Bone Marrow Hematopoietic Stem Cells Research Articles

Nonclinical safety assessment of AMG 553, an investigational anti-FLT3 CAR-T therapy.

7032 Background: FMS-like tyrosine kinase 3 (FLT3) is a tyrosine-protein kinase involved in hematopoiesis. In acute myeloid leukemia (AML), higher FLT3 transcript levels, independent of the presence of FLT3 mutations, correlate with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells. FLT3 protein is detectable on the cell surface of more than 80% of leukemia isolates from adult AML patients whereas FLT3 mutations are present only in approximately one third of patients. AMG 553 is a novel, investigational, adoptive cellular immunotherapy for the treatment of relapsed refractory AML, consisting of autologous T cells genetically modified ex vivo to express a transmembrane chimeric antigen receptor (CAR) to target FLT3 protein on the surface of AML cells irrespective of FLT3 mutational status. Methods: The nonclinical safety evaluation of AMG 553 includes assessment of FLT3 expression in normal tissues; in vitro assessment of cytotoxicity against human cells with or without FLT3 expression; toxicology studies in cynomolgus monkeys administered autologous T-cells transduced with an anti-FLT3 CAR or administered anti-FLT3 bi-specific T-cell engager (BiTE) antibodies. Results: Assessment of FLT3 expression in normal tissues indicates that FLT3 protein is detected on the cell surface of a subpopulation of human bone marrow hematopoietic stem and progenitor cells; expression in other tissues is cytoplasmic and not anticipated to be accessible to AMG 553. AMG 553-induced cytotoxicity was only observed in a percentage of primary bone marrow CD34+ cells. There was no evidence of CAR-T cell-mediated toxicity, expansion, or persistence in cynomolgus monkeys likely due to restricted cell surface FLT3 protein expression in healthy animals which demonstrates the limited value of such studies for this target. All findings in toxicology studies with anti-FLT3 BiTEs were consistent with the targeted killing of cells expressing FLT3 on the plasma membrane. Conclusions: The nonclinical safety data support AMG 553 as a novel therapeutic to target FLT3 protein on AML cells irrespective of FLT3 mutational status, while only affecting a percentage of normal hematopoietic stem and progenitor cells.

Bone marrow recovery of hematopoietic stem cells and microenvironment after chemotherapy in childhood acute lymphoblastic leukemia: consecutive observations according to chemotherapy schedule

We investigated bone marrow (BM) recovery of hematopoietic stem cells (HSC) and hematopoietic microenvironment after chemotherapy in childhood acute lymphoblastic leukemia (ALL). Twenty-nine de novo childhood ALL patients were enrolled and BM biopsy sections at diagnosis (BM0), after induction (BM1), consolidation (BM3), interim maintenance (BM5) and delayed intensification (BM7) chemotherapy were obtained. Expressions of CD133, CD34, CD117, osteopontin, osteonectin, CXCL12, and CXCR4 were evaluated by semiquantitative immunohistochemical stains. All markers recovered significantly following chemotherapy while highest values at BM3 (for CD133/CD117/CXCL12/CXCR4), BM5 (for CXCL12/CD34/osteonectin), and BM7 (for osteopontin). Patients with cytogenetic good risk expressed significantly more CD133+/CD34+ cells than those with standard and poor risk in BM5. Patients without aberrant immunophenotype expressed significantly more CD133+ cells in BM1, and more CD117+ cells in BM5 than those with aberrant immunophenotype. Patients treated with standard risk-average chemotherapeutic protocol expressed significantly more CXCR4+ cells than those treated with other protocols in BM7. Patients who showed lowest ANC ≥ 200/µL during induction chemotherapy expressed significantly more CXCR4+ cells at from BM1 to BM5, and more CD133+ cells in BM3 than those who did not. Early and full recovery of BM HSC is most vigorous at BM3 and BM5, respectively. Reconstruction of BM niche and stromal cell recovery is mostly active at BM5, and hematopoietic activity of BM niche recovers mostly at BM7. Patients with cytogenetic good risk, nonaberrant immunophenotype, standard risk-average chemotherapeutic protocol and less BM suppression during induction chemotherapy show prompt recovery of some BM HSC and microenvironment markers compared to others.

Bone Marrow Adipocytes: The Enigmatic Components of the Hematopoietic Stem Cell Niche.

