formation of mature osteoclasts, identified as TRAP+ multinucleated cells, in control cultures containing non-leukemic cells and osteoclast precursors. Leukemic cells significantly increased TRAP+ mono-nucleated osteoclast precursors (No. TRAP+ mononucleated cells/well: 34±3.3 vs 20±6.0 in non-leukemic cells, p=0.0136). Under this culture condition, we did not observe increased mature osteoclast formation by leukemic cells. Surprisingly, we found that osteoclast precursors strongly prolonged the survival of leukemia cells. In control cultures without a feeder layer of osteoclast precursors there were no viable leukemia cells present after 6 days in culture while in the co-culture system viable leukemia cells were still abundant after 6 days in culture, identifiable by their expression of GFP/YFP (No. GFP+/YFP+/high power field: 0 vs 142±6.4, p

Description: Background/Rationale: Hematologic malignancies are known to remodel the bone marrow microenvironment, reducing support for normal hematopoiesis while increasing support for the malignant clone. The chemokine CCL3 has been demonstrated to play a role in microenvironmental dysfunction in multiple malignancies including myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome. In addition, CCL3 has been shown to be critical for the progression of chronic mylogenous leukemia in murine models. However, to consider anti-CCL3 therapy as an option for hematologic malignancies we must first understand its role in the regulation of normal hematopoiesis. To date the role of CCL3 in this process is poorly understood. Methods/Results: In these experiments we utilized genetically altered mice with a global loss of CCL3 (CCL3KO) on a C57bl/6 background. Peripheral blood counts revealed that monocytes, granulocytes, and red blood cells were all significantly decreased in the peripheral blood of CCL3KO mice as compared to WT controls at 12 weeks of age (9.78 ± 0.3 vs. 8.06 ± 0.2 RBCs*106/μl, WT vs. CCL3KO p≤0.001 n=8 mice/group). CCL3KO mice also demonstrate a 2-fold increase in the frequency and number of phenotypic long-term hematopoietic stem cells (LT-HSCs: Lin-sca1+ckit+flt3-CD150+CD48-) at 12 weeks of age in the bone marrow by flow cytometric analysis (0.0053 ± 0.0005 vs. 0.0106 ± 0.0007 % of cells, WT vs. CCL3KO p≤0.0001 n=8 mice/group). A significant increase was also seen in short-term HSCs (ST-HSCs), but not in multipotent progenitor (MPP) populations (data not shown), suggesting that CCL3 regulates the most immature hematopoietic cells. To quantify functional hematopoietic stem cells in the marrow of CCL3KO mice competitive transplants were performed using whole bone marrow cells. In primary transplants CCL3KO mice demonstrated a small but significant decrease in engraftment over 22 weeks when compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Decreased engraftment was seen in B cells, T cells, and myeloid cells in the peripheral blood. Upon secondary transplantation the decrease in engraftment of HSCs from CCL3KO donor mice was much more profound. At 16 weeks post-transplant HSCs from CCL3KO donors contributed to hematopoiesis at a rate 5 times lower than WT littermate controls (64.67 ± 1.967 vs. 11.97 ± 5.322 % of cells, WT vs. CCL3KO p≤0.0001 n=10 mice/group). These results were seen in both male and female mice and suggest that, although phenotypic HSCs were increased in the bone marrow of CCL3KO mice, those HSCs were defective. To test this hypothesis we sorted Lineage-Sca1+Ckit+Flt3- (Flt3-LSK) bone marrow cells enriched for LT-HSCs in order to establish stem cell activity on a per cell basis through competitive transplantation. As with the whole bone marrow transplants, primary transplant of sorted Flt3-LSK cells resulted in reduced engraftment of CCL3KO cells as compared to WT littermate controls (2-way ANOVA, p≤0.0001 over 22 weeks, n=8 mice/group). Surprisingly, upon secondary transplantation, CCL3KO Flt3-LSK donor cells performed better than the WT littermate controls (2-way ANOVA, p

Description: 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. Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

