BM Cellularity Research Articles

Cxcl10 and Cxcr3 regulate self-renewal and differentiation of hematopoietic stem cells

BackgroundThe function of hematopoietic stem cells (HSC) is regulated by HSC internal signaling pathways and their microenvironment. Chemokines and chemokine ligands play important roles in the regulation of HSC function. Yet, their functions in HSC are not fully understood.MethodsWe established Cxcr3 and Cxcl10 knockout mouse models (Cxcr3−/− and Cxcl10−/−) to analyze the roles of Cxcr3 or Cxcl10 in regulating HSC function. The cell cycle distribution of LT-HSC was assessed via flow cytometry. Cxcr3−/− and Cxcl10−/− stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. To study the effects of Cxcr3 or Cxcl10 deficient bone marrow microenvironment, we transplanted CD45.1 donor cells into Cxcr3−/−or Cxcl10−/− recipient mice (CD45.2) and examined donor-contributed hematopoiesis.ResultsDeficiency of Cxcl10 and its receptor Cxcr3 led to decreased BM cellularity in mice, with a significantly increased proportion of LT-HSC. Cxcl10−/− stem/progenitor cells showed reduced self-renewal capacity in the secondary transplantation assay. Notably, Cxcl10−/− donor-derived cells preferentially differentiated into B lymphocytes, with skewed myeloid differentiation ability. Meanwhile, Cxcr3-deficient HSCs demonstrated a reconstitution disadvantage in secondary transplantation, but the lineage bias was not significant. Interestingly, the absence of Cxcl10 or Cxcr3 in bone marrow microenvironment did not affect HSC function.ConclusionsThe Cxcl10 and Cxcr3 regulate the function of HSC, including self-renewal and differentiation, adding to the understanding of the roles of chemokines in the regulation of HSC function.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p < 0.0001) and MEP (p < 0.0001), but skewing towards GMP (p<0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p < 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

Lymphoid aggregates in the bone marrow biopsies of patients with myelodysplastic syndromes - A potential prognostic marker?

Lymphoid aggregates (LA) are occasionally seen in bone marrow biopsies (BMB) of myelodysplastic syndromes (MDS) patients. Our aim was to evaluate their incidence and association with prognosis. We compared BMB reports of MDS patients treated at the Tel Aviv Sourasky Medical Center (2011-2018), and controls (2015-2017, normal BMB), and examined the charts of the MDS patients (LA+ and LA-). Categorical, normally and non-normally distributed continuous variables were compared using Fisher's exact, independent t and Mann-Whitney tests respectively. Adjusted [age, gender, lymphocytes, white blood cells (WBC) and diabetes mellitus (DM)] Cox proportional hazard model examined survival at 12 and 24 months. MDS patients (N=140) were older than controls (N=38; 74.1 vs 69.2 years, p=0.005); 34 MDS (24.3%) and 5 controls (13.2%) had LA+ (P=0.141). CD20/CD3 staining suggested LA polyclonality. MDS/LA+ (vs MDS/LA-) patients were younger, with a trend (not statistically significant) towards poor prognostic parameters: lower Hb, WBC, and platelets, higher LDH, BM cellularity, and IPSS-R score. The incidence of cardiovascular disease was similar, but MDS/LA+ had twice the incidence of DM (38.2% vs 19.0%, p=0.022). Similar trend for cancer (26.5% vs 14.3%, p=0.102). Twelve-month survival: 24/34 (70.6%) MDS/LA+; 88/106 (83.0%) MDS/LA- (p=0.140). This trend, seen in Kaplan-Meier curves, disappeared at 24 months. The hazard ratio for LA was 2.283 (p=0.055) for 12 months. These preliminary data suggest LA are relatively common (24%) in MDS BMB, and might indicate poor prognosis. This may reflect involvement of the immune system in MDS. Future studies will examine larger groups, to clarify the incidence, significance and the pathophysiology.

Obesity-Induced Inflammation Cooperates with Loss of DNA Methyltransferase 3A to Develop Early-Onset of Leukemia

Obesity is an increasing epidemic disease world-wide responsible for enhancing the risk for developing Type 2 diabetes mellitus (T2DM) as well as cancer. However, it is unclear if and how obesity contributes to the transformation of pre-leukemic stem and progenitors (pre-LHSC/Ps) into full-blown leukemia such as acute myeloid leukemia (AML) or severe form of myeloproliferative neoplasm (MPN). We hypothesized that obesity induced chronic inflammation might be responsible for clonal selection of pre-LHSC/Ps bearing pre-leukemic mutations such as DNA methyltransferase 3A (DNMT3A) and for promoting the progression of early-onset MPN towards severe forms of AML/leukemia. To test this hypothesis, we genetically crossed pre-leukemic Dnmt3a+/-;Mx-Cre+ mice with leptin deficient obese (LepOb/Ob) mice to obtain Ob/Ob;Dnmt3a+/-;Mx-Cre+ compound mutant mice. Further, the Dnmt3a gene was deleted by giving the PolyIC and the deletion was confirmed through PCR. After 12 days of post-PolyIC the myeloid cells (neutrophils and monocytes) were expanded in Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice compared to Dnmt3a+/-;Mx-Cre+, Dnmt3a+/-;Mx-Cre-, Ob/Ob and WT mice. We have harvested and analyzed all these mice after 26 days of post-PolyIC. Interestingly, Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice showed increased BM cellularity, both the frequency of lineage negative, Sca-1+ and c-KIT+ (LSK) cells, short-term hematopoietic stem cells (ST-HSCs; LSK/CD48+/CD150-), granulocyte macrophage progenitor (GMPs; LSK/CD16+/CD34+), and reduction in LT-HSCs (LT-HSCs; LSK/CD48-/CD150+) compared to other groups. Flow cytometry analysis of PB, BM and spleen from Ob/Ob;Dnmt3a+/-;Mx-Cre+ mice demonstrated a significant increase in the frequency of mature myeloid cells (Gr-1+/Mac-1+) and a profound reduction in B220+ B cells compared to other groups. Remarkably, these mice also showed splenomegaly, elevated heart size and early signs of AML blasts as reflected by the presence of c-KIT+/CD11b+ double positive cells in the BM, consistent with severe MPN/AML development. Taken together, these results demonstrate that obesity induced inflammation cooperates with pre-leukemic Dnmt3a+/- mutation to induce an early-onset of severe MPN/AML like disease.

