Whole Bone Marrow Research Articles

Granulocyte-Macrophage-Colony-Stimulating-Factor Combined with Prostaglandin E1 Create Dendritic Cells of Leukemic Origin from AML Patients' Whole Blood and Whole Bone Marrow That Mediate Antileukemic Processes after Mixed Lymphocyte Culture.

Although several (chemotherapeutic) protocols to treat acute myeloid leukemia (AML) are available, high rates of relapses in successfully treated patients occur. Strategies to stabilize remissions are greatly needed. The combination of the (clinically approved) immune-modulatory compounds Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) and Prostaglandine E1 (PGE-1) (Kit-M) converts myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu ex vivo, leukemia-specific antileukemic immune cells are activated. Therefore, Kit-M treatment may be an attractive immunotherapeutic tool to treat patients with myeloid leukemia. Kit-M-mediated antileukemic effects on whole bone marrow (WBM) were evaluated and compared to whole blood (WB) to evaluate the potential effects of Kit-M on both compartments. WB and WBM samples from 17 AML patients at first diagnosis, in persisting disease and at relapse after allogeneic stem cell transplantation (SCT) were treated in parallel with Kit-M to generate DC/DCleu. Untreated samples served as controls. After a mixed lymphocyte culture enriched with patients' T cells (MLC), the leukemia-specific antileukemic effects were assessed through the degranulation- (CD107a+ T cells), the intracellular IFNγ production- and the cytotoxicity fluorolysis assay. Quantification of cell subtypes was performed via flow cytometry. In both WB and WBM significantly higher frequencies of (mature) DCleu were generated without induction of blast proliferation in Kit-M-treated samples compared to control. After MLC with Kit-M-treated vs. not pretreated WB or WBM, frequencies of (leukemia-specific) immunoreactive cells (e.g., non-naive, effector-, memory-, CD3+β7+ T cells, NK- cells) were (significantly) increased, whereas leukemia-specific regulatory T cells (Treg, CD152+ T cells) were (significantly) decreased. The cytotoxicity fluorolysis assay showed a significantly improved blast lysis in Kit-M-treated WB and WBM compared to control. A parallel comparison of WB and WBM samples revealed no significant differences in frequencies of DCleu, (leukemia-specific) immunoreactive cells and achieved antileukemic processes. Kit-M was shown to have comparable effects on WB and WBM samples regarding the generation of DCleu and activation of (antileukemic) immune cells after MLC. This was true for samples before or after SCT. In summary, a potential Kit-M in vivo treatment could lead to antileukemic effects in WB as well as WBM in vivo and to stabilization of the disease or remission in patients before or after SCT. A clinical trial is currently being planned.

Inflammatory Signaling Mediated By Gasdermin D Underlies Tet2-Mutant Clonal Hematopoiesis-Associated Tissue Dysfunction

Aging is characterized by the progressive accumulation of somatic mutations. Acquisition of certain somatic mutations in hematopoietic stem cells (HSCs) confers on them a fitness advantage enabling them to outcompete other HSCs by clonal expansion in a phenomenon called clonal hematopoiesis (CH). CH is associated with a 40% increase in all-cause mortality linked to elevated risk for aging-associated morbidities such as cardiovascular disease (CVD), chronic kidney disease, COPD, and chronic liver disease. However, the mechanism by which CH exerts its adverse effects on disparate tissues is poorly understood. Here, we use CH-associated cardiac dysfunction as a paradigm to uncover general principles by which CH leads to the decline of tissue function. CH was modeled in mice by transplanting whole bone marrow (WBM) cells deficient for Tet2, a frequently mutated CH gene, into irradiated WT mice. We observed an elevated incidence of atrial fibrillation (AF) in Tet2 -/- WBM recipients relative to WT WBM recipients without any surgical or dietary intervention (Fig. A). The Tet2 -/- WBM recipients also had reduced left ventricular ejection fraction (LVEF), an indicator of heart failure. Mice exhibiting elevated incidence of AF and reduced LVEF are defined as having cardiac dysfunction. In addition, we observed that Tet2 -/- WBM recipients had increased systemic inflammation compared to controls, as evidenced by elevated levels of proinflammatory cytokines in the serum. Furthermore, their cardiac tissue exhibited upregulation of the inflammasome signaling pathway including the activation of Gasdermin D, the effector of pyroptotic cell death. Inflammation in the cardiac tissue was concomitantly associated with increased cardiac fibrosis in Tet2 -/- WBM recipients. Since inflammation is a known driver of CVD, we hypothesized that abrogating Gasdermin D-mediated inflammatory signaling could ameliorate CH-induced cardiac dysfunction. Indeed, Gsdmd -/- recipient mice were protected from cardiac dysfunction upon transplantation with Tet2 -/-WBM cells (Fig. A). Importantly, expansion of mutant hematopoietic cells was also mitigated in the absence of GSDMD: transplantation of Tet2 -/- WBM cells along with either WT or Gsdmd -/- WBM cells revealed that GSDMD deficiency restrained the rate of clonal expansion of Tet2 -/- cells (Fig. B). This suggests that instead of being mere bystanders, WT cells in the environment may play an active role in regulating the rate of mutant cell expansion via intercellular communication mediated by GSDMD. Identification of proinflammatory factors mediating clonal expansion and cardiac dysfunction is of significant interest for potential therapeutic intervention. Thus, we sought to determine factors that were upregulated in CH mouse models while being downregulated in Gsdmd -/- mice. Luminex multiplexed assay enabled us to identify several understudied proinflammatory cytokines present in serum satisfying the above criteria. In addition to elevated levels in the serum, some of these cytokines were upregulated in the cardiac tissue of Tet2 -/- WBM recipient mice but not in WT WBM recipients. Furthermore, some are reported to induce inflammasome signaling. CH, even in the absence of known drivers, is associated with higher CVD-associated mortality, suggesting the existence of pathways common to multiple forms of CH; systemic inflammation is one potential common mechanism. We tested whether aged WT WBM, which has low levels of mutant clone expansion but exhibits several features of CH, could cause cardiac dysfunction. We transplanted either aged or young WT WBM into young WT recipients and found that the aged WT WBM recipients exhibited elevated AF and reduced LVEF. These data suggest that aging-associated systemic inflammation may be sufficient for cardiac dysfunction. Importantly, one of the cytokines we uncovered was also upregulated in aged WT mice and has been associated with inflammation and fibrosis in multiple tissues. This cytokine is being further investigated as a top candidate for therapeutic intervention since it is a common feature of CH with and without identified drivers. Overall, our findings demonstrate the critical role of Gasdermin D-mediated inflammatory signaling in the pathogenesis of Tet2-mutant CH-induced cardiac dysfunction. We have also uncovered a cytokine that has emerged as a promising therapeutic target for CH-associated pathologies.