Bone marrow adipocytes (BMA) exert pleiotropic roles beyond mere lipid storage and filling of bone marrow (BM) empty spaces, and we are only now beginning to understand their regulatory traits and versatility. BMA arise from the differentiation of BM mesenchymal stromal cells, but they seem to be a heterogeneous population with distinct metabolisms, lipid compositions, secretory properties and functional responses, depending on their location in the BM. BMA also show remarkable differences among species and between genders, they progressively replace the hematopoietic BM throughout aging, and play roles in a range of pathological conditions such as obesity, diabetes and anorexia. They are a crucial component of the BM microenvironment that regulates hematopoiesis, through mechanisms largely unknown. Previously considered as negative regulators of hematopoietic stem cell function, recent data demonstrate their positive support for hematopoietic stem cells depending on the experimental approach. Here, we further discuss current knowledge on the role of BMA in hematological malignancies. Early hints suggest that BMA may provide a suitable metabolic niche for the malignant growth of leukemic stem cells, and protect them from chemotherapy. Future in vivo functional work and improved isolation methods will enable determining the true essence of this elusive BM hematopoietic stem cell niche component, and confirm their roles in a range of diseases. This promising field may open new pathways for efficient therapeutic strategies to restore hematopoiesis, targeting BMA.

Dickkopf-1 Treatment Stimulates Hematopoietic Regenerative Function in Infused Endothelial Progenitor Cells.

Acute high-dose radiation injury damages the bone marrow hematopoietic stem and progenitor cell compartment. This damage compromises the functional ability of the bone marrow to produce mature blood cells and results in an increased risk of death due to hematopoietic complications. Past work has shown that the bone marrow endothelium provides critical cues, which promote hematopoietic stem cell regeneration after injury. Additionally, transfusion of endothelial cells after radiation injury has been shown to promote recovery of both the bone marrow vasculature and hematopoietic systems. In this work, we examined the regenerative capacity of intravenous infusion of umbilical cord-blood derived endothelial progenitor cells (EPCs) since this is a cell source which is easy to obtain, expand and cryopreserve. We show that pre-treatment with the Wnt-antagonist Dickkopf1 (Dkk1) augments EPC regenerative function in an allogeneic mouse transplant model. Here, hematopoietic recovery was assessed in Balb/c mice after 5 Gy total-body irradiation and transplantation with C57/BL6-derived EPCs either with or without Dkk1 pre-treatment. The Dkk1-treated EPC group had significantly faster recovery of peripheral white blood cells, total bone marrow cellularity, bone marrow progenitors and BM endothelial cells compared to EPC treatment alone or saline controls. Importantly, after an LD50/30 dose of 8 Gy in the Balb/c mouse, Dkk1-treated EPCs were able to rescue 100% of irradiated mice versus 80% in the EPC control group and only 33% in the saline-treated group. To understand how Dkk1 induces regenerative function in the EPCs, we screened for pro-regenerative factors secreted by the EPC in response to Dkk1. Dkk1-treated EPCs were observed to secrete high levels of the anti-fibrotic protein follistatin as well as several proteins known to promote regeneration including EGF, VEGF and G-CSF. This work demonstrates the potential for Dkk1-treated EPCs as a rescue therapeutic for victims of acute radiation injury.

Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

AbstractThe myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice

AbstractDendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8– LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8– and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8– LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery.

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, exvivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34+ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P<0·01; platelets, P<0·01) and aggregates (platelet/granulocytes, P<0·01; mononuclear/red cells, P<0·01) were higher in SCD BM. Total CD34+ cell count was lower in SCD BM (P<0·05), however CD34+ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P<0·05). Greater than 50% of CD34+ HSPCs from SCD BM are CD34dim due to higher erythroid lineage expression (P<0·01) as single cell CD34+ CD45+ GPA+ (P<0·01) and CD34+ CD45- GPA+ (P<0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.

Uniquely human CHRFAM7A gene increases the hematopoietic stem cell reservoir in mice and amplifies their inflammatory response

A subset of genes in the human genome are uniquely human and not found in other species. One example is CHRFAM7A, a dominant-negative inhibitor of the antiinflammatory α7 nicotinic acetylcholine receptor (α7nAChR/CHRNA7) that is also a neurotransmitter receptor linked to cognitive function, mental health, and neurodegenerative disease. Here we show that CHRFAM7A blocks ligand binding to both mouse and human α7nAChR, and hypothesized that CHRFAM7A-transgenic mice would allow us to study its biological significance in a tractable animal model of human inflammatory disease, namely SIRS, the systemic inflammatory response syndrome that accompanies severe injury and sepsis. We found that CHRFAM7A increased the hematopoietic stem cell (HSC) reservoir in bone marrow and biased HSC differentiation to the monocyte lineage in vitro. We also observed that while the HSC reservoir was depleted in SIRS, HSCs were spared in CHRFAM7A-transgenic mice and that these mice also had increased immune cell mobilization, myeloid cell differentiation, and a shift to inflammatory monocytes from granulocytes in their inflamed lungs. Together, the findings point to a pathophysiological inflammatory consequence to the emergence of CHRFAM7A in the human genome. To this end, it is interesting to speculate that human genes like CHRFAM7A can account for discrepancies between the effectiveness of drugs like α7nAChR agonists in animal models and human clinical trials for inflammatory and neurodegenerative disease. The findings also support the hypothesis that uniquely human genes may be contributing to underrecognized human-specific differences in resiliency/susceptibility to complications of injury, infection, and inflammation, not to mention the onset of neurodegenerative disease.