Assessing the Immune Tumour Microenvironment (iTME) Using Multiplex Immunoflourescence Histochemistry (mIHC) Demonstrates Close Proximity of Cytotoxic T-Cells to Plasma Cells (PC) in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Aim: A dysfunctional iTME facilitates disease progression in MM. Studies have demonstrated the association between the spatial distribution of immune cells and progression of various cancers. Using mIHC we aim to describe quantitative and qualitative changes in CD3+CD8+ T-cells (T cytotoxic) in patients with MGUS, ND and relapsed/refractory MM (RRMM) and assess spatial proximity to PCs.Method: Formalin-fixed, paraffin-embedded trephine sections from pts with MGUS (n=32), NDMM (n=65) and RRMM (n=59) were sequentially stained for CD138, CD3, CD8 and checkpoint receptors (CPs) Tim3, Lag-3 and PD-1 (Figure 1). Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of T cytotoxic populations and analysis of proximity to PCs. Descriptive statistics and ordinary one-way ANOVA were applied as appropriate.Results: Patient demographics, disease characteristics, treatment (including prior therapies, where applicable), best response, duration of response, median progression free (PFS) and overall survival (OS) will be presented for all cohorts. There was no difference in BM cellularity or total number of nucleated cells assessed across the cohorts (p=0.16 and p=0.25). PC % was higher in the ND and RRMM compared to MUGS cohort (p<0.001). The average distance between T cytotoxic and PCs was similar between the cohorts (p=0.38), but a higher proportion of T cytotoxic were within 50μm of a PC in the ND cohort (p=0.0036, 90.8±15.8% (ND) vs. 77.6±19.5% (MGUS) and 80.1±25.9% (RR)). The % of unique PCs with a single T cytotoxic within 100μm is higher in patients with MGUS and RRMM than NDMM (p=0.0007). There was no difference in the %CD3+, %CD3+CD8+ or %CD3+ cells expressing CD8 (p=0.22, p=0.62, p=0.48). CP expression on T cytotoxic was similar (Tim3 p=0.46, Lag-3 p=0.35; PD-1 p=0.54) with no difference in dual or triple CP expression. Sub-analyses assessing CP expression patterns and T cytotoxic/PC proximity within individual cohorts based on response to treatment/disease progression are to follow.Conclusion: The infiltration of cytotoxic T cells into tumours is a critical factor in immunotherapy efficacy. Here we clearly demonstrate the feasibility of mIHC to describe the spatial context of the iTME and we plan to implement it for predictive value in future studies of immunotherapies in patients with MM.Figure 1: Bone marrow FFPE trephine section stained with mIHC using the Opal TM workflow demonstrating plasma cells (CD138, green), T cells (CD3, yellow; CD8, pink) and checkpoint receptors (PD-1, orange; Lag-3, magenta; Tim3, light blue) with colocalisation of some signals. DisclosuresHarrison: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/ Juno/ BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche/Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Haemalogix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eusa: Consultancy, Honoraria, Speakers Bureau; Terumo BCT: Consultancy, Honoraria. Quach: Janssen/Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Antengene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

P-074: Assessing the immune microenvironment with multiplex immunofluorescence histochemistry demonstrates proximity of cytotoxic T-cells to plasma cells in patients with newly diagnosed multiple myeloma

<h3>Background</h3> A dysfunctional immune tumour microenvironment (iTME) facilitates disease progression in MM. Using multiplex immunohistochemistry (mIHC) we aim to describe quantitative and qualitative changes in CD3+CD8+ T-cells (Tcytotoxic) in patients with MGUS, ND and relapsed/refractory MM (RRMM) and assess spatial proximity to PCs. <h3>Method</h3> Formalin-fixed, paraffin-embedded trephine sections from pts with MGUS (n=32), NDMM (n=65) and RRMM (n=59) were sequentially stained for CD138, CD3, CD8 and checkpoint receptors (CPs) Tim3, Lag-3 and PD-1 (Figure 1). Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of Tcytotoxic populations and analysis of proximity to PCs. Descriptive statistics and ordinary one-way ANOVA were applied as appropriate. <h3>Results</h3> Patient demographics, disease characteristics, treatment (including prior therapies, where applicable), best response, duration of response, median progression free (PFS) and overall survival (OS) will be presented for all cohorts. There was no difference in BM cellularity or total number of nucleated cells assessed across the cohorts (p=0.16 and p=0.25). PC % was higher in the ND and RRMM compared to MUGS cohort (p<0.001). The average distance between Tcytotoxic and PCs was similar between the cohorts (p=0.38), but a higher proportion of Tcytotoxic were within 50μm of a PC in the ND cohort (p=0.0036, 90.8±15.8% (ND) vs. 77.6±19.5% (MGUS) and 80.1±25.9% (RR)). The % of unique PCs with a single Tcytotoxic within 100µm is higher in patients with MGUS and RRMM than NDMM (p=0.0007). There was no difference in the %CD3+, %CD3+CD8+ or %CD3+ cells expressing CD8 (p=0.22, p=0.62, p=0.48). CP expression on Tcytotoxic was similar (Tim3 p=0.46, Lag-3 p=0.35; PD-1 p=0.54) with no difference in dual or triple CP expression. Sub-analyses assessing CP expression patterns and Tcytotoxic/PC proximity within individual cohorts based on response to treatment/disease progression are to follow. <h3>Conclusion</h3> Multiplex IHC is a novel technique to assess the iTME. While there is no discernible quantitative difference in cytotoxic T-cells or checkpoint receptor expression, we demonstrate increased proximity of cytotoxic T-cells to plasma cells in newly diagnosed patients suggestive of a more robust immune system compared to multiply treated patients.

TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-β plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-β1 than TGF-β2 and TGF-β3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-β1/TGF-β3 protein trap, to block the excessive TGF-β signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-β1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-β1–induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-β1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Hypoplastic Myelodysplastic Syndrome: Clinical, Histopathological and Molecular Characterization

Introduction and AimsHypoplastic Myelodysplasic Syndrome (h-MDS) has distinct features compared to normo- or hypercellular MDS, with a higher response rate to immunosuppressive therapy and more favorable prognosis. However, to date, reproducible diagnostic criteria for h-MDS have not been clearly defined, making differential diagnosis from other bone marrow failure (BMF) syndromes challenging. Based on a large and well-annotated consecutive cohort of patients with BM hypocellularity from two tertiary centres, we aimed to delineate the clinical, histopathological and molecular features of h-MDS.MethodsOf 1262 consecutive adult patients analyzed at King's College Hospital, London, UK and IRCCS Policlinico San Matteo & University of Pavia, Italy: 533 patients had a hypocellular BM, including 205 MDS patients with BM cellularity ≤25%, 77 MDS with reduced age-adjusted BM cellularity, 139 with Aplastic Anemia (AA), 97 with a diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) and 15 with congenital BMF (c-BMF) and 729 with normo- or hypercellular MDS (n-MDS).ResultsComparison of clinical features and outcome of patients with h-MDS as defined by a BM cellularity ≤25% (n=205) and those with a reduced age-adjusted cellularity (n=77) did not detect any significant differences. Therefore, a reduced age-adjusted BM cellularity was adopted as a criterion to define h-MDS (n=282). Compared with patients with n-MDS, those with h-MDS were significantly younger, displayed higher levels of hemoglobin (p=.01), lower neutrophil and platelet counts and lower percentage of BM blasts, ring sideroblasts (RS) and CD34+cells as determined by immunohistochemistry (all p<.001). No significant difference was observed in the distribution of chromosomal abnormalities. A significantly higher prevalence of PNH clones was observed in h-MDS (24% vs 2%, p<.001). Those patients with h-MDS showed a significantly lower risk of leukemic evolution compared with n-MDS (4-year cumulative incidence 14% vs 26%, p=.003). Comparing h-MDS with AA, h-MDS showed a significantly higher percentage of RS (p=.004), BM blasts, dysmegakaryopoiesis and dysgranulopoiesis (p<.001), BM fibrosis (p=.002), clusters of CD34+ cells and chromosomal abnormalities (p<.001). H-MDS patients showed a significantly higher risk of leukemic evolution compared with AA (4-year cumulative incidence 14% vs 0%, p<.001).Targeted sequencing of 24 commonly mutated myeloid genes was performed on 358 patients (73 h-MDS, 45 AA and 240 n-MDS). Twenty-five patients with h-MDS (34%) showed one or more somatic mutation (median 1; range 1-4). Comparing h-MDS to n-MDS revealed a significantly lower number of mutations per subject and variant allele frequency (VAF) (p<.001). Moreover, a significantly lower number of mutations per patient and VAF were observed in AA compared to h-MDS (p=.031 and p=.003, respectively). Thirty-seven patients with a hypocellular BM had a peripheral blood telomere length (TL) ≤1st percentile and/or a germline mutation consistent with a diagnosis of c-BMF, leading to an estimated prevalence of unidentified c-BMF of 13% in cases without robust morphological evidence of MDS.Based on these findings, we defined a diagnostic score (h-score), including variables with the highest specificity for MDS: dysmegakaryopoiesis, dysgranulopoiesis, BM fibrosis, clusters of CD34+ cells, RS 2-14%, BM blasts 2-4% (score 1); RS≥15% and BM blasts ≥5% (score 2). By applying ROC analysis, a cut off value of 2 was associated with the highest specificity for MDS (0.98). Notably, 71% of patients with a hypoplastic BM and low h-score (0-1) had no evidence of clonal disease by cytogenetic or mutation analyses or had a mutation pattern consistent with age-related clonal hematopoiesis. These patients showed a significantly better OS and lower risk of leukemic evolution (P<.001).ConclusionsIntegration of cyto-histological and genetic features in patients with a hypocellular BM identifies two distinct groups, one with clinical and genetic features highly consistent with a clonal disease with high risk of leukemic evolution, and one with features more consistent with a non-malignant bone marrow failure. Furthermore, we identified relevant enrichment of patients with previously undiagnosed c-BMF. DisclosuresKulasekararaj:Akari Therapeutics Plc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria; Ra Pharmaceuticals: Consultancy, Honoraria; Achillion pharmaceuticals: Consultancy, Honoraria.

CXCL12-Expressing Bone Marrow Niches Differentially Regulate CML Compared to Normal Stem Cells

CXCL12 plays a major role in hematopoietic stem cells (HSC) localization to regulatory niches in the bone marrow microenvironment (BMM). Previous studies indicate that CXCL12 deletion from Prx+ mesenchymal stem cells (MSC) reduces murine HSC numbers, and from Tek+ endothelial cells (EC) modestly reduces their repopulating activity. In contrast, CXCL12 deletion from Ocn+ osteoblasts or Osx+ osteoprogenitors does not affect HSC function (Greenbaum et al. Nature. 2013 Mar 14;495(7440):227-30; Ding et al. Nature. 2013 Mar 14;495(7440):231-5.). These studies identified MSC and endothelial cells as important HSC niche components. However, niche cells for CML leukemic stem cells (LSC) remain poorly characterized. To evaluate the contribution of CXCL12-expressing populations to LSC regulation, we transplanted normal and CML BM cells (CD45.1/2+; 2*106/mouse) into CXCL12f/f (loxP sites flanking exon 2) mice crossed with Tek-Cre and Prx-Cre mice ( reported in ASH 2016, Blood, Abstract: 26) . We showed that CXCL12 deletion from Prx+ MSC resulted in reduction in normal HSC (LSK Flt3- CD150+ CD48- cells) numbers and function compared to controls, but did not observe significant reduction of HSC with CXCL12 deletion from Tek+ EC. Interestingly, transplantation of CML cells into mice with CXCL12 deleted from Prx+ MSC led to reduced survival compared to control mice associated with increased WBC counts, BM cellularity, LSC numbers, and secondary repopulation capacity. In contrast, CXCL12 deletion from Tek+ cells led to enhanced survival, associated with reduced BM cellularity and reduced LSC numbers. In further studies, we transplanted mixtures of equal numbers of normal and CML cells into Prx-Cre and Tek-Cre mice, and found a marked growth advantage of LSC compared with HSC in Prx-Cre mice, and a growth advantage of HSC over LSC in Tek-Cre knockout mice. We also show that both HSC and LSC demonstrate increased cell cycling via EdU labeling. Consistent with this, we found that Nilotinib treatment leads to significantly greater reduction in BM cellularity, LSC numbers and secondary repopulation potential in Prx-Cre mice, compared to Tek-Cre or WT mice. To evaluate the effects of CML on CXCL12-expressing BMM cells, we crossed CXCL12GFP (GFP reporter knocked into the CXCL12 locus) and SCL-tTA-BCR-ABL mice to generate CXCL12GFP-BCR-ABL mice. CML development lead to significantly reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) but increased GFP+ endothelial cells (CD45-Ter119-CD31+), and within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased compared to WT mice. To further understand niche mediated interactions that regulate LSC fate, we studied gene expression profiles of LSC from mice following Cre-mediated targeting of CXCL12, compared to LSC from Cre-negative mice. Our preliminary analysis indicates significant enrichment of genes associated with cell cycle progression and Myc activation, and negative enrichment of genes related to TGF-b, STAT3, and Polycomb Repressive Complex 2 (PRC2) activity in LSC from Prx1+Cre CXCL12f/f mice. Reduced TGF-b, STAT3 and PRC2/EZH2 activity represent potential mechanisms for regulation of CML LSC maintenance and/or TKI resistance, and may potentially contribute to Prx1+ MSC-mediated LSC regulation. We conclude that CXCL12+ BM MSC inhibit CML development, maintain LSC quiescence, and enhance TKI resistance, while CXCL12+ EC enhance LSC maintenance and disease development. Leukemia associated reduction in CXCL12+MSC and increase in CXCL12+EC may enhance CML development. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM MSC and EC for CML LSC compared with normal HSC. DisclosuresNo relevant conflicts of interest to declare.

MTOR Restricts Chromatin Access and Genomic Activity to Maintain Hematopoietic Stem Cell Quiescence and Engraftment

mTOR kinase is an anti-cancer drug target that integrates signals from nutrients, growth factors, and cellular energy status to control cell growth and proliferation, but its biological function in hematopoietic stem cells remains unclear. Numerous studies by using rapamycin or mTOR kinase inhibitors have indicated that mTOR inhibition suppresses chromatin access and gene expression and inhibits cell growth. Consistent with the notion that mTOR targeting suppresses gene activation, hyperactivation of mTOR by deletion of negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and exhaustion of HSCs. To directly examine the function of mTOR and its kinase activity in HSC regulation, we used the conditional Mx-Cre;mTORflox/flox and the Mx-Cre;mTORflox/knockin mice that produce the mTOR-/- and the mTOR kinase-activity deficient D2338A mutant knockin genotype, respectively, upon poly I:C induced Cre recombination. We have determined the phenotypic, genomic and molecular effects of their HSCs. Our previous studies have found that depletion of mTOR drastically affected hematopoiesis, with a marked reduction in total BM cellularity and transient increase in the number of HSCs in bone marrow. Remarkably, mTOR-/- HSCs displayed a loss of quiescence, increased proliferation and a normal survival index (Guo et al., Haematologic a 98:1353). Our current study has found similar phenotypes of the HSCs in increased number and proliferation in the Mx-Cre;mTORflox/knockin mice where the mTOR kinase activity is abolished. Both mTOR-/- and mTORKI/- HSCs are defective in engraftment upon competitive transplant into recipient mice, with a clear molecular signature of drastically reduced downstream effectors p-S6K and p-4E-BP. ATAC-seq reveals that loss of mTOR or mTOR kinase activity results in selectively increased chromatin access and global gene activation, in contrast to the reported effects by mTOR inhibitors such as rapamycin or pp242/AZD2014. ChIP-seq of two active histone markers, H3K27Ac and H3K4Me3, yields consistent results that chromosome of the HSCs are activated upon the loss of mTOR or its kinase activity. In particular, increased H3K27Ac and H3K4Me3 ChIP-seq activities are observed in both the upstream and downstream region of c-myc locus, consistent with an activated c-myc expression. RNA-seq analysis reveals that upon mTOR KO or KI, thousands of genes exhibit differential expression. The up-regulated genes are particularly enriched in functions involved in ribosome biogenesis/protein translation, cell cycle progression, cell fate determination, and survival. Analysis using the STRING functional interaction network to search for the compensatory genes/pathways discovers a number of likely compensatorily upregulated genes, upon mTOR KO or KI, that share a significant number of interacting genes with mTOR, including c-myc, Fos, and Jun. These genes are enriched in pathways such as EGFR-MAPK pathway, PI3K-Akt pathway, Jak-Stat pathway, and NFkB pathway, which may compensate for mTOR loss. RT-PCR and Western blotting further verify that loss of mTOR or its kinase activity causes elevated mRNA and protein expression of selected compensatory genes, including c-myc, Fos, Jun, and Pik3cd. In addition, broad compensatory elevations in ribosome and translation machinery gene expression, activation of RNA pol-II, and activation of several interesting compensatory pathways are seen. The BET inhibitor, JQ1, that inhibits c-myc activity readily suppresses the hyper-proliferative phenotype of mTOR-/- and mTORKI/-HSCs. Thus, contrary to dogma established mostly by transient rapamycin or kinase inhibitor usages, our genetic and genomic studies demonstrate that mTOR, through its kinase activity, plays a restrictive role to maintain chromosomal access and genomic activity of HSCs. Loss of mTOR or its kinase activity unleashes a compensatory chromosomal and gene activation effects that involve master regulators of gene activities such as c-myc and Fos, protein translation and ribosomal proteins, and cell growth signaling pathways. These genomic changes may cause the HSC loss of quiescence and engraftment phenotypes seen upon mTOR loss, and need to be taken into consideration in the development of new generations of mTOR targeted therapy and combinatory therapy.Disclosures: No relevant conflicts of interest to declareASH Members: Yi Zheng DisclosuresNo relevant conflicts of interest to declare.

MiR-199b-5p Differentially Modulates Hematopoietic Stem Cell Self Renewal in Steady State and Stress Hematopoiesis

The emergence of microRNAs (miRNAs) is providing novel insights into the molecular control networks underlying hematopoiesis and diseases that stem from aberrations in hematopoiesis. We, amongst others, have shown that miR-199b is significantly downregulated in acute myeloid leukemia (AML) and is a promising prognostic factor for FAB-M5 subtype. Herein, to determine miR-199b's role in steady state and stress hematopoiesis, we utilized our recently generated miR-199b knockout (KO) mice (CRISPR approach) to assess frequencies of hematopoietic stem progenitor cells (HSPCs) in WT and miR-199b KO mice through flow cytometry. Under steady state conditions, deletion of miR-199b resulted in significant reduction of Lin- Sca-1+ c-Kit+ (LSK), long-term hematopoietic stem cells (LT-HSCs) (Lin− CD117+ Sca1+ CD150+ CD34low/− CD48−) and increases in multipotent progenitors (MPPs) (Lin− CD117+ Sca1+ CD150− CD34+). However, 5FU induced stress hematopoiesis resulted in significant increases in frequencies of LSK cells, LT-HSCs, and Lin- Sca-1- c-Kit+ (KL) cells compared to wild-type (WT) litter mates. Furthermore, silencing of miR-199b resulted in a significant increase of granulocyte monocyte progenitors (GMP), which was reversed during stress hematopoiesis. The highest expression levels of miR-199b were restricted to the LT-HSC fraction. Mir-199b levels remained moderately high in MPPs and LSK cells. Primarily within the LSK population, miR-199b expression was most pronounced in LT-HSCs and to a lesser extent, MPPs, indicating that miR-199b may play a functional role in LT-HSC activity. Gene profiling studies in HSPCs identified Integrin alpha 8 (Itga8) as a conserved target of miR-199b. We then examined Itga8 transcript levels in HSCs from miR-199b knockout (-/-) mice and found significant elevation of Itga8 transcripts compared to WT mice. One possible mechanism for the differential expansion of miR-199b-/- HSPCs under steady state and stress is an increased or decreased rate of cell cycling. To test this, we examined cell proliferation using Ki67 and Hoechst 33342 markers. MiR-199b-/- LT-HSC at steady state showed significantly decreased proliferation whereas miR-199b-/- LT-HSCs under 5FU induced stress showed significant expansion when compared to wild-type. Next, we tested the adhesion abilities of LT-HSCs from miR-199b KO mice under both steady state and stress hematopoiesis. At steady state, miR-199b-/- LT-HSCs significantly elevated adhesion to vitronectin and fibronectin. Interestingly, like HSPC frequencies, stress hematopoietic-derived miR-199b-/- LT-HSCs showed significantly decreased adhesion to both vitronectin and fibronectin. These findings imply that miR-199b intrinsically regulates HSC behavior, including cell adhesion. To elucidate whether miR-199b's effects on LT-HSCs are mediated by Itga8, we performed an Itga8 knockdown in WT and miR-199b-/- LT-HSCs. Cell proliferation assessed by Ki67 positive cells revealed that downregulation of Itga8 in miR-199b KO LT-HSCs reverts to a miR-199b-induced decrease of LT-HSC proliferation. Intriguingly, we observed that silencing of Itga8 reversed the miR-199b-induced increase of cell adhesion. To corroborate that miR-199b-/- LT-HSCs are fully functional, we performed a competitive repopulation assay. BM derived lineage negative hematopoietic progenitor cells were further purified via immunomagnetic selection for SLAM marker purified (CD150+, CD244+ and CD48-) to enrich the LT-HSC fraction. MiR-199b-/- or miR-199b+/+ LT-HSCs (n=100 LT-HSCs) were transplanted intravenously together with 2.5 X 105 competitor recipient cells. Overall donor BM cellularity was markedly increased in LT-HSCs derived from 5FU treated miR-199 KO mice and overall donor BM cellularity was markedly decreased in LT-HSCs derived from steady state miR-199 KO mice. This suggested that under stress, miR-199b-/- LT-HSCs self-renew extensively after transplantation to repopulate the recipient. The analyses of HSPCs in BM of primary recipients revealed a significant increase in donor LT-HSCs in recipients that were transplanted with LT-HSCs derived from the 5FU induced stress model. Intriguingly, this didn't translate into subsequent increases in MPP, GMP or MEPs, suggesting that miR-199b may govern the self renewal of LT-HSCs and also have progenitor cell specific roles as evident from its expression during early hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Show Safety and Early Clinical Signal in Primary Myelofibrosis

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Show Safety and Early Clinical Signal in Primary Myelofibrosis

Combined Heterozygous Loss of Tet2 and Dnmt3a Along with Expression of Flt3ITD/WT results in Acute Myeloid Leukemia Which Responds to a Combination of FLT3 Inhibitor, APE1 Inhibitor and Decitabine

Heterozygous mutations in FLT3ITD, TET2 and DNMT3A are associated with human neoplasms. Whole exome sequencing of patients demonstrate co-occurrence of several of these mutations in myeloid malignancies; however it is unclear if and how these mutations cooperate and how does this cooperation manifest in disease. We assessed the consequence of co-existence of TET2, DNMT3A and FLT3ITD/WT mutations on AML development and overall survival. Mice were bred to obtain eight distinct genotypes WT, Tet2+/-, Flt3ITD/WT, Dnmt3aFlox/- MxCre, double het Tet2+/-; Dnmt3aFlox/- MxCre (TD), double het Tet2+/-; Flt3ITD/WT (TF), double het Flt3ITD/WT: Dnmt3aFlox/- MxCre (FD) and triple het Tet2+/-; Flt3ITD/WT; Dnmt3aFlox/+ MxCre (TFD) mice. These mice were treated with poly:IC and monitored for survival. Of the 8 genotypes examined, only TFD mice succumbed by 150 days. Peripheral blood (PB) counts, BM cellularity and splenomegaly were significantly higher in TFD mice compared to FD and TF mice. The absolute number of LSK cells in the BM was highest in TFD mice, although TF and FD mice also showed an increase relative to controls. A similar increase in the frequency of GMPs was noted in both double and triple heterozygous mice relative to controls as well as single heterozygous groups. An increase in Gr-1/Mac-1 cells was observed in TF and FD mice as well as in TFD mice relative to controls. To assess if the observed AML phenotype in TFD mice is transplantable, we transplanted BM cells from the 8 genotypes into lethally irradiated hosts. Recipients with TFD BM succumbed to rapid AML development and died within 45 days of transplantation. In contrast, none of the other recipients died during the entire monitoring period. A significant increase in PB neutrophil and monocyte counts with a significant reduction in red blood cells, and platelets counts was noted in TFD mice compared to other groups. Furthermore, a significant increase in BM cellularity, Lin- Kit+ progenitors, frequency and absolute number of LSK cells was also noted in mice with TF and FD BM but most significantly in recipients that received TFD BM. Given that the disease manifestation was qualitatively similar between FD, TF and TFD mice, although the severity greatest in TFD mice, we asked if these differences were due to quantitative or qualitative differences in gene expression between the various groups. RNA-seq analysis revealed distinct differences between WT and FD, TF and TFD BM cells. We observed 2328, 2168 and 1787 up-regulated genes and 1861, 1770 and 1430 downregulated genes in FD, TF and TFD vs. WT, respectively. The variations among FD, TF and TFD groups was smaller compared to the differences between WT and FD, TF and TFD groups, in which TF and TFD were more similar to each other compared to DF. Most of the cytokines regulating the differentiation of myeloid cells were downregulated in FD, TF and TFD, suggesting that all the mutant groups lost their differentiation ability. Of note, cytokines specific to stem cells and those involved in the differentiation of GMPs were all upregulated. Given the RNA-seq profile, we assessed the impact of using a combination of drugs that target Flt3ITD, inflammation as well as methylation status of cells in TFD mice. A cohort of mice was treated with a combination of AC220 (10 mg/kg, orally; Flt3 inhibitor), E3330 (20 mg/kg, i.p.; APE1 inhibitor; regulates the redox function of multiple transcription factors including NFkB and Stat3) and 2-dexoy-aza-cytidine (0.5 mg/kg, i.p). A significant correction in all PB, spleen and BM parameters was observed including in the frequency of Lin- Kit+ and LSK cells, HSCs, HPC1 and peripheral Gr1/Mac1 double positive cells in TFD mice. Collectively, these results demonstrate that a combination of three drugs targeting three different aspects of disease manifestation in TFD mice can significantly impact the leukemia progression both at the level of very primitive stem and progenitor cells as well as more mature myeloid and lymphoid cells. Disclosures No relevant conflicts of interest to declare.

Higher Intensity of Cell Surface Glucose-Regulated Protein 78 (csGRP78) Expression Is Seen in Patients with Early Progressive Disease/Mortality in a Cohort of Relapsed, Refractory Multiple Myeloma Patients Treated with Carfilzomib, Thalidomide and Dexamethasone

Background: GRP78, an endoplasmic reticulum stress-inducible molecular chaperone, is up-regulated at times of cellular stress to limit proteotoxicity and promote cell survival. Translocation of GRP78 to the cell surface (csGRP78) is emerging as a critical step providing tumour cells with a survival advantage. Here we quantified, monitored and correlated plasma cell (PC) csGRP78 expression in patients (pts) with relapsed/refractory multiple myeloma (RRMM) treated with carfilzomib, thalidomide and dexamethasone (KTd). Method: Patients enrolled in the single arm, multicentre, phase II Australasian Leukaemia &amp; Lymphoma Group MM018/Asian Myeloma Network 002 study were treated with KTd as described previously (Quach et al. Blood 2018 132:1955). Formalin-fixed, paraffin-embedded BM trephine sections collected at baseline (n=29), after 6-months (mo) of KTd (n=19) and at time of disease progression (PD; n=5) were stained for CD138 and GRP78 by multiplex immunofluorescence histochemistry using the OpalTM workflow. Membrane expression of CD138 and GRP78 was extracted using inForm® software, compared across timepoints and correlated to disease characteristics and treatment outcomes. Descriptive statistics, paired/unpaired two-tailed t-test, Pearson's or Spearman's correlation were applied as appropriate. Results: Correlative BM biopsies were collected for 29 pts [male = 18, mean age = 65.0 years (range 41.9-83.2), 2 median prior lines of therapy (range 1-3)] at baseline, 21 pts after 6 months of KTd (7 had PD/died prior to cycle 6, 1 came off study due to grade 4 AE after 5 cycles) and 5 pts at time of PD. There was no difference in the number of fields, BM cellularity (%) or number of nucleated cells (NCs) assessed at baseline and 6mo (p=0.927, 0.331 and 0.491 respectively). PC burden (%; mean±SD) reduced significantly following 6mo KTd (28.3±28.1 vs. 2.12±2.37; p=0.0007). The number of plasma cells expressing csGRP78 (% of all NCs) was reduced following 6mo KTd treatment (27.04±26.83 vs. 2.05±2.32; p=0.0007) while the % of CD138-ve BM cells expressing csGRP78 (% of all NCs; mean±SD) increased (62.61±27.55 vs. 87.46±10.11; p=0.0005). Globally, there was a trend for reduced intensity of csGRP78 expression after 6mo KTd (H-score 70.79±62.16 vs. 53.53±51.45; p=0.2073). There was no correlation between baseline BM NC GRP78 H-score and baseline paraprotein level, involved/uninvolved serum free light chain ratio or depth of response to KTd. Pts with early (&lt;6mo) disease progression/mortality (n=7) had a significantly higher baseline H-score (136±78.8 vs. 75.1±65.2; p=0.049). There was a separation of survival curves, but no significant difference regarding risk of early PD/mortality based a baseline H-score &gt;75th percentile of the cohort (&gt;152); p=0.1472 (Figure 1). Conclusion: Here we demonstrate that cell surface expression of GRP78 is prominent in both the plasma cells and cells of the tumour microenvironment in patients with RRMM and persists in the TME cells in patients on treatment. Early (&lt;6mo) disease progression or mortality is associated with higher baseline intensity of global cell surface GRP78 expression. Additional studies are being performed to evaluate the promise of cell surface GRP78 expression on plasma cells as a potential biomarker of response to therapy. Disclosures Harrison: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding. Quach:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees.

Protein Arginine Methyltransferase 1 Is Required for Maintenance of Normal Adult Hematopoiesis

Protein arginine methyltransferase 1 (PRMT1) is the predominant asymmetric methyltransferase in mammalian cells. Mounting evidence suggested that PRMT1 is essential to embryonic development and tumor pathogenesis, but its role in normal adult hematopoiesis is less studied. In this study, we determined the function of PRMT1 in normal adult hematopoiesis using a Prmt1 conditional knockout (KO) mouse model (PRMT1f/f/Mx1-Cre). Our overall analysis in peripheral blood of the 6 to 8-week-old mice revealed a significant cell number decrease in red blood cells, hemoglobin, white blood cells including neutrophils and lymphocytes at 12 weeks post pIpC induction. Importantly, BM cellularity of PRMT1-KO mice was decreased compared with littermate controls. We also determined the frequency of erythroid (Ter119+), mature myeloid (Mac1+/Gr1+), B cells (B220) and T cells (CD3) in the BM of PRMT1-deleted mice and found that Ter119+ and B220+ subsets were significantly decreased compared with littermate controls (p&lt;0.01), while the myeloid and T cell populations were relatively intact. We also calculated the absolute numbers of erythroid, myeloid, T and B cells on the basis of their frequencies and BM cellularity and observed overall decreases in all lineage cells. In PRMT1 KO mice, we observed significantly enriched primitive erythroid subsets including S0 (CD71-/Ter119-) as well as S1 (CD71hi/Ter119-) populations, accounting for a relative decrease of mature erythroid cells (S5, CD71-Ter119hi), thereby indicating erythroid maturation block. Interestingly, our histology analysis of BM showed hypocellularity with megakaryocytosis, indicating more differentiation to the lineage of megakaryocytes. We further analyzed the frequency and absolute cell number of BM HSPC populations from PRMT1-KO mice by flow cytometry. The frequencies of phenotypic progenitor subsets in BM, including LSK, MPP and LMPP was not affected upon PRMT1 KO. Notably, the frequency of the ST-HSC population and GMPs was significantly lower in PRMT1-KO BM compared with littermate controls (p&lt;0.05). Moreover, the absolute cell numbers of ST-HSCs and the GMPs subset was significantly decreased compared with littermate control mice (p&lt;0.01). We next evaluated the function of PRMT1 KO hematopoietic progenitor cells using an in vitro serial replating assay. PRMT1 KO progenitors generated lower numbers of CFUs in all passages relative to controls. Moreover, to assess the long-term consequences of Prmt1 deficiency in adult mouse hematopoiesis under the transplant context, we performed a competitive repopulation assay using unfractionated BM cells. We transplanted approximately 1 × 106 BM cells isolated from Prmt1f/f, or PRMT1f/f/Mx1-CRE mice (which are CD45.2), together with equal numbers of WT CD45.1 competitor cells, into lethally irradiated CD45.1 recipient mice. Post-pIpC induction, PRMT1-deleted CD45.2 cells were dramatically reduced compared to control donor cells, in both the peripheral blood and BM of recipient mice. Consistently, the chimerism contribution of PRMT1-deficient HSPCs or lineages were reduced relative to PRMT1-intact cells. To evaluate the long-term repopulation capacity of Prmt1null HSCs, we performed secondary BM transplantation (BMT). While analyzing BM at 16 weeks post BMT, we found PRMT1 KO markedly reduced CD45.2+ donor chimerism of multiple lineages as well as HSPCs subsets relative to those of controls. In BM, the absolute numbers of HSPCs subsets and lineages were markedly reduced compared to littermate control mice. showed a significant decrease compared with similarly treated littermate control mice. In summary, our data suggest PRMT1 is required at multiple stages of hematopoietic differentiation and HSPCs self-renewal. Our results indeed reveal that PRMT1 serves as a key regulator of normal adult hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Heterozygous loss of Srp72 in mice is not associated with major hematological phenotypes.

Familial cases of hematological malignancies are associated with germline mutations. In particular, heterozygous mutations of SRP72 correlate with the development of myelodysplasia and bone marrow aplasia in two families. The signal recognition particle 72kDa protein (SRP72) is part of the SRP complex, responsible for targeting of proteins to the endoplasmic reticulum. The main objective of this study is to investigate the role of SRP72 in the hematopoietic system, thus explaining why a reduced dose could increase susceptibility to hematological malignancies. We developed an Srp72 null mouse model and characterized its hematopoietic system using flow cytometry, bone marrow transplantations, and gene expression analysis. Heterozygous loss of Srp72 in mice is not associated with major changes in hematopoiesis, although causes mild reductions in blood and BM cellularity and minor changes within the stem/progenitor compartment. We did not observe any hematological disorder. Interestingly, gene expression analysis demonstrated that genes encoding secreted factors, including cytokines and receptors, were transcriptionally down-regulated in Srp72+/- animals. The Srp72+/- mouse model only partially recapitulates the phenotype observed in families with inherited SRP72 lesions. Nonetheless, these results can provide mechanistic insights into why SRP72 mutations are associated with aplasia and myelodysplasia in humans.

PB1727 OUTCOME OF CORE BINDING FACTOR ACUTE MYELOID LEUKEMIA IN ADULT PATIENTS

Background:Core binding factor acute myeloid leukemia (CBF AML) encodes two recurrent cytogentic abnormalities, t(8;21) and inv (16) and carries an overall good prognosis, yet some cases relapse. Aims:is to define unfavorable group of CBF AML by analysis of (C‐KIT and FLT3‐ITD) and to correlate with outcome of therapy.Methods:Prospective study started at Jan 2015 till June 2017, and included 70 patients CBF AML diagnosed and managed at medical oncology department of National Cancer Institute (NCI), Cairo University. All patients received standard of care protocol at NCI (“3+7”induction followed by 3‐4 courses of high dose cytarabine consolidation. The study was approved by the Institutional Review Board (IRB), of NCI, Egypt.Results:the median age was 31 years (18‐60, with a male/female ratio of 4/3. 42(60%) patients had t(8;21) and 28 patients had inversion 16(40%). C‐KIT mutations (exon 8 and exon 17) were detected in 10/52 tested patients and FLT3‐ITD was detected in 3/70 patients. While patients with Inv.16 had more lymphadenopathy and splenomegaly, median initial leucocytic count and exclusive gum hyperplasia, HCV‐Ab positivity (8/42) was exclusively present in patients with t(8;21). Clinically, lymphadenopathy, pallor and dyspnea were associated with worse overall survival (OS)(&lt;0.05). BM cellularity at day 28 of induction had a significant impact on survival (P = 0.002). Median OS was 19.5 month while median disease free survival (DFS) was not reached for the whole group. Patients with Inv.16 had non‐significant better DFS than patients with t(8;21)(P = 0.07). Neither C‐ KIT D816 V mutation nor FLT3‐ITD mutation had significant impact on OS or DFS, but when taken together (either C‐KIT or FLT3‐ITD mutant) had negative impact on DFS (P = 0.04).Summary/Conclusion:C‐KIT or FLT3‐ITD mutation had negative effect on DFS but not OS. HCV‐Ab positivity may be associated with t(8;21)AML, while Inv.16 is associated with more extramedullary diseaseimage

Myelodysplastic Syndromes with Hypocellular Marrow: Clinical Characteristics and Evaluation of Outcome

Background. MDS may be characterized by hypocellular marrow, irrespective of their WHO classification or molecular characteristics. Their prognostic weight must still be completely evaluated. MDS with hypocellular marrow tend to be considered an aplastic anemia "overlap syndrome" or a pre-aplastic anemia stage. There are no strong specific therapy recommendations, and large studies analyzing the outcome of hypocellular MDS are lacking. While selective sensitivity to immunosuppressive therapy is suggested, evidence in this sense is controversial. Aims. We wanted to evaluate the clinical characteristics, outcome and choice of therapy of patients with hypocellular MDS and compare them with normocellular MDS. Methods. We analyzed 2559 MDS cases with complete clinical annotations and with evaluable bone trephine biopsy, enrolled in our Italian National Registry FISMonlus. In this cohort of patients, 438 had a bone marrow cellularity Results. In FISM cohort median age was 72.5 yrs in the hypocellular group and 72,3 yrs in the normocellular group; M/F were 53.2%/46.8% for hypocellular MDS vs 62.6%/37.4% in normocellular MDS. IPSS-R risk categories were distributed as follows: Hypocellular MDS Very Low 15.5%, Low 35.1%, Intermediate 30.1%, High 11.3%, Very high 8%; Normocellular MDS Very Low 12.8%, Low 37.2%, intermediate 23.7%, High 15.5%, Very High 11.4%. Global median overall survival (OS) was 77 months for hypocellular MDS and 56 months for normocellular MDS. When OS was evaluated in the different IPSS-R risk groups, Lower risk MDS cases with hypocellular BM had a median OS of 125 mos while normocellular had a median OS of 74 mos (p Conclusion. Our results are based on an unbiased analysis of "real life" MDS patients with hypocellular BM compared to normocellular ones. Clinical characteristics between the two groups were not significantly different in terms of age, gender, and distribution in the various IPSS-R risk categories. The outcome of the hypocellular marrow-MDS cases was better in comparison with that of normocellular MDS, with significantly longer OS in IPSS-R lower risk cases. The advantage in OS for hypoplastic MDS wasn9t present for IPSS-R higher risk cases. Finally, the choice of first line therapy doesn9t seem to be influenced by the BM cellularity, with a surprising very low proportion of patients receiving immunosuppressive agents, despite several guideline recommend of this treatment for hypoplastic MDS. Disclosures Santini:Novartis: Honoraria; Otsuka: Consultancy; Celgene: Honoraria, Research Funding; AbbVie: Membership on an entity9s Board of Directors or advisory committees; Amgen: Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Finelli:Novartis: Consultancy, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Gaidano:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria. Oliva:Celgene: Consultancy, Other: Royalties, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; La Jolla: Consultancy; Sanofi: Consultancy, Speakers Bureau. Cilloni:celgene: Membership on an entity9s Board of Directors or advisory committees; Janssen: Membership on an entity9s Board of Directors or advisory committees.