Understanding the Functional Differences between PRC2.1 and PRC2.2 in Hematopoiesis

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell (HSPC) fate through the modification of chromatin architecture. Polycomb repressive complex 2 (PRC2) is an indispensable epigenetic modifier that catalyzes histone 3 lysine 27 tri-methylation (H3K27me3) repressive mark through its catalytic subunit EZH2. PRC2 is divided into two independent complexes, PRC2.1 and PRC2.2, based on the specific interacting accessory proteins. PRC2.1 comprises one of the three polycomb-like proteins (PCL1/PHF1, PCL2/MTF2, or PCL3/PHF19), as well as one of the two recently identified accessory proteins, EPOP or PALI1. However, PRC2.2 contains either JARID2 or AEBP2 as co-factors. These accessory proteins aid in the recruitment of the PRC2 complexes to different sites in the genome to catalyze H3K27me3. Recent studies performed on mouse embryonic stem cells showed that PRC2.1 and PRC2.2 are involved in distinct lineage specification and commitment through their specific co-factors. However, the functional differences between PRC2.1 and PRC2.2 in HSPC differentiation and lineage commitment has not been thoroughly characterized. To compare the complete loss of PRC2 versus PRC2.2-specific loss of function in normal hematopoiesis, we generated Mx1-Cre: Jarid2fl/fl and Mx1-Cre: Ezh2fl/fl mouse strains. Two-month old Mx1-Cre(+) control, Mx1-Cre(+): Jarid2fl/fl, and Mx1-Cre(+): Ezh2fl/fl mice were injected with six doses of polyinosinic:polycytidylic acid (pIpC) to promote conditional deletion of Jarid2 and Ezh2. Two months post-pIpC injection, whole bone marrow (WBM) cells were used to perform competitive transplantation (5x10 5 CD45.2 test WBM plus 5x10 5 CD45.1 competitor WBM cells). Jarid2-KO primary recipients showed the highest CD45.2 peripheral blood (PB) engraftment (~80%), and the highest tri-lineage (myeloid 80%, B cell 70%, T cell 80%) chimerism compared to the control (myeloid 50%, B cell 45%, T cell 25%) and Ezh2-KO (myeloid 65%, B cell 5%, T cell 5%) recipients. Ezh2-KO recipients showed the lowest CD45.2 PB engraftment (~20%), and were deficient for PB lymphoid cell production. Jarid2-KO recipients showed the highest bone marrow (BM) engraftment (~ 90%), while Ezh2-KO recipients showed significant BM lymphoid engraftment (B cell 55% and T cell 40%) inverse to what was observed in the PB (B cell 5% and T cell 5%). This suggested that complete loss of PRC2 function promotes blockage in the migration of mature lymphoid cells from BM to the PB. Jarid2-KO and Ezh2-KO recipients showed significant reductions in phenotypically defined HSC cell number and frequency compared to control recipients suggesting that PRC2.2 function through its co-factor Jarid2 to maintain HSC pool. To further investigate the functional effect of both genes' loss of function at the molecular level, we performed single cell transcriptomic analysis from KSL (lineage-, c-Kit+, Sca1+, CD45.2+) cells sorted from the aforementioned recipients. Based on transcriptomic analysis, Ezh2 loss of function promoted an increase in MPP4 and precursor B cell populations in the BM of recipient mice. However, no significant changes was observed between control and Jarid2-KO clusters. Analysis showed that the Mtf2 co-factor has the highest expression level throughout the whole KSL clusters compared to other PRC2.1 and PRC2.2 accessory proteins. These results suggested that Mtf2-associated PRC2.1 is necessary to restrict MPP4 self-renewal and to repress B cell priming in HSPCs. Although no significant change was observed in the HSC cluster between Jarid2-KO and Ezh2-KO genotypes, Jarid2 and Ezh2 loss of functions showed significant differential gene expression in the HSC population when compared to each other and to the control suggesting that Jarid2-associated PRC2.2 regulates a unique molecular program in HSCs independently from PRC2.1. Combined with our functional data, these results suggested that PRC2.2 restricts HSC-specific gene expression in non-self-renewing progenitor cells through its co-factor Jarid2; while PRC2.1 restricts MPP4 fate and B cell priming through its co-factor Mtf2. Ongoing studies are aimed to delineate the distinct molecular programs regulated by PRC2.1 and PRC2.2 in HSPCs using CRISPR-Cas9 sgRNA lentiviral system targeting each of the PRC2.1 and PRC2.2 accessory proteins independently and validation in lineage-specific functional assays.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=<0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

JAK2 V617F Impairs Lymphoid Differentiation in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2 V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2 V617F clone does not contribute to mature lymphocytes. We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2 V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2 V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2 V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2 V617F alleles were largely absent in T and B cells relative to HSCs. Lymphopoiesis declines with age, potentially explaining the absence of JAK2 V617F in lymphocytes from older patients with MPN even if JAK2 V617F has no effect on lymphopoiesis. This “JAK2 V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2mutated lymphocytes. However, we found that JAK2 V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2 V617F neutral” hypothesis are not supported by patient data. The alternative explanation is that JAK2 V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2 V617F adverse” hypothesis by tracking JAK2 V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2 V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2 V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2 V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2 V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2 V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation. To further define the extent to which JAK2 V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2 V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2 V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2 V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis. To determine the effect of JAK2 V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2 V617F or wild type JAK2 (JAK2 WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2 V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2 V617F HSCs displayed a competitive disadvantage relative to their JAK2 WT counterparts during lymphopoiesis in vivo. Myeloid proliferation in MPN driven by JAK2 V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2 V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2 V617F interferes with T cell differentiation.