Description: Abstract 642 HSCs are rare immature cells capable of reconstituting all blood cell lineages throughout the life of an individual. We have previously shown that intermittent treatment with PTH is sufficient to increase the number of HSCs in the marrow of mice. This PTH effect is blocked in vitro with inhibition of gamma-secretase, the mediator of a required step in Notch signaling. Osteoblastic cells are a critical component of the HSC niche and are likely mediators of the PTH-induced increase in HSCs. Specifically the Notch ligand Jag 1 is expressed on osteoblasts and is therefore implicated as a mechanism through which PTH acts on HSCs. Therefore we investigated in vivo the role of osteoblastic Jag 1 in the PTH-dependent increase in HSCs. We utilized the 2.3kb collagen 1 promoter driven cre recombinase to specifically excise Jag 1 from osteoblastic cells in mice (OBJag1 mice). As we previously reported treatment of wild type (WT) controls with PTH 3 times daily for 10 days resulted in a significant increase in phenotypic HSC populations including Lin-Sca1+cKit+CD48-CD150- short-term HSCs (ST-HSCs) (VEH/PTH 0.0405±0.001 vs 0.0650±0.0038, p≤0.0001) and Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (LT-HSCs) (VEH/PTH 0.0077±0.0008 vs 0.0125±0.00096, p≤0.01) as determined by flow cytometric analysis. In contrast treatment of OBJag1 mice did not result in a phenotypic increase in these populations. Despite the lack of a phenotypic increase in HSCs in OBJag1 mice, when HSC function was assessed by competitive repopulation assay, OBJag1 marrow cells demonstrated the same increased repopulating ability as WT mice (WT: VEH/PTH 12.16±2.7 vs 22.32±2.4, p≤0.01, OBJag1: VEH/PTH 13.6±1.8 vs 31.6±5.9, p≤0.01). Upon secondary transplantation however, HSCs from OBJag1 donors treated with PTH resulted in a lower engraftment rate than VEH treated controls (VEH/PTH 14.61±3.8 vs 4.38±0.9, p≤0.05). This result suggests that osteoblastic Jag 1 is necessary for the increase in phenotypic HSCs resulting from PTH treatment and is required to maintain LT-HSC self-renewal. However these data also suggest an osteoblastic Jag 1 independent mechanism that mediates a transient increase in repopulating ability. Decreased apoptosis is a potential mechanism by which PTH may functionally increase HSCs in the absence of increased self-renewal. To determine if PTH treatment decreases the apoptosis rate of HSCs, WT mice were treated intermittently with PTH once a day for 7 days. Despite a lack of increased HSCs by phenotypic analysis at 7 days, marrow from PTH treated mice displayed an increase in LT-HSC function as measured by competitive transplantation. We determined to measure the effect of PTH on apoptotic rates of HSCs using Annexin V membrane expression. By the 7th day of PTH treatment, LT-HSC apoptotic rates were decreased in the PTH treated group (VEH/PTH 10.482±2.25 vs. 6.27±1.93, p≤0.01) suggesting that changes in apoptotic rate of LT-HSCs precedes the HSC increase. These results were confirmed by flow cytometric measurement of activated caspase 3. PTH treatment decreased the percentage of LT-HSCs that were positive for activated caspase 3 (VEH/PTH 4.3±0.5 vs. 2.4±0.3, p≤0.01). PTH induced micro-architectural changes in trabecular bone at day 7 of treatment suggesting bone involvement despite the lack of an increase in bone volume. These results suggest for the first time that PTH may exert its beneficial effect on bone marrow reconstitution through both Jag 1 dependent and independent effects. Additionally, HSCs demonstrate decreased apoptotic rates and increased reconstitution ability prior to a demonstrable phenotypic increase, mimicking the effect seen in the absence of osteoblastic Jag 1. Together these results suggest that the decreased apoptotic rate may be mediated by an osteoblastic Jag 1 independent mechanism. Whether osteoblasts are required for the observed osteoblastic Jag 1 independent effects remains to be seen as these effects could be mediated by a Jag 1 independent osteoblastic mechanism or by an altogether different cellular component of the HSC niche. Further, since stressful manipulation of HSCs ex vivo is essential for their use in transplantation, defining factors regulating and decreasing their apoptosis may improve their engraftment efficiency, expanding their clinical use when their numbers are limited. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points An in vivo model of MDS displays time-dependent defects in HSPCs and in microenvironmental populations. Normalization of the marrow microenvironment alters disease progression and transformation and improves hematopoietic function.