Sclerostin expression in distinct osteolineage cell types differentially regulates hematopoietic stem cell and B lymphocyte development

Abstract Investigation of the reciprocal effects of hematopoietic cell types and osteolineage cells on each other’s cell fates have clinical applications to improve bone fracture healing, prevent age-related bone loss and immune deficiencies. A significant knowledge gap exists as to the contribution of different osteolineage cells to the differentiation, proliferation, and long term survival of B cells in the BM. In global sclerostin knockout (SostKO) mice, we previously uncovered a cell-extrinsic requirement for sclerostin on B lymphocyte development. It is widely accepted that sclerostin is transcribed primarily in osteocytes (OCYs), but whether is it expressed and/or active in other bone progenitors has been controversial. In order to identify the Sost-expressing osteolineage cell type that is responsible for B cell support, we analyzed hematopoietic differentiation in mice lacking Sost in mesenchymal stem cells (MSCs) [Prx1-Cre], osteoblasts (OBs) [Col1a-Cre] and OCYs [Dmp1-Cre] through the use of conditional Sost-KO (cKO) mice. Loss of Sost expression in MSCs resulted in reduced BM cellularity, whereas this was unaffected by Sost-deficiency in OB and OCYs. Furthermore, MSC-specific depletion of Sost caused an accumulation of early B cell progenitors. In contrast, loss of Sost in OBs and OCYs resulted in a reduction of mature “recirculating” B cells in the BM. Remarkably, the percentage of long-term CD150+ CD48− HSCs was significantly reduced in MSC-cKO mice, but was not affected in OB- and OCY-cKOs. The data described herein suggest that Sost deficiency in MSCs, OBs, and OCYs differentially regulates hematopoietic stem cell retention, survival, maintenance and B cell differentiation in the bone microenvironment.

Role of CXCL12-Expressing Bone Marrow Populations in Leukemic Stem Cell Regulation

CXCL12 is the major chemoattractant for hematopoietic stem cells (HSC) in the bone marrow microenvironment (BMM), and plays a major role in HSC localization to their regulatory niches. Studies using genetic drivers/reporters have shown that CXCL12 deletion from perivascular mesenchymal stem cells (MSC) and CAR cells (using Prx1-Cre line) leads to loss of HSC quiescence, frequency and self-renewal potential, while deletion from endothelial cells (using Tek-Cre line) results in modest loss of HSC long-term repopulating activity. In contrast, deletion of CXCL12 from osteoprogenitors (using Osx-Cre line) resulted in HSC mobilization without any effect on HSC function, while deletion from mature osteoblasts (using OCN-Cre line) had no effect on HSC function (Greenbaum et al. Nature. 2013;495(7440):227-30; Ding et al. Nature. 2013;495(7440):231-5.). These studies have been useful in identifying MSC/CAR cells and endothelial cells as important HSC niche components but the BM niches for LSC remain poorly characterized. In the present study, we examined alterations in CXCL12-producing niche cells in the CML BMM, and their role in regulating LSC growth, using the SCL-tTA BCR-ABL mouse model of CML. Our previous studies indicated that CXCL12 levels are reduced in CML compared to normal BM (Zhang et al. Cancer Cell. 2012; 21(4):577). To evaluate the effect of leukemia development on specific CXCL12-expressing cell populations in the BMM, we crossed CXCL12GFP mice (GFP reporter knocked into the CXCL12 locus) with SCL-tTA-BCR-ABL mice to generate CXCL12GFP-SCL-tTA-BCR-ABL mice. CXCL12-expressing cells in the BM were identified by GFP expression. Mice developing CML after BCR-ABL induction by tet-withdrawal demonstrated significantly increased numbers of GFP+ endothelial cells (CD45-Ter119-CD31+) and reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) compared to WT mice. Within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased. To evaluate the contribution of CXCL12-expressing populations towards LSC regulation, we crossed CXCL12f/f mice (loxP sites flanking exon 2) with Tek-Cre, Prx1-Cre, OCN-Cre and OSX-Cre transgenic lines. CML BM cells (CD45.1/2+; 2*106/mouse) were transplanted into lethally irradiated (8Gy) WT (CD45.2) Cre- or Cre+ CXCL12f/f knockout animals, and followed for CML development. When compared to WT mice, CXCL12f/f-Tek-Cre+ animals exhibited significantly reduced engraftment of CML cells (CD45.1/2+) in the BM, with associated reduction in CML myeloid cells (Gr-1+Mac-1+), MEP (CD16/32- CD34-), CMP (CD16/32lowCD34+), MPP (LSK+CD48+) and LTHSC (CD150+CD48-) numbers. No changes in splenic engraftment were seen. To evaluate long-term reconstitution, BM cells from primary transplanted WT or knockout animals were transplanted into lethally irradiated (8Gy) WT secondary recipients. CML engraftment in secondary mice receiving BM from Tek-Cre+ and WT animals was similiar at 12 weeks, indicating that residual LTHSC retained repopulating potential. In contrast, CXCL12f/f-OCN-Cre and CXCL12f/f-Osx-Cre mice did not demonstrate significant differences in total CML cell or CML LTHSC engraftment, but showed increased LMPP engraftment in the BM. Interestingly CXCL12f/f-Prx1-Cre+ animals exhibited significantly increased leukocytosis and BM cellularity, and increased MEP, CMP, LMPP, MPP, STHSC and LTHSC numbers in the BM compared to WT mice. Increased numbers of CML myeloid cells and LSK were seen in the peripheral blood, but no change in splenic engraftment was seen. CML engraftment in secondary mice receiving BM from Prx1-Cre+ animals was significantly increased at 12 weeks compared to WT animals, indicating that the expanded LTHSC population maintained repopulating potential. These results suggest that loss of endothelial cell expressed CXCL12 reduced CML LTHSC in BM, whereas loss of MSC/CAR cell expressed CXCL12 enhances CML LTHSC numbers in BM, in association with increased mobilization to PB. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM endothelial cell and MSC/CAR cells in CML, and indicate significant differences in niche regulation of CML LSC compared with normal HSC. We expect that improved characterization of BM niches in CML will facilitate further dissection of key niche interactions underlying LSC maintenance and expansion. DisclosuresNo relevant conflicts of interest to declare.