Increased Expression of SETDB1 Predicts Poor Prognosis in Multiple Myeloma

Several genes on chromosome 1q21 region predict a high risk of multiple myeloma (MM); however, the underlying molecular pathology remains elusive. Overexpression, amplification, or activation of SET Domain Bifurcated 1 (SETDB1), which is located on 1q21, is closely associated with poor prognosis of many human solid malignancies. In our study, upregulation of SETDB1 might indicate an unfavorable prognosis of MM using bioinformatics analysis from GEO databases and MMRF-CoMMpass. Here, increased SETDB1 expression was observed in the plasma cells from newly diagnosed multiple myeloma patients compared to those from the normal controls. Meanwhile, SETDB1 overexpression was the result of increased copy numbers of SETDB1 gene. In MM patients, the Kaplan-Meier analysis was employed to demonstrate that increased SETDB1 expression was associated with shorter overall survival (OS) and event-free survival (EFS). Besides, we conducted multifactorial cox regression analysis to state that SETDB1 expression was an independent biomarker for OS and EFS. MM patients with higher SETDB1 expression showed higher levels of beta-2 microglobulin (β2M), lactate dehydrogenase (LDH), and bone marrow biopsy plasma cells (BMPC) and lower levels of haemoglobin (HGB). Functional enrichment analysis suggested that SETDB1 could promote cell cycle progression in myeloma. Finally, we observed that SETDB1 was distinctly correlated with tumor immunity in MM. SETDB1 expression in myeloma cells was positively correlated with CD56dim natural killer cells but negatively correlated with infiltrating levels of type17 T helper cells, effector memory CD8 T cells, activated dendritic cells, and natural killer T cells from whole bone marrow (WBM) biopsies. Taken together, these results indicated that SETDB1 could be used as a novel biomarker for predicting the prognosis of MM patients.

Hematopoietic Stem Cell Potential within the Primary Sorted Lineage Positive Population: What Are We Losing with Hematopoietic Stem Cell Purification?

Lineage positive cells within murine whole bone marrow (WBM) are classically considered to be devoid of stem cell capacity. As such, lineage depletion prior to further selection for stem cell marker positive, lineage negative cells dominates virtually all hematopoietic stem cell (HSC) purification protocols. Here, we present data showing a significant population of long-term multi-lineage hematopoietic stem cells within the primary sorted lineage positive (Lin+) murine marrow population. Using limiting dilution competitive bone marrow transplantation assays, we found significant long-term multi-lineage engraftment capacity within the total primary sorted Lin+ population (cells positive for erythroid (TER119), myeloid (GR1, CD11b) or lymphoid (B220, CD4, CD8) markers). When 0.25 x 106 Lin+ cells were competed against 1 x 106 competitor WBM cells, average percent donor chimerism in recipient mice at 6 months post-transplant was 7.2% ± 2.9%. Donor chimerism was multi-lineage. There was still 1.9% ± 0.5% donor chimerism when only 62,500 primary sorted Lin+ cells were competed against 1 x 106 competitor WBM cells at 3 months post transplant, and further limiting dilution assays are ongoing to quantitate the stem cell compartment within the primary sorted Lin+ population. These levels of engraftment were similar to that found within un-separated donor WBM at the same dilutions, and multi-lineage engraftment capacity within the primary sorted Lin+ population persisted in secondary transplant. When further sub-fractionated into erythroid, myeloid or lymphoid sub-populations, every primary sorted individual sub-population contained long-term multi-lineage engraftment capacity at 6 months post transplant. Primary sorts were between 96-97.5% pure. When primary Lin+ sub-sets (B220+ cells, GR1+ cells, or CD11b+ cells) were double sorted, there was loss of engraftment potential within the double sorted persistently positive Lin+ subsets, but surprisingly high engraftment within those cells negative for only the single Lin+ marker on double sort (31%-59% donor chimerism at 6mo post-transplant; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Immunophenotype analysis showed that these populations were not enriched in c-Kit+/Sca-1+/CD150+ cells. The high engraftment capacity of cells within the primary sorted Lin+ population, further isolated solely based on their being negative for a single Lin+ marker on the double sort prompted us to directly compare their engraftment capacity to that of purified Lineage negative (Lin-) cells. Interestingly, although isolated solely based on the presence or absence of GR1 on double sort, we found that the average percent donor chimerism at 6 months post-transplant was equal between purified Lin- cells (58.3% ± 17%) and the population of GR1 positive cells that became negative for GR1 on double sort (58.3% ± 26%; 1 x 105 donor cells + 3 x 105 competitor WBM cells). Using tritiated thymidine suicide, we found that the stem cell activity within these populations isolated from the primary sorted Lin+ fraction was due to actively cycling cells. These data further indicate that the engraftment capacity within the primary sorted Lin+ population is not simply due to conventionally purified quiescent HSCs contaminating our primary sort. In sum, there is a cycling population of stem cells within the primary sorted Lin+ fraction that appears to be distinct from quiescent highly purified HSCs. As primary sorted Lin+ cells, routinely discarded in most HSC studies, comprise up to 98% of WBM, engraftment capacity within this population represents a potentially large and understudied stem cell population within WBM worthy of further characterization. Work is currently underway to further quantitate and define this population, and delineate how cell cycle-related changes influence the phenotype and function of this cycling murine HSC population. DisclosuresNo relevant conflicts of interest to declare.

Deconvolution of the Immune Microenvironment Can Predict the Outcome of Myeloma Patients and Inform Potential Intervention Strategies

IntroductionThe microenvironment of cells around multiple myeloma (MM) tumors strongly influences patient outcomes, as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate pro- and anti-myelomatous cell types within the bone marrow.MethodsThe population of immune and other non-tumor cell types was estimated from RNA microarray data derived from paired whole bone marrow (WBM) core biopsies and CD138-enriched (CD138+) bone marrow aspirates at presentation from 405 newly diagnosed MM patients who underwent similar treatments. Expression profiles from CD138+ cells were used to infer the residual transcriptome contribution of immune and stromal cells in the WBM array data. We performed unsupervised clustering of the estimated microenvironment gene expression profiles and calculated the statistical significance of cluster splitting using the R packages ConsensusClusterPlus and sigclust, respectively. Individual cell type composition of these microenvironments was estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) that we augmented with phenotypic profiles of 5 bone marrow-specific cell types (myelomatous plasma cells, plasma memory cells, osteoclasts, osteoblasts, and adipocytes).ResultsThis deconvolution approach accurately estimated the percent of tumor cells in WBM core biopsy as estimated by both microscopy and flow cytometry (PCC = 0.57, RMSE = 10.9%). Unsupervised consensus clustering of whole bone marrow microenvironment gene expression data revealed 5 significantly different subtypes. The two largest subtypes, Cluster 1 (N=136) and Cluster 2 (N=128), had the most divergent outcomes with median PFS values of 78 months and 53 months, respectively. Cluster 1 was significantly enriched for ISS-I patients (FDR < 0.02) while Cluster 2 was enriched for ISS-III patients (FDR < 7x10^-5). Cluster 2 showed a significant increase in estimated M1 macrophages, CD8+ T cells, adipocytes, and dendritic cells with a decrease in estimated osteoclasts versus all other clusters combined (FDRs < 0.05). Cluster 5 (N=63) had the second worst PFS at 55 months, however, it had 44% fewer than expected ISS-III patients (FDR = 0.04). Cluster 5 had fewer estimated T cells and adipocytes, and it was the only cluster with a significant (3-fold) increase in estimated NK cells compared to the other clusters combined (FDRs < 0.05). No clusters were significantly enriched for older individuals, hyperdiploidy or common chromosomal translocations (t(4;14), t(11;14), t(14;16)). No clusters were significantly enriched for CD138+ gene expression-defined high risk patients (UAMS GEP-70) or CD138+ gene expression-based molecular subtypes (Zhan et al., 2006).ConclusionsWe demonstrated the technical feasibility to accurately delineate CD138+ expression profiles from a bulk transcriptome admixture and to use the residual microenvironment transcriptome to cluster patients into distinct microenvironment subtypes. Within these subtypes, the clinical outcomes are not explained by CD138+ molecular features nor ISS stage. For example, Cluster 5 has patients with poor prognosis compared to the rest of the cohort, yet it is not significantly enriched for high risk translocations, ISS-III patients, nor GEP-70 high risk patients. Cluster 5 patients show significant decrease in both CD8+ T cell infiltrate and activated dendritic cells, perhaps reflective of a high risk immune state that would not be responsive to checkpoint inhibition. Moreover, Cluster 5 patients are the only ones to have an increase in NK cells, suggesting one could try to stimulate these NK cells to reinvigorate their anti-tumor immunity. Cluster 2, with the poorest prognosis patients and highest tumor burden (by ISS stage), shows increased CD8+ T cells and dendritic cells (where all other clusters are neutral or decreased in these cell types). Cluster 2 patients may thus benefit from checkpoint blockade, and we plan to further explore relevant checkpoint-related pathways in this cohort. Adding this layer of immune-microenvironment to the analysis of myeloma patient samples enables us to formulate biological hypotheses independent of CD138+ biology and may eventually guide more tailored therapeutic interventions and improve outcomes for patients. [Display omitted] DisclosuresDanziger:Celgene Corporation: Employment. Dervan:Twinstrand Biosciences: Equity Ownership; Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment. Copeland:Celgene Corporation: Employment. Fitch:Celgene Corporation: Employment. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene Corporation: Consultancy, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding. Foy:Celgene Corporation: Employment, Equity Ownership. Trotter:Celgene Corporation: Equity Ownership; Celgene Institute for Translational Research Europe: Employment. Hershberg:Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Fraizer Healthcare Partners: Consultancy; NanoString Technologies: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment. Morgan:Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Jarid2 Restricts Long-Term Repopulating Stem Cell Capacity in Multipotent Progenitors and Acts As Tumor Suppressor in Chronic Myeloid Neoplasms

Myeloid disorders including myeloproliferative neoplasms (MPN) are clonal diseases of hematopoietic stem or progenitor cells that arise from genetic and epigenetic alterations which perturb key processes such as self-renewal, proliferation and differentiation. The most common genetic mutations found in the MPN initiating clone occur in the genes JAK2, MPL and CALR, and these mutations are likely responsible for initiation of disease. Acquisition of additional co-operating mutations in genes such as TET2, IDH1/2, and NRAS in the MPN clone can give rise to secondary acute myeloid leukemia (sAML), a much more aggressive disease with an average survival time after transformation less than six months. Recent genome sequencing studies identified chromosomal deletions of JARID2, a Polycomb Repressive Complex 2 (PRC2) co-factor involved in implementing H3K27me3 marks, in the leukemic phase of post-MPN sAML but not in chronic MPN phase of the disease. These data indicate that JARID2 might act as a tumor suppressor in chronic myeloid disorders and genetic deletion of JARID2 could be a sAML-specific transforming event.To investigate the role of Jarid2 as a tumor suppressor, we utilized an inducible mouse model of the prototypical MPN driver mutation Jak2V617F. The absence of Jarid2 in a Jak2V617F/+ background accelerated MPN progression, with a median survival of 23 and 43 days post Jarid2 -deletion for Jarid2-/-- Jak2V617F/+ and Jarid2+/-- Jak2V617F/+ mice, respectively, compared to 113 days for Jak2V617F/+ control mice. Together, these data suggest that loss of Jarid2 in Jak2V617F/+ background accelerates MPN pathogenesis.To more completely understand the tumor suppressor role of Jarid2 in MPNs, we aimed to define its role in normal hematopoiesis. We deleted Jarid2 using the Vav-CRE driver to facilitate conditional inactivation of Jarid2 in hematopoietic system. Competitive transplantation was employed to assess the hematopoietic stem cell (HSC) potential. Either 100 phenotypically defined HSCs (Lineage-cKit+ScaI+CD48-CD150+) or 2.5x105 whole bone marrow (WBM) cells were transplanted into lethally irradiated recipient mice along with 2.5 x 105 WBM cells from genetically distinguishable WT mice. At 16 weeks post-transplant, the analysis of test cell contribution in recipient mice revealed that Vav-CRE: Jarid2 -KO HSCs did not contribute to peripheral blood (PB) chimerism. In contrast, Vav-CRE: Jarid2 -KO WBM contributed to PB lineages at levels comparable to control WBM. These data suggested that a population of cell(s) outside phenotypically-defined HSCs contribute(s) to hematopoiesis in the absence of Jarid2 . To find the population(s) that reside in Vav-CRE: Jarid2 -KO WBM with long-term multi-lineage capacity, we transplanted the following sorted progenitor populations: multipotent progenitors (50 MPPs; Lineage-cKit+ScaI+CD48-CD150-) and restricted progenitors (250 RPs; Lineage -cKit+ScaI+CD48+CD150-) in a competitive setting as described above. As expected, neither control MPPs or RPs were capable of long-term multi-lineage reconstitution. Strikingly, Vav-CRE: Jarid2 -KO MPPs engrafted in primary and secondary recipients, generating tri-lineage hematopoiesis effectively, whereas Vav-CRE: Jarid2 -KO RPs did not engraft long-term in any recipients. Additionally, MPPs isolated from Jak2V617F - Jarid2KO mice can propagate MPN in transplanted recipients, unlike MPPs transplanted from Jak2V617F mice. RNA-seq expression profiling revealed upregulation of Runx1t1 and Mycn in Jarid2-KO MPPs, genes which have been used to reprogram committed blood cells to a multipotent state. We hypothesized that the inability to silence Runx1t1 and Mycn in Jarid2-KO MPPs was a mechanism that permits self-renewal in this normally transiently-repopulating cell fraction. Enforced expression of Runx1t1 and Mycn in WT MPPs endowed long-term repopulating capacity in vivo, phenocopying the behavior of Jarid2-KO MPPs. Cumulatively, our data suggest that the normal role of Jarid2 in hematopoiesis is to restrict self-renewal capacity to long-term HSCs, and that dysregulation of this process can lead to hematopoietic malignancies. DisclosuresNo relevant conflicts of interest to declare.

Age-Associated Changes in Bone Marrow-Derived Extracellular Vesicles May Alter Their Effects on Murine Hematopoietic Stem Cell Function

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the bone marrow niche and have been implicated in numerous features of aging. However, their role in natural hematopoietic stem cell (HSC) aging has not been fully elucidated. The goal of this work was to test the hypothesis that EVs from whole bone marrow (BM-EVs) can modulate the HSC aging phenotype in vivo. With respect to HSC aging, our prior work showed that, in contrast to the well-known reduced functional capacity and prominent myeloid skewing displayed by old immunophenotypically-defined HSCs, old unseparated whole bone marrow (WBM) (24-26-mo-old) had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM in limit dilution competitive bone marrow transplantation, and showed minimal to no myeloid skewing. In order to test the ability of BM-EVs derived from this total WBM population to alter HSC aging phenotype, we first isolated EVs from WBM flushed from old (24-26-month old) and young (6-8-week old) C57/BL6 (CD45.2) mice by differential centrifugation (2000 × g for 30 min, supernatant centrifuged 100,000 × g for 1 hour, BM-EV pellet collected). Utilizing nanoparticle tracking analysis, we found no difference in mean particle size between old and young BM-EVs, but there was an approximately 2-fold increase in the number of EVs from old WBM compared to young WBM. To test the ability of these BM-EVs to alter HSC function in vivo, we injected old CD45.2 mice with 2 x 109 young BM-EVs and young CD45.2 mice with 2 x 109 old BM-EVs mice via tail vein, daily x 3 days. Control mice were injected with age-matched BM-EVs or vehicle alone. At one-month post injection, we isolated total WBM and Lineage negative/c-Kit+/Sca-1+/CD150+ cells (LSK-SLAM) from the EV-exposed or vehicle control mice. We injected either 3 x 105 WBM cells or 400 LSK-SLAM mixed with 3 x 105 healthy WBM competitor cells from young B6.SJL (CD45.1) mice into lethally irradiated young CD45.1 hosts and measured peripheral blood chimerism and lineage contribution by flow cytometry up to 6 months post-transplant. For the young marrow, exposure to old BM-EVs had no appreciable effects on engraftment capacity or lineage distribution. However, old WBM exposed to young BM-EVs exhibited a significant decrease in engraftment (15% ± 5%) when compared to old WBM exposed to age-matched old BM-EVs (61% ± 14%) or vehicle control (47% ± 7%) (% average donor chimerism ± SEM, n=4-5 mice/group, p&amp;lt;0.04). Similarly, there was a trend toward decreased engraftment capacity by old LSK-SLAM after young BM-EV exposure (7% ± 4%) and increased engraftment capacity by the old LSK-SLAM after old BM-EV exposure (27% ± 10%) compared to vehicle control (15% ± 2%) (% average donor chimerism ± SEM, n=4-5 mice/group, p=not significant (ns)). These results are consistent with our prior data discussed above in which old un-separated WBM, the source of the old BM-EVs, had increased engraftment capacity compared to young WBM. Interestingly, there was also a trend toward reversal of classic myeloid skewing when old LSK-SLAM were exposed to young BM-EVs (36% ± 11%) compared to vehicle control (64% ± 3%) (average myeloid % of donor-derived peripheral blood ± SEM, n=4-5mice/group, p=ns). In addition, consistent with the known increase in LSK-SLAM with age, our preliminary data showed that old mice exposed to young BM-EVs had an approximately 7-fold decrease in the number of LSK-SLAM in marrow, indicating that BM-EVs within the young bone marrow microenvironment may modulate HSC population size. Finally, microRNA (miR) expression profiling (NanoString Technologies) indicated that a number of miRs known to be involved in hematopoiesis, proliferation, self-renewal, differentiation, senescence and inflammation were differentially represented in old and young BM-EVs, with miR-29a, miR-24, and miR-21 significantly increased and miR-105 significantly decreased in old BM-EVs compared to young BM-EVs. Together, these data indicate that young BM-EVs, via transfer of differentially age-related cargo, may alter engraftment capacity, modify the lineage commitment and may regulate LSK-SLAM population size during natural aging. Future studies more clearly delineating age-associated BM-EV effects on HSCs and defining the molecular mechanisms underlying these effects could yield key insights into the natural aging of HSCs and facilitate restoration of healthy hematopoiesis in the elderly. Disclosures No relevant conflicts of interest to declare.

3079 – MURINE WHOLE BONE MARROW MAY CONTAIN A NONCONVENTIONAL POPULATION OF LONG-TERM MULTI-LINEAGE REPOPULATING CELLS WITH ALTERED SUSCEPTIBILITY TO AGING

Studies in hematopoietic aging have shown that classically defined, highly purified hematopoietic stem cells (HSCs) increase in number but have impaired function and exhibit myeloid skewing. However, we identified a cycling population of HSCs in young mice that appear to be preferentially lost during standard HSC isolations and therefore poorly represented within the purified HSC population. Here, we sought to determine how aging may alter this total HSC population in unseparated murine whole bone marrow (WBM). In limit dilution competitive bone marrow transplantation, we found that, at 6 months post-transplant, old (24-26-mo-old) WBM had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM. Unlike purified old HSCs, old WBM showed only minimal myeloid skewing (myeloid: 26%±11% of old donor peripheral blood vs.19%±16% of young donor peripheral blood; average percent donor peripheral blood ± SD; n=26 mice). Interestingly, in two experiments, old WBM retained full engraftment capacity in secondary transplant with no myeloid skewing, while in a third experiment, old WBM engraftment was significantly decreased when compared to young WBM (15%±9% vs. 57%±3%, respectively; average% donor chimerism ± SD), and exhibited clear myeloid skewing (myeloid: 65%±7% of old donor peripheral blood vs. 20%±10% of young donor peripheral blood; 4-5mice/grp/exp). These data suggest that only those old HSCs in unseparated WBM with high engraftment capacity may retain youthful myeloid/lymphoid ratio patterns. Preliminary data in female mice were similar, showing that HSCs were 7.8-fold increased in old vs. young WBM, retained repopulation capacity in secondary transplantation, and displayed no significant myeloid skewing. These patterns of robust reconstitution without myeloid skewing by old WBM are quite distinct from those of the classically-defined, purified old HSCs, suggesting the presence of a unique HSC population in WBM with differential susceptibility to aging. Further study of this population may yield novel insights into the effects of aging on HSC function and lineage fate decisions and facilitate innovative rejuvenation strategies.

A Novel Class of Bifunctional Immunotherapeutic That Exploit a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cells Expressing CD38 Demonstrates Anti-Multiple Myeloma Activity in Vitro and Ex Vivo against Patient Tumor Cells

Background: Antibody recruiting molecules (ARM) are novel, immunotherapeutic bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus, called universal antibody binding terminus (uABT), recruit all endogenous IgG antibodies independent of their antigen binding specificity. As a result, the target cell is "opsonized" by antibodies which then bring the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms. Kleo Pharmaceuticals has developed a series of CD38-ARM mlecules which target human CD38 highly expressed by multiple myeloma cells. CD38-ARM compounds are able to mediate ADCC without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab. Methods: Cyclized peptides containing natural and non-natural amino-acid that selectively bind to human CD38 were identified using Peptidream Flexizyme-based, cell free Peptide Discovery Translation System. These peptides were linked to uABT antibody binder via a linker to generate the final CD38-ARM molecules Binding of CD38-ARM was tested by ternary complex formation between CD38 expressing cells, CD38-ARM and labelled human IgG1. To confirm the activity of CD38-ARM, surrogate CD16a binding and signaling assays were performed using the NFAT Promega system. Antibody dependent cellular cytotoxicity (ADCC) assays using purified NK cells from multiple donors with polymorphism variants (V/V, F/F, and V/F) of CD16a were performed to confirm activity. Live cell imaging was utilized to assess the dynamics of NK-RAJI cell interactions mediated by CD38-ARM +/- IgG. We evaluated the ability of compounds to mediate complement dependent cytotoxicity (CDC). We tested the effect of CD38-ARM on human immune cell populations within PBMC and whole bone marrow (WBM) by flow cytometry. Lastly, ex vivo samples from WBM of MM patients at diagnosis or relapse were used to evaluate CD38-ARM anti-tumor activity as well as off-target effects, without the addition of external source of IgG, through multiparametric flow-cytometry (CD45, CD19, CD38, CD138, CD56, CD27, CD8, CD117). Results: The CD38-ARM were shown to have the ability to bind to CD38 with a 7nM affinity and to human IgG1 and IgG2 with affinity of 15nM and 11nM by SPR. Activity of KP compounds was observed in all assays except for CDC. In ternary assay, KP-6 had an apparent EC50 of 16nM while KP-7's EC50 was 6nM. Both KP-6 and 7 had comparable EC50s in the single digit nanamolar range in the NFAT activation assay induced by CD16a binding was confirmed using human IgG to induce, while Daratumumab had an apparent EC50 of 0.04nM. In the ADCC assay, both KP-6 &amp; KP-7 had EC50s of 7 &amp; 6nM respectively, while Daratumumab had an EC50 of 0.1nM. In addition, no NK cell depletion was observed when PBMC were treated with KP compounds, whereas a profound reduction in both percentages and absolute numbers in this cell subset was observed with Daratumumab treatment. Increasing dose of CD38-ARM (range 0.1uM- 25uM) were tested in ex vivo WBM samples from MM patients together with a negative control and Daratumumab. At concentrations of 10uM and 25uM, CD38-ARM induced a significant reduction of MM cells achieving results comparable to those of Daratumumab activity (p &gt;0.05 in both cases), while sparing all other CD38+ normal cells such as NK, T lymphocytes, monocytes and granulocytes, which are always reduced in the presence of Daratumumab. Conclusions: CD38-ARMs are able to kill MM cells by ADCC without depleting CD38 expressing immune cells contrary to existing antibodies such as Daratumumab. CD38-ARMs do not activate complement, which might be involved in the infusion reaction seen with Daratumumab. Most importantly, CD38-ARMs kill multiple myeloma cells ex vivo in patient bone marrow samples as well as plasma cell leukemia in patient blood. Combined with the in vivo efficacy data presented elsewhere, this data establishes the therapeutic potential of CD38-ARM. They also represent the first demonstration of the ARM platform ability to generate therapeutic agents tailored to a specific indication, by varying target binding moiety of the molecule. Disclosures Rossi: Kleo pharmaceuticals: Employment, Equity Ownership. Bunin:Kleo pharmaceuticals: Employment, Equity Ownership. Iben:Kleo Pharmaceuticals: Employment, Equity Ownership. Welsch:Kleo pharmaceuticals: Employment, Equity Ownership. Berbasova:Kleo Pharmaceuticals: Employment, Equity Ownership. Riillo:Kleo Pharmaceuticals: Research Funding. Ohuchi:Peptidream Inc.: Employment. Alvarez:Kleo pharmaceuticals: Employment, Equity Ownership. Kawakami:Peptidream Inc.: Employment. Nagasawa:Peptidream Inc.: Employment. Spiegel:Kleo pharmaceuticals: Equity Ownership. Rastelli:Kleo pharmaceuticals: Employment, Equity Ownership.

Treatment of shoulder osteoarthritis and rotator cuff tears with bone marrow concentrate and whole bone marrow injections

AbstractBackground: Human bone marrow has shown promise as a minimally invasive approach in treating a variety of musculoskeletal conditions due to the presence of stem cells, platelets, and growth factors in solution. This study examines the clinical effect of whole bone marrow (WBM) and bone marrow concentrate (BMC) injections in patients who were diagnosed with rotator cuff tears or shoulder osteoarthritis. Methods: Forty-seven patients and fifty shoulders with rotator cuff tears or shoulder osteoarthritis underwent one or two BMC or WBM treatments. These patients were grouped based on number of treatments and pathology for analysis. The patients who were injected twice received them approximately 22.33 days apart. Outcomes of resting pain, active pain, upper extremity functionality scale and overall improvement percentage were compared to baseline and between groups. Results: Patients who received either one and two treatments reported significant improvements in resting pain, active pain, and functio...

Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Nuclear Factor I-X May Regulate a Myeloid-Biased Hematopoietic Stem Cell Population during Stress Hematopoiesis

Hematopoietic stem and progenitor cells (HSPC) are routinely exploited in the clinic to treat patients with cancers or other hematologic diseases. The transcription factor nuclear factor I-X (NFIX) has a proven role regulating migration, adhesion, differentiation and quiescence in neural stem cells. Importantly, Nfix was recently shown to be upregulated in FLT3-ITD+ acute myeloid leukemia (AML) patient samples and the NFI binding motif was protected during DNase hypersensitivity screening. We recently identified Nfix as a novel regulator of HSPC repopulating potential. Using our mouse models, Nfixflox/floxRosa26-CreERT2 and Nfix+/+Rosa26-CreERT2, we induced the deletion of Nfix and performed competitive transplants; here we observe a 50% increase in chimerism 20 weeks post-transplant accompanied by a 10% increase in reconstitution of peripheral blood (PB) B cells. Primary recipient whole bone marrow (WBM) was then transplanted into secondary recipients. Beginning four weeks post-transplant, a 2.5-fold loss of PB myeloid reconstitution was observed in secondary recipients of Nfix-deficient WBM. This effect persisted beyond 20 weeks post-secondary transplant, suggesting that a self-renewing myeloid-biased HSPC is functionally perturbed by the loss of Nfix. To identify genes transcriptionally regulated by NFIX, we recently validated an anti-NFIX monoclonal antibody that is currently being used for chromatin immunoprecipitation (ChIP) followed by paired-end sequencing. Future efforts will focus on investigating Nfix-deficient myeloid-biased HSC subsets. This study will illuminate the transcriptional control of specific HSC subsets. DisclosuresNo relevant conflicts of interest to declare.

3016 - JARID2 Restricts Self-Renewal Capacity in Hematopoietic Progenitors and is A Myeloid Tumor Suppressor

The most common genetic mutations in myeloproliferative neoplasms (MPN) occur in JAK2, MPL and CALR, while additional mutations in genes such as TET2, IDH1/2, and NRAS drive transformation to secondary acute myeloid leukemia (sAML). Genome sequencing studies identified chromosomal deletions of JARID2, a Polycomb Repressive Complex 2 (PRC2) co-factor, in post-MPN sAML. To investigate Jarid2 as a tumor suppressor, we utilized an inducible mouse model of Jak2V617F combined with floxed alleles of Jarid2 and the inducible Mx1-CRE. The absence of Jarid2 accelerated MPN, with a median survival of 23 and 43 days post Jarid2-deletion for Jarid2-/–Jak2V617F/ + and Jarid2+/–Jak2V617F/ + mice respectively, compared to 113-days for Jak2V617F/ + control mice. These data establish Jarid2 as a bona fide tumor suppressor in MPN. To understand the tumor suppressor role of Jarid2, we defined its role in normal hematopoiesis. Transplantation of hematopoietic stem cells (HSCs) or whole bone marrow (WBM) showed a striking difference. Vav-CRE:Jarid2-KO HSCs did not contribute to peripheral blood (PB) chimerism, whereas Vav-CRE:Jarid2-KO WBM contributed to PB lineages at levels comparable to control WBM. These data suggested that a population of cell(s) other than HSCs contributes to hematopoiesis in the absence of Jarid2. Strikingly, Vav-CRE:Jarid2-KO multipotent progenitors (MPPs) engrafted in primary and secondary recipients, generating tri-lineage hematopoiesis effectively. Additionally, MPPs from Jak2V617FJarid2KO mice can propagate MPN, unlike MPPs transplanted from Jak2V617F mice. RNA-seq analysis revealed upregulation of Runx1t1 and Mycn in Jarid2-KO MPPs, genes which can reprogram mature blood cells to a multipotent state, which was associated with depletion of H3K27me3. Expression of Runx1t1 and Mycn in wild-type MPPs endowed long-term repopulating capacity in vivo, phenocopying Jarid2-KO MPPs. Our data suggest that the biological role of Jarid2 in hematopoiesis is to restrict self-renewal capacity in lineage-committed progenitor cells, and that dysregulation of this process can contribute to hematopoietic malignancies.

Chronic Infection Depletes Hematopoietic Stem Cells through Stress-Induced Terminal Differentiation

Chronic infections including tuberculosis, hepatitis C, and HIV are estimated to affect over a third of the world's population and are associated with significant health implications including bone marrow suppression and an increased risk for cancer (Jian et al., 2014; Ramos-Casals et al., 2003; Scadden et al., 1989). Pancytopenia, a suppression of blood counts across multiple lineages, can affect as many as 12% of people with miliary tuberculosis and increases risk of death from the infection (Achi et al., 2013). However little is known about the mechanisms by which infections affect hematopoietic stem cells (HSCs). Here we show that chronic infection depletes HSCs and we identify terminal differentiation as the major route of HSC loss. Furthermore, we define Batf2 as a potent new mediator of inflammation-induced differentiation.Using an established model of chronic infection, we conducted repeated monthly infection of C57Bl/6 WT mice with 2 x 106 cfu Mycobacterium avium, generating a sustained chronic IFNg response. Mice became pancytopenic after 4-6 months of repeated infection. The number of HSCs in the bone marrow was depleted to just 5% of the starting number by 4 months following initial infection, without evidence of extensive myelofibrosis. Poor engraftment upon transplant of whole bone marrow (WBM) from mice repeatedly infected with M. avium confirmed that functional HSCs were lost. When WBM from Ifngr1-deficient mice infected with M. aviumwas transplanted, a similar engraftment defect was not seen, suggesting that HSC loss is IFNg-dependent.In addition to loss of HSCs, secondary HSC transplants revealed a defect in HSC self-renewal capacity following repeated M. aviuminfection. Mathematical modeling demonstrated that the rate of HSC loss after chronic infection must increase by 57% from steady state to account for the observed decay in total HSCs.In order to define the mechanism of HSC loss during infection, we conducted mobilization and apoptosis assays after infection or IFNg treatment but found no evidence of increased displacement or death. Next we performed RNAseq profiling of HSCs from infected and control animals. GSEA analysis reflected increased myeloid differentiation rather than apoptosis during infection, consistent with our previous findings. We confirmed that IFNg treatment alone promotes myeloid differentiation of human CD34+ progenitors in vitro, with a reciprocal decrease in the persistence of CD34+CD38- HSPCs. Out of 4 transcription factors among the 151 differentially regulated genes identified by RNAseq, Batf2 stood out as one whose role in HSC biology has not been studied. We confirmed that Batf2 is induced in murine HSCs during infection, and that BATF2 is upregulated in human CD34+ hematopoietic progenitor cells following IFNg treatment. Consistent with a role in differentiation, retroviral overexpression of Batf2 in Sca1+ murine progenitor cells resulted in increased myeloid production upon transplant. Further, knockout of BATF2 in human CD34+ progenitors using CRISPR-Cas9 gene editing resulted in impaired myeloid differentiation in response to IFNg in vitro.Our studies demonstrate that chronic infection depletes the HSC pool by promoting HSC differentiation via an IFNg-dependent mechanism, and we identify the transcription factor Batf2 as a key player in infection-induced myeloid differentiation. These findings may provide a therapeutic opportunity to protect or restore hematopoiesis in patients with aplastic anemia, hemophagocytic histiocytosis (also associated with high IFNg levels), and chronic infections. DisclosuresNo relevant conflicts of interest to declare.

Arthroscopic micro fracture and intra articular whole bone marrow injection for the treatment of articular cartilage lesions in the knee joint.

Background: Articular cartilage lesions in the knee joint are frequently seen,its treatment represents a challenge for orthopedic surgeons especially in young age patients and if left untreated,then they can progress to advanced osteoarthritis that may affect the functional status of the patients.Many treatment modalities had developed for these lesions, in the current study arthroscopic micro fracture plus intra articular whole bone marrow(WBM) injection was used as modality of treating these lesions.Objectives: to assess the effectiveness of arthroscopic micro fracture plus WBM intra articular injection as a treatment modality for knee articular cartilage lesions.Patients and Methods : a prospective experimental study was performed on 30 patients (14 females,16 males ) with a mean age of 38.6 years who had chondral lesions in their knee joint that failed to respond to conservative treatment.Arthroscopic micro fracture plus WBM intra articular injection was done to them.The functional assessment of the patients and their knee pain were evaluated by modified Cincinnati knee rating score (CKRS) and Visual Analogue Scale (VAS) respectively, both preoperatively and six months postoperatively. These results were compared.Results: there was statistically significant improvement in a modified CKRS and VAS six months after surgery.26 patients out of 30 patients (86.7 %) were improved.The improvement was statistically significant for those patients ≤ 40 years of age and also for those above 40 yeas of age, but it was more pronounced in those patients ≤ 40 years.Conclusion : Arthroscopic micro fracture plus intra articular WBM injection is an effective treatment modality for small,well defined,full thickness chondral lesions in the knee joint especially for those ≤ 40 years old on short term follow up. A long term follow up is needed.

Hematopoietic Stem Cell Purification Leads to Loss of a Stem Cell Population within the Lineage Positive Cellular Fraction

Background: Hematopoietic stem cells (HSCs) have tremendous self-renewal and differentiation capacity. The majority of murine hematopoietic stem cell studies have focused on rare purified populations of HSCs, conventionally described as negative for lineage-specific markers and positive for particular cell surface epitope profiles, including c-Kit, Sca-1, and CD150. However, our data indicate that such purifications lead to the loss of a significant population of actively cycling marrow cells with long-term multi-lineage stem cell potential. In the studies presented here, we tested the hypothesis that this discarded stem cell population lies, in part, within the lineage positive (Lin+) fraction of marrow.Methods: We flushed whole bone marrow (WBM) from B6.SJL mice and incubated it with allophycocyanin-tagged antibodies against erythroid (TER119), myeloid (CD11b, GR1), B-lymphoid (B220), or T-lymphoid (CD3, CD4, CD8) markers. Different doses of each specific Lin+ subset isolated by fluorescence activated cell sorting were competitively engrafted into lethally irradiated C57BL/6 host mice. At 1,3, and 6 months post-transplant, peripheral blood was analyzed for donor contribution to chimerism and lineage specificity.Results: Although typically considered to be without stem cell activity, we found that all Lin+ sub-fractions upon single sorting were able to contribute to marrow repopulation in competitive bone marrow transplants. For example, when lethally irradiated recipient mice received 3x105 C57BL/6J competitive whole bone marrow cells in combination with single-sorted GR1+ ± CD11b+ cells (2x106 cells/mouse), peripheral blood showed 15% donor chimerism at 6 months. Similarly, if single sorted CD3+ ±CD4+ ±CD8+ cells (70,000 cells/mouse), B220+ cells (1x106 cells/mouse), or Ter119+ cells (1x106 cells/mouse) were competitively engrafted with 3x105 C57BL/6 WBM cells, the donor Lin+ sub-fractions contributed to 2%, 15%, and 35% peripheral blood chimerism at 6 months post-transplant, respectively. This contribution was multi-lineage in all cases. When we performed double sorting of the Lin+ subsets, there was a dramatically reduced engraftment capacity between 1-6% donor chimerism for all subgroups. However, we do not think the loss of stem cell capacity with double sorting seen in these studies is due merely to the loss of classical hematopoietic stem cells (Lineage-/stem cell marker+). In our earlier studies, we showed that the total Lin+ population contains long-term multi-lineage engraftment capacity due almost entirely to actively cycling cells. Therefore, if the engraftment capacity within the single sorted Lin+ sub-fractions was due solely to the presence of classical HSCs lost with double sorting, the engraftment capacity found within the Lin+ compartment should be due only to quiescent cells in keeping with the cell cycle status of engrafting highly purified stem cells.Conclusions: Based on these data, we predict that a cycling population of stem cells exists within this single sorted, Lin+ enriched fraction discarded with conventional HSC purification. Future studies are ongoing to further characterize the subsets of Lin+ cells that both remain Lin+ and are found to be Lin- upon double sorting. We will analyze these populations for engraftment capacity, concomitant stem cell marker expression and cell cycle status, in order to fully characterize the total stem cell potential within whole bone marrow that is not included in the purified HSC populations. DisclosuresNo relevant conflicts of interest to declare.

Biological Effects of Different Extracellular Vesicles Population on Reversal of Marrow Cells Radiation Damage

We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin).Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cellsThere are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles).Acknowedgements: This work was support by the NIH grant 5UH2TR00080 DisclosuresNo relevant conflicts of interest to declare.