Effect of Microvascular Endothelial Cells on the Proliferation of Bone Marrow Hematopoietic Stem Cells under Different Culture Conditions

To investigate the effect of microvascular endothelial cells (MEC) on the proliferation of hematopoictic stem cells (HSC) under different culture conditions in vitro. The MEC from lung tissue of C57BL/6 mice and the HSC from bone marrow of GFP mice were used for non-contact co-culture, 2 D contact co-culture, at same time the single MEC and single HSC culture were seted up and were used as control group. The cell counting and CCK-8 method were used to detect and compare the proliferation levels of suspension cells in different groups on day 1, 3, 5 and 7. MEC presented adherent growth. In process of cell culture in vitro, the number of suspension cells in MEC and HSC co-culture group and single HSC culture group increased, the suspension cells in 2D contact and non-contact co-culture groups more early gated into logarithmic growth phase as compared with suspension cells in control group, the proliferation level of suspention cells in 2D contact culture group was higher than that in non-contact co-culture group and single HSC culture group (P<0.05), the proliferation level of suspension cells in non-contact co-culture group was higher than that in single HSC culture group (P<0.05). The culture of HSC in vitro can proliferate HSC, MEC can promote the proliferation of HSC, MEC also can promote the HSC proliferation by non-contact co-culture in vitro, which relates with the effect of cytokines secreted from MEC; the effect of MEC and HSC contact co-culture on the proliferation of HSC is stronger than that of non-contact co-culture, which relates with the regulation of cell-cell contact.

Metabolic Targets for Improvement of Allogeneic Hematopoietic Stem Cell Transplantation and Graft-vs.-Host Disease.

Utilization of the adaptive immune system against malignancies, both by immune-based therapies to activate T cells in vivo to attack cancer and by T-cell therapies to transfer effector cytolytic T lymphocytes (CTL) to the cancer patient, represent major novel therapeutic advancements in oncologic therapy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is a form of cell-based therapy, which replaces the HSC in the patient's bone marrow but also serves as a T-cell therapy due to the Graft-vs.-leukemia (GVL) effect mediated by donor T cells transferred with the graft. Allogeneic HSCT provides one potentially curative option to patients with relapsed or refractory leukemia but Graft-vs.-Host-Disease (GVHD) is the main cause of non-relapse mortality and limits the therapeutic benefit of allogeneic HSCT. Metabolism is a common cellular feature and has a key role in the differentiation and function of T cells during the immune response. Naïve T cells and memory T cells that mediate GVHD and GVL, respectively, utilize distinct metabolic programs to obtain their immunological and functional specification. Thus, metabolic targets that mediate immunosuppression might differentially affect the functional program of GVHD-mediating or GVL-mediating T cells. Components of the innate immune system that are indispensable for the activation of alloreactive T cells are also subjected to metabolism-dependent regulation. Metabolic alterations have also been implicated in the resistance to chemotherapy and survival of malignant cells such as leukemia and lymphoma, which are targeted by GVL-mediating T cells. Development of novel approaches to inhibit the activation of GVHD-specific naïve T cell but maintain the function of GVL-specific memory T cells will have a major impact on the therapeutic benefit of HSCT. Here, we will highlight the importance of metabolism on the function of GVHD-inducing and GVL-inducing alloreactive T cells as well as on antigen presenting cells (APC), which are required for presentation of host antigens. We will also analyze the metabolic alterations involved in the leukemogenesis which could differentiate leukemia initiating cells from normal HSC, providing potential therapeutic opportunities. Finally, we will discuss the immuno-metabolic effects of key drugs that might be repurposed for metabolic management of GVHD without compromising GVL.

Dynamic Changes in the Niche with N-Cadherin Revisited: The HSC "Niche Herein".

Recently in Cell Reports, Zhao etal. report that chemo-resistant (reserve) hematopoietic stem cells (HSCs) are maintained in the endosteal niche under the influence of N-cadherin+ bone-lining cells (Zhao etal., 2019). These findings further indicate that niches for bone marrow HSCs may differ dynamically during physiological and stress hematopoiesis.

Translating HSC Niche Biology for Clinical Applications

Over the final 3 decades of the twentieth century, the advent and evolution of hematopoietic stem cell transplantation (HSCT) to cure refractory malignancies and severe non-malignant diseases created a need to better understand basic scientific mechanisms underlying the maintenance and migration of hematopoietic stem cells (HSC). Over the past 20 years, researchers have discovered complex overlapping bone marrow (BM) HSC niches that utilize a number of signaling pathways to tightly regulate HSC physiology. Here, we review recent efforts to define critical mediators of HSC niche function in health and disease, and how these discoveries are now enabling the development of the next generation of cellular therapies for cancer and severe non-malignant diseases. A number of cellular interactions and molecular pathways critical for HSC mobilization, donor HSC engraftment after HSCT, and HSC/progenitor cell recovery following chemotherapy have recently been identified. Additional studies have defined mechanisms by which cancer and bone marrow failure states disrupt the normal function of these pathways. Translational investigators are now leveraging these discoveries to develop preclinical and clinical strategies to target the niche for regenerative and cancer therapy. Ongoing research to define fundamental aspects of HSC niche biology will lead to further refinements and precision approaches to improve the safety and efficacy of clinical cell therapy.

Oxaliplatin Treatment Alters Systemic Immune Responses.

Purpose Oxaliplatin is a platinum-based chemotherapeutic agent demonstrating significant antitumor efficacy. Unlike conventional anticancer agents which are immunosuppressive, oxaliplatin has the capacity to stimulate immunological effects in response to the presentation of damage associated molecular patterns (DAMPs) elicited upon cell death. However, the effects of oxaliplatin treatment on systemic immune responses remain largely unknown. Aims of this study were to investigate the effects of oxaliplatin treatment on the proportions of (1) splenic T cells, B cells, macrophages, pro-/anti-inflammatory cytokines, gene expression of splenic cytokines, chemokines, and mediators; (2) double-positive and single-positive CD4+ and CD8+ T thymocytes; (3) bone-marrow hematopoietic stem and progenitor cells. Methods Male BALB/c mice received intraperitoneal injections of oxaliplatin (3mg/kg/d) or sterile water tri-weekly for 2 weeks. Leukocyte populations within the spleen, thymus, and bone-marrow were assessed using flow cytometry. RT-PCR was performed to characterise changes in splenic inflammation-associated genes. Results Oxaliplatin treatment reduced spleen size and cellularity (CD45+ cells), increased the proportion of CD4+, CD8+, and Treg cells, and elevated TNF-α expression. Oxaliplatin was selectively cytotoxic to B cells but had no effect on splenic macrophages. Oxaliplatin treatment altered the gene expression of several cytokines, chemokines, and cell mediators. Oxaliplatin did not deplete double-positive thymocytes but increased the single-positive CD8+ subset. There was also an increase in activated (CD69+) CD8+ T cells. Bone-marrow hematopoietic progenitor pool was demonstrably normal following oxaliplatin treatment when compared to the vehicle-treated cohort. Conclusion Oxaliplatin does not cause systemic immunosuppression and, instead, has the capacity to induce beneficial antitumor immune responses.

Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1

Abstract The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells delayed EL4 WT tumor growth but not EL4 Qa-1 KO tumor growth when inoculated in the same hosts. Tumor-infiltrating CD8 T-cells displayed a more activated phenotype in WT tumors compared to KO tumors, indicating that retrogenic T-cells recognize and target EL4 cells in a Qa-1-dependent manner. Citation Format: Hidetoshi Nakagawa, Hye-Jung Kim, Harvey Cantor. FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A039.

Recent advances in mechanisms of megakaryopoiesis and platelet formation

Platelets are specialized cells, which are functioned to prevent bleeding and minimize blood vessel injury, and are also the predominating factor in the processes of hemostasis and thrombosis, while implicate in other processes including inflammation, innate immunity, angiogenesis and tumor metastasis. Among current mechanisms of platelet formation, platelets are produced by megakaryocytes, which are themselves generated by a process of controlled differentiation and maturation of bone marrow hematopoietic stem cells (HSC) and progenitor cells. Megakaryocytes release platelets by extending long, branching proplatelets, into sinusoidal blood vessels. Bone marrow cavity and extracellular matrix composition together with cytokines as thrombopoietin (TPO) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet formation. Understanding the mechanisms of megakaryocyte differentiation and platelet formation can provide a solid theoretical basis for the treatment of platelet-related diseases. This review summarizes the current scientific research progress in the mechanisms of megakaryopoiesis and thrombopoiesