Decreased Bone Marrow Cellularity Research Articles

TGF-β signaling in myeloproliferative neoplasms contributes to myelofibrosis without disrupting the hematopoietic niche.

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre–targeted MSCs prevented the development of myelofibrosis in both MPLW515L and Jak2V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPLW515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPLW515L was intact in Osx-Cre Smad4fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPLW515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.

ERK2 Substrate Binding Domains Perform Opposing Roles in Pathogenesis of a JAK2V617F-Driven Myeloproliferative Neoplasm

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines.To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients.To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction.Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. DisclosuresLevine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.

JAK inhibition for murine HLH requires complete blockade of IFN-γ signaling and is limited by toxicity of JAK2 inhibition

AbstractHemophagocytic lymphohistiocytosis (HLH) is an inflammatory disorder in which numerous cytokines are elevated, though interferon-γ (IFN-γ) is central to disease pathogenesis and a key therapeutic target. Experimental and early clinical reports have shown that ruxolitinib, a small molecule inhibitor of Janus kinases (JAKs), which are essential for cytokine signaling, may be therapeutic in HLH. In contrast, we found that intermittently administered ruxolitinib at various dose levels failed to prevent HLH development or treat established murine HLH. High doses of ruxolitinib blocked IFN-γ signaling only transiently after administration, consistent with human pharmacokinetics, and only continuously administered drug could prevent HLH development or treat established HLH. Continuously administered ruxolitinib was therapeutic in only a narrow dose range and intermittently dosed ruxolitinib worsened survival and decreased bone marrow cellularity of animals concurrently treated with anti-IFN-γ antibody, indicating a narrow therapeutic window and potential toxicity. Because JAK2 is essential for hematopoietic cytokine signaling, we also tested a JAK1-selective inhibitor and observed therapeutic benefit without apparent toxicity, though it did not improve survival when combined with anti-IFN-γ. We conclude that continuous blockade of IFN-γ signaling is necessary for optimal control of HLH and that JAK2 inhibition may be toxic in this disorder.

Gaining a Better “Sense” for ASXL1 Mutations in Myelofibrosis: Assessing the Variable Impact of Frameshift, Nonsense, and Missense Mutations

Introduction: ASXL1 gene mutations commonly occur in myeloid diseases and are associated with inferior prognoses. They can occur as frameshift, nonsense or missense mutations. In myelofibrosis (MF) specifically, ASXL1 mutations are associated with worse overall survival (OS) and leukemia-free survival (LFS). While the general impact of ASXL1 mutations has been well-studied, the variable impact based on type of mutation is less clear. In CMML and MDS, frameshift and nonsense mutations correlate with shorter survival and increased risk of leukemic transformation. In this study, we aimed to analyze the role of ASXL1 mutations in MF while stratifying for type of mutation.Methods: We retrospectively analyzed MF patients seen at our institution who had next-generation sequencing (NGS) performed as part of routine clinical care between 9/1/2013 and 5/1/2017. Laboratory and clinical parameters calculated from first presentation. OS defined from time of NGS assessment.Results: We evaluated 182 MF patients with available molecular profiling. ASXL1 mutations were present in 71 (39%) patients. Among ASXL1 mutations, 44 were frameshift (FS) mutations (62%), 14 were nonsense (NS) mutations (20%) and 13 were missense (MS) mutations (18%). Demographic and clinical features of these groups can be seen in figure 1. Grouped together, ASXL1 mutations were associated with high-risk clinical features such as decreased hemoglobin (p = 0.003), decreased platelet count (p = 0.01), increased bone marrow fibrosis (p = 0.02), transfusion dependence (p = 0.005), increased LDH (p = 0.02), and higher MF-specific prognostic scores (IPSS, DIPSS, and DIPSS+) (p < 0.001, p < 0.001, p = 0.003, respectively). Excluding MS mutations, the correlation with high-risk features was strengthened and new correlations between ASXL1 mutations and leukocytosis (p = 0.006), monocytosis (p = 0.02), and the presence of EZH2 (p = 0.04), NRAS (p = 0.008), and SRSF2 (p = 0.01) mutations emerged. ASXL1 NS mutations were associated with presence of splenomegaly (p = 0.01), increased immature myeloid cells (p = 0.03), and high IPSS (p = 0.04) and DIPSS (p = 0.04) scores when compared to ASXL1 wild-type (WT) patients. ASXL1 MS mutations correlated with decreased hemoglobin (p = 0.03) and decreased bone marrow cellularity (p = 0.007), but did not correlate with prognostic scores.High molecular risk (HMR) mutations (SRSF2, EZH2, IDH1/2) occurred more commonly with ASXL1 FS mutations than with ASXL1 MS mutations (p = 0.003) or ASXL1 WT (p = 0.002). SRSF2 mutations occurred more commonly in patients with ASXL1 FS mutations compared to ASXL1 MS mutations (p = 0.02) and ASXL1 WT (p = 0.03). EZH2 mutations occurred more commonly with ASXL1 FS mutations than with ASXL1 WT (p = 0.02).Including all ASXL1 mutations, there was a trend towards inferior LFS (p = 0.12) when compared to ASXL1 WT. Excluding NS and MS mutations, ASXL1 FS mutations were associated with decreased LFS (p = 0.02). The rate of transformation to blast-crisis was 14% with FS mutations, 0% with NS and MS mutations, and 4% in ASXL1 WT patients. ASXL1 mutations were associated with inferior OS (p = 0.005). When assessed by type of mutation, only FS mutations were associated with inferior OS (p = 0.003). NS mutations were not associated with OS (p = 0.39).Conclusion: The impact of ASXL1 mutations on clinical phenotype and outcome is most clearly demonstrated with frameshift mutations. Nonsense mutations appear clinically significant given their correlation with phenotype and higher-risk disease, but their impact on survival is less clear. Missense mutations do not have an apparent impact in MF. Increased presence of additional high-risk mutations in patients with ASXL1 frameshift mutations suggests a more unstable genome and likely contribute to inferior outcomes. [Display omitted] DisclosuresSallman:Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Lancet:Pfizer: Other: Institutional research funding; Biopath, Biosight, Boehringer Ingelheim, Celator/Jazz, Celgene, Janssen, Karyopharm Therapeutics, and Novartis: Consultancy. Komrokji:Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau.

Exploring the B cell microenvironment in von-Hippel Lindau (Vhl) Dmp1-specific knockout mice

Abstract Osteoblasts (OBs) and osteocytes (OCYs) have been shown to support B cell development. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor degradation and is involved in responses to low oxygen environments. We utilized Dmp1-Cre;Vhl conditional knockout mice (cKO), in which Vhl is deleted in OBs and OCYs. The cKO display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to wild-type (WT) mice. B cell progenitors were significantly increased in frequency and numbers whereas Hardy Fractions B, C, D, E and F were significantly decreased in the cKO BM. cKO Fraction B-C cells showed an increase in apoptosis and decreased proliferation. Reciprocal BM chimeras showed significant reduction in Fractions B-C and later Fractions in the WT→cKO, while B cell development was normal in the cKO→WT chimeras, indicating a cell-extrinsic effect of Vhl on B cells within the cKO BM microenvironment. We hypothesize that B cell development is not supported due to reduction of key niche cells such as stromal cells, OBs, mesenchymal stem cells and decreased production of key cytokines. In preliminary studies, we found that CXCL12 and SCF, key cytokines for B cell development, were reduced in the cKO BM serum. We have begun longitudinal studies using intravital and ex vivo imaging of the cKO BM to identify temporally dependent changes in oxygenation, blood vessel type, structure, permeability, and location of stromal cells. Ex vivo long bone imaging of cKO BM in 6-month-old mice revealed an increase in blood vessel diameter compared to WT. These studies will reveal details and novel molecular mechanisms by which Vhl in Dmp1 expressing cells may serve as a B cell niche.

Deletion of von-Hippel Lindau in Dmp1-expressing cells impairs B cell development

Abstract Our objective is to characterize how altered bone homeostasis influences B lymphocyte development. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor degradation and is involved in responses to low oxygen environments. Osteoblasts (OBs) support B cell development and mature into osteocytes (OCYs). We utilized Dmp1-Cre;Vhl conditional knockout mice (cKO), in which Vhl is deleted in OBs and OCYs. The cKO display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and an overall decrease in BM cellularity compared to wild-type (WT) mice. B cell progenitors is significantly increased in frequency and numbers whereas Hardy Fractions B, C, D, E and F were significantly decreased in the BM of cKO mice. Analysis of BM B cells 16 weeks after whole BM transplantation, showed a significant reduction in Fractions B-C and later Fractions in the WT→cKO mice, while B cell development was normal in the cKO→WT chimeras, indicating a cell-extrinsic effect of Vhl on B cells within the cKO BM microenvironment. We hypothesize that B cell development is not supported due to reduction of key niche cells and decreased production of IL7, SCF, CXCL12, and Flt3. To define the defective niche cells, we will use in vitro B cell development assays in which WT B cells will be co-cultured with sorted cKO OBs, MSCs, and OCYs. B cell apoptosis, proliferation and turnover rates will be measured. We have begun preliminary studies using intravital and ex vivo imaging of the cKO BM to identify changes in oxygen tension, blood vessel type and structure, the presence and location of stromal cells. These studies have the potential to reveal novel molecular mechanisms by which Vhl in Dmp1 expressing cells may serve as a B cell niche.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.

Erythropoietin-Induced Changes in Bone and Bone Marrow in Mouse Models of Diet-Induced Obesity

Obesity remodels bone and increases bone marrow adipocytes (BMAT), which negatively regulate hematopoiesis and bone. Reduced BMAT could restore altered hematopoiesis and bone features. We analyzed the potential of erythropoietin (EPO), the cytokine required for erythropoiesis, to inhibit BMAT in C57BL6/J mice fed four weeks of a high-fat diet (HFD). Acute EPO administration markedly decreased BMAT in regular chow diet (RCD) and HFD-fed mice, without affecting whole body fat mass. Micro-CT analysis showed EPO reduced trabecular bone in RCD- and HFD-fed mice, but EPO-treated HFD-fed mice maintained cortical bone mineral density and cortical bone volume, which was reduced on RCD. Despite achieving similar increased hematocrits with BMAT loss in RCD- and HFD-fed mice treated with EPO, decreased bone marrow cellularity was only observed in RCD-fed mice concomitant with an increasing percentage of bone marrow erythroid cells. In contrast, in HFD-fed mice, EPO increased endothelial cells and stromal progenitors with a trend toward the normalization of marrow homeostasis. EPO administration increased c-terminal FGF23 and intact serum FGF23 only in HFD-fed mice. These data demonstrate the distinct EPO responses of bone and marrow in normal and obese states, accompanying EPO-induced loss of BMAT.

Hematological Disorders in Human Patients with Son Mutations

Precise regulation of transcription and RNA splicing is critical for controlling hematopoietic cell fate determination and lineage differentiation. Alteration of expression of lineage-specific transcription factors and several core spliceosome components in hematopoietic malignancies highlight the significance of abnormal transcription and RNA splicing as disease-causing factors. Our group previously demonstrated that SON, a large nuclear speckle protein possessing dual abilities to bind both DNA and RNA, functions as a splicing factor as well as a transcriptional repressor. We recently identified heterozygous loss-of-function mutations in the SON gene from children with intellectual disability and developmental delay often with a broad spectrum of other congenital anomalies. The disorder caused by SON haploinsufficiency has been designated as ZTTK syndrome (Zhu-Tokita-Takenouchi-Kim syndrome; OMIM #617140). The majority of the mutations found in these patients are frameshift or nonsense mutations which cause degradation of the mutation-bearing transcript. While the most prominent features of these patients are brain malformations and musculoskeletal abnormalities, we identified various hematological disorders from children with ZTTK syndrome. Notable symptoms include bone marrow failure, severe anemia, immunoglobulin deficiency, thalassemia, polycythemia, polycythemia vera, stroke due to blood clots, and leukocytopenia. Apart from the ZTTK syndrome, SON is known to be upregulated in acute myeloid leukemia (AML) patients and is correlated to altered hematopoietic differentiation. To investigate how altered SON expression affects hematopoiesis, we generated a mouse line with the Son gene deleted specifically in the hematopoietic lineage. Homozygous deletion of Son in hematopoietic lineage led to embryonic lethality, indicating that SON expression in blood cells is indispensable during development. Mice with heterozygous deletion of Son in the hematopoietic lineage were viable and born without notable defects or sign of diseases. However, there is a significant decrease in bone marrow cellularity in the mice with heterozygous deletion of Son. Furthermore, Son haploinsufficiency decreased the size of the lineage negative (Lin-) cell population and short-term hematopoietic stem cell (ST-HSC) population with a concurrent increase in megakaryocyte/erythrocyte lineage-biased multipotent progenitors (MPP2) within hematopoietic stem/progenitor cells. These findings suggest that the level of Son expression potentially affects stem cell maintenance and MPP lineage bias, and the distortion of the subpopulation balance within hematopoietic stem/progenitors is possibly linked to multiple hematological disorders. Our ongoing analyses of hematopoiesis and gene expression changes using this mouse model will expand our knowledge about the role of SON in several hematological disorders and benefit clinical practice for ZTTK syndrome patients. Disclosures No relevant conflicts of interest to declare.

Disease Phenotype and Clonal Amplification in Calreticulin del52 and ins5 Knock-in Mice Are Dependent on the Type of Mutations and Gene Dosage

Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustrated by the presence of reticulin fibers stained with silver, mainly in spleen. Presence of fibrosis was not as pronounced in heterozygous del52 mice and more rarely observed in spleen of homozygous ins5 KI mice. In homozygous del52 KI mice, there was a significant amplification of the HSC compartment in both BM and spleen that was stronger than in homozygous ins5 mice. To study whether del52 and ins5 could provide a competitive advantage to HSCs, we performed BM transplantation with increasing percentages of non-induced homozygous del52 or ins5 cells with wild-type GFP+ cells into lethally-irradiated recipient mice. The homozygous del52 BM cells strongly competed wild-type hematopoiesis from an initial engraftment as low as 10% of mutated clones, reaching 100% in both blood myeloid cells and BM HSC compartments at 4 months. In contrast, out-competition of wild-type hematopoiesis by homozygous ins5 cells was slower, especially when less than 50% of mutated cells were initially engrafted suggesting that del52 provides a stronger advantage to the HSCs than ins5. Conclusion In conclusion, these results demonstrate that modeling CALRdel52 and ins5 mutations in mice can successfully recapitulate the differences in phenotype observed in patients, i.e del52 KI mice recapitulate an ET progressing to MF while ins5 KI mice only mimic an ET. This might be explained by a more profound effect of del52 than ins5 at the level of HSC. These KI mice offer solid in vivo models to investigate the mechanism of action of both types of mutations on HSCs and MKs and will be used to test new therapeutic approaches. Disclosures: No relevant conflicts of interest to declare. Disclosures Constantinescu: AlsaTech: Other: Co-Founde; Wiley &amp; Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

The role of the von-Hippel Lindau gene in osteocytes on B lymphocyte maturation and activation

Abstract The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation, which is involved in cellular adaptation to low oxygen environments. Conditional deletion of Vhl in osteoblasts and in hematopoietic progenitors have demonstrated a role for VHL in these cell types, but how changes in bone homeostasis affects immune development are not fully understood. Osteoblasts (OBs) support B cell development and mature into osteocytes (OCYs). To test if OCYs also support B cell development, we generated OCY-specific (Dmp1-Cre) conditional VHL knockout (cKO) mice. We previously found that cKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to controls. In line with this, the frequencies and numbers of B cell precursors (B220+ IgM−), immature B (B220+ IgMint) and recirculating B (B220+ IgMhigh) cells in the bone marrow are significantly decreased. Although the absolute numbers of B cells in the spleens of cKOs were normal, frequency of B220+ CD19+ cells are decreased, while B1a (IgM+ CD5+) cells are increased. To define the mechanisms by which changes in the bone microenvironment affect B cells, we are investigating oxygenation status, vascular architecture and metabolic profiles of bone and B cells. We hypothesize that loss of VHL in OCYs could permanently and adversely affect B cell functional response. To test this, responses in T-independent and T-dependent antigens will be assessed in cKOs. Taken together, our data suggest that OCY-specific VHL deletion might be helpful to increase bone density, but at the expense of B cells.

Lifelong Residual bone Marrow Damage in Murine Survivors of the Hematopoietic Acute Radiation Syndrome (H-ARS): A Compilation of Studies Comprising the Indiana University Experience.

Accurate analyses of the delayed effects of acute radiation exposure in survivors of the hematopoietic acute radiation syndrome are hampered by low numbers of mice for examination due to high lethality from the acute syndrome, increased morbidity and mortality in survivors, high cost of husbandry for long-term studies, biological variability, and inconsistencies of models from different laboratories complicating meta-analyses. To address this, a compilation of 38 similar hematopoietic acute radiation syndrome studies conducted over a 7-y period in the authors' laboratory, comprising more than 1,500 irradiated young adult C57BL/6 mice and almost 600 day-30 survivors, was assessed for hematopoietic delayed effects of acute radiation exposure at various times up to 30 mo of age. Significant loss of long-term repopulating potential of phenotypically defined primitive hematopoietic stem cells was documented in hematopoietic acute radiation syndrome survivors, as well as significant decreases in all hematopoietic lineages in peripheral blood, prominent myeloid skew, significantly decreased bone marrow cellularity, and numbers of lineage-negative Sca-1+ cKit+ CD150+ cells (KSL CD150+; the phenotype known to be enriched for hematopoietic stem cells), and increased cycling of KSL CD150+ cells. Studies interrogating the phenotype of bone marrow cells capable of initiation of suspension cultures and engraftment in competitive transplantation assays documented the phenotype of hematopoietic stem cells in hematopoietic acute radiation syndrome survivors to be the same as that in nonirradiated age-matched controls. This compilation study adds rigor and validity to our initial findings of persistent hematopoietic dysfunction in hematopoietic acute radiation syndrome survivors that arises at the level of the hematopoietic stem cell and which affects all classes of hematopoietic cells for the life of the survivor.

Differential Toxicity Profile of Busulfan and Treosulfan on Endothelial Cells In Vivo - Relevance to Hematopoietic Stem Cell Transplantation

The success of Hematopoietic stem cell transplantation (HSCT), an established curative treatment option for hematological malignancies and non-malignant diseases, is often limited by regimen related toxicity (RRT) caused by conditioning regimen drugs. Despite their intended marrow toxicity, the conditioning drugs also potentially damage endothelium of different organs leading to RRT such as sinusoidal obstruction syndrome. Among different conditioning drugs, busulfan (Bu) and treosulfan (Treo) (a structural analog of Bu), although widely used in HSCT, exhibit differences in their toxicity profile, the mechanism of which is still unclear. Here we compared the effect of Bu and Treo in mice model to identify factors causing the differences in toxicity. BALB/c mice (6-8 weeks old) were injected with myeloablative doses of either Bu (4 × 25 mg/Kg) or Treo (3 × 1000 mg/kg) intraperitoneally. Groups of animals were euthanized on day +1 & day +7 post drug administration. WBCs, CD31+ bone marrow endothelial cells (BMECs), CD146+ Liver sinusoidal endothelial cells (LSECs) were enumerated by flow cytometry. Liver, spleen and bone marrow sections were evaluated by histology and immunofluorescence. Mice treated with Bu showed decrease in bone marrow WBCs on day +1 that persisted till day +7. Treo showed moderate decrease in WBC compared to untreated controls on day +1, the WBCs recovered on day +7. Hematoxylin & eosin (H&E) staining of bone marrow sections showed loss of bone marrow cellularity in Bu & Treo treated mice on day +1, while on day +7, mice treated with Treo alone resulted in increased marrow cellularity indicating probable recovery. BMECs were decreased by Bu on day +1 and day +7 while Treo slowly decreased BMECs from day +1 to day +7. Similarly, LSECs were highly damaged by Bu treatment on day +1 & day +7 while Treo resulted in relatively less damage on both day +1 & day +7. This was confirmed by immunofluorescence staining of liver sections with LSEC specific SE-1 antibody. Further, CD31, a marker for capillarization of LSECs were observed more in Bu compared to Treo treatment as evidenced by immunofluorescence. Treo also upregulated angiogenesis related genes [BMX, EGFL7, MMRN2, LYL1] in liver possibly explaining lesser damage. Histology of liver & spleen corroborated the findings of severe damage mediated by Bu than Treo. Bu treatment resulted in decreased bone marrow cellularity, increased damage to BMECs and capillarization of LSECs while Treo resulted in moderate decrease in bone marrow cellularity, lesser damage to BMECs & LSECs. These results indicate that although Bu & Treo had profound myeloablative effect, their toxicity profile on endothelial cells differs. Further studies are warranted to elucidate the mechanism of differential endothelial toxicity and probable hematopoietic recovery by Treo treatment.

Continuous One Year Oral Administration of the Radiation Mitigator, MMS350, after Total-Body Irradiation, Restores Bone Marrow Stromal Cell Proliferative Capacity and Reduces Senescence in Fanconi Anemia (Fanca-/-) Mice.

We quantitated age-related accumulation of senescent cells in irradiated Fanconi anemia (FA) (Fanca-/- mouse cell lines in vitro, and monitored the effect of continuous administration (via drinking water) of the water-soluble radiation mitigator, MMS350, on tissues in vivo over one year after 7.5 Gy total-body irradiation (TBI). Irradiated Fanca-/- mouse bone marrow stromal cell lines showed increased numbers of beta-galactosidase- and p21-positive senescent cells compared to Fanca+/+ cell lines, which was reduced by MMS350. One week after 7.5 Gy TBI, Fanca-/- mice showed increased numbers of senescent cells in spleen compared to Fanca+/+ controls, decreased bone marrow cellularity, failure of explanted bone marrow to proliferate in vitro to form a hematopoietic microenvironment and no detectable single stromal cell cloning capacity. There was no detectable amelioration by MMS350 administration at one week. In contrast, one year post-TBI, Fanca-/- mice demonstrated fewer senescent cells in brain and spleen compared to Fanca+/+ controls. While Fanca-/- mouse bone marrow stromal cells explanted one year post-TBI still failed to proliferate in vitro, continuous oral administration of 400 µ M, MMS350 in drinking water restored explanted stromal cell proliferation. The data indicate that continuous administration of MMS350 modulated several properties of TBI-accelerated aging in Fanca-/- mice as well as control mice, and support further study of MMS350 as a modulator of radiation late effects.

PI3Kinase Alpha and Delta Promote Hematopoietic Stem Activation Under Stress

Hematopoietic stem cells (HSCs) are a rare population in the bone marrow (BM) that mostly exists in a quiescent state. Upon environmental stresses such as infection, inflammatory signals are released and induce HSCs to proliferate to quickly re-establish homeostasis and maintain the blood system. Many hematopoietic growth factors and chemokines signal through PI3K pathway. In hematopoietic cells, 3 Class IA PI3K isoforms are expressed (p110α, β, and δ), each encoded by a different gene. While p110α and p110β are ubiquitously expressed, p110δ is enriched in leukocytes. We previously showed that p110α is dispensable for HSC function (Gritsman et al, J Clin Invest 2014 124:1794-1809), suggesting redundancy between Class I PI3K isoforms in HSCs.To test for potential redundancy between p110α and p110δ in HSCs, we generated mice with conditional deletion of p110α and germline deletion of p110δ (DKO mice). DKO mice have anemia, leukopenia and decreased BM cellularity. While there is no change in HSC numbers, the number of lymphoid-primed multipotent progenitors (LMPPs) is significantly decreased. In the primary competitive repopulation assay, DKO BM cells fail to reconstitute the B cell lineage, while the myeloid and T cell lineages were relatively preserved. However, in a secondary competitive transplantation setting, we also observed a significant decrease in myeloid reconstitution, suggesting that p110α and p110δ play redundant roles in emergency myelopoiesis. To examine effects of p110α and p110δ deletion on gene expression, we performed microarray analysis of WT, DKO, p110δ-/-, and p110α-/- hematopoietic stem and progenitor cells (HSPCs) after bone marrow transplantation. Gene set enrichment analysis revealed negative enrichment of gene sets associated with inflammatory response pathways in both DKO HSCs and LMPPs. In DKO HSCs, we also observed negative enrichment of gene sets associated with cell cycle progression.To further examine the roles of p110δ and p110α in the hematopoietic stress response, we injected DKO, p110δ-/-, and WT; Mx1-Cre mice with 5-fluorouracil (5-FU). We observed decreased survival of 5-FU-treated DKO mice associated with impaired hematopoietic recovery, and with the failure of HSCs to enter the cell cycle. Given the important roles of inflammatory signaling pathways in HSC activation and emergency myelopoiesis, we examined the roles of p110α and p110δ in signal transduction in HSPCs in response to IL1β or TNFα. Our phospho-flow cytometry analysis revealed a decrease in p38-MAPK phosphorylation in both p110δ-/- and DKO HSPCs, both at baseline and after stimulation with either IL1β or TNFα. To confirm these results, we stimulated p110δ KO and DKO cKit-enriched bone marrow cells with IL1β or TNFα. We observed a significant decrease in both p38-MAPK phosphorylation and phosphorylation of Akt at Ser473 in DKO cells, but not in p110δ-/- cells, both at baseline and with IL1-β or TNF-α stimulation. This suggests that both p110α and p110δ are required for optimal transduction of IL1β or TNFα in hematopoietic progenitors. Surprisingly, we found that DKO HSPCs can enter the cell cycle normally upon in vivo stimulation with lipopolysaccharide (LPS), which simulates bacterial infection. Our results suggest that p110α and δ act in a redundant fashion to transduce specific inflammatory signals in HSPCs, such as IL1β and TNFα, in response to hematopoietic stress. Our findings have important implications for the use of PI3K inhibitors in combination with chemotherapy and in the setting of infection or inflammation. DisclosuresNo relevant conflicts of interest to declare.

Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hemostasis at Extremes of Body Weight.

Extremes of body weight are not uncommon in the modern world and include anorexia nervosa (AN) and obesity. Both conditions are associated with increased morbidity and mortality: AN has the highest mortality rate of all mental illnesses and unfortunately obesity has reached epidemic proportions and is a well-recognized risk factor for cardiovascular disease including venous thromboembolism (VTE). This article summarizes the current understanding of hemostatic changes of these extremes of body weight. The hemostatic changes of AN have not been well described. Severe AN is associated with pancytopenia with decreased bone marrow cellularity, which causes a mild thrombocytopenia. Platelet hyperaggregability has been recognized in AN and has been attributed at least in part to increased adrenoceptor density. Obesity and the metabolic syndrome are associated with prothrombotic changes, which have been well characterized and related to complex adipocyte-induced inflammatory changes, including increased levels of plasminogen activator inhibitor type 1, von Willebrand factor, fibrinogen, and other evidence of increased coagulation and platelet activation. Accumulating evidence suggests a significant role for increased tissue factor expression and signaling in this relationship, with increased tissue factor expression present in adipose and possibly systemic tissues, induced by adipose-generated cytokines. Intriguingly, the hemostatic changes do not seem to increase with increasing BMI, although the risk of VTE increases with BMI, suggesting that decreased venous flow due to venous enlargement may play the most important role in increased VTE risk with obesity.

Preclinical evidence for management of thrombocytopenia associated with bromodomain extra-terminal (BET) inhibition therapy.

e24207 Background: Thrombocytopenia is a potential dose-limiting toxicity associated with many oncology agents including BET inhibitors (BETi). Thrombocytopenia in rats closely mirrors clinical dose-limiting thrombocytopenia, enabling the use of this model to explore potential supportive care options. Methods: A pan-BETi molecule A-1550592 (1mg/kg) was administered by oral gavage for 4 days to Sprague Dawley rats and hematologic parameters measured on Day 5. Recombinant human erythropoietin (rhEPO 150 IU) was administered subcutaneously 4 days before and concomitantly with BETi. Folic Acid (FA, 3mg/kg and 30 mg/kg) was administered subcutaneously 4 days before and concomitantly with BETi. Romiplostim (30 micrograms) was administered prior to (2 doses) and concomitantly (1 dose) with BETi on days 2, 4 and 6. Results: rhEPO administration resulted in reticulocyte increases as compared to BETi alone (BETi, n = 3: 54x109; BETi + rhEPO, n = 3: 272x109; Control, n = 3: 346X109cells/mL). Interestingly, rhEPO administration was also associated with platelet increases in the rhEPO+ BETi group compared to BETi alone (BETi, n = 3: 465x109; BETi + EPO, n = 3: 808x109; Control, n = 3: 1175x109cells/mL). rhEPO was also noted to reverse the moderate decreased bone marrow cellularity seen with BETi administration. FA partially mitigated thrombocytopenia in the FA+ BETi group when compared with BETi alone (BETi, n = 3: 529x109; BETi + 3 mg FA, n = 3: 585x109; BETi + 30 mg FA, n = 3: 973x109; Control, n = 3: 1138x109). Romiplostim partially mitigated thrombocytopenia in the Romiplostim + BETi group when compared with BETi alone (BETi, n = 5: 808x109; Romiplostim, n = 5: 2678x109; BETi + Romiplostim, n = 5: 1150x109cells/mL). Conclusions: The observed hematologic responses in the rodent model to BET inhibition and various supportive care agents (rhEPO, FA, Romiplostim) suggests that these agents may be effective in mitigating clinical thrombocytopenia associated with BETi.

Loss of the von-Hippel Lindau gene in osteocytes alters B lymphocyte development and immune response

Abstract The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation. Conditional-deletion of VHL in osteoblasts and in hematopoietic progenitors have demonstrated a role for VHL in these cell types, but how changes in bone homeostasis affects immune development are not fully understood. Osteoblasts (OBs) support B cell development. OBs mature into osteocytes (OCYs) and to test if OCYs also support B cell development, we generated OCY-specific (Dmp-Cre) conditional VHL knockout (cKO) mice. cKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to controls. In line with this, the frequencies and numbers of B cell precursors (B220+ IgM−), immature B (B220+ IgMint) and recirculating B (B220+ IgMhigh) cells in the bone marrow are significantly decreased. These data suggest that the change in bone homeostasis adversely affects B cell development in a cell-extrinsic manner. Although the absolute numbers of B cells in the spleens of cKOs were normal, we hypothesized that loss of VHL in OCYs could permanently and adversely affect B cell functional response. To test this, VHL cKO mice were immunized with a T-independent antigen (NP-Ficoll). IgM and IgG3 antigen-specific titers to NP-Ficoll in VHL cKOs were similar to WT mice. Testing of B cell responses to T-dependent antigens (NP-OVA) is ongoing. Taken together, our data suggest that OCY-specific VHL deletion might be helpful to increase bone density, but at the expense of B cell numbers. Further investigation is required to understand the molecular mechanisms by which VHL in OCYs contribute to its putative role as a B cell niche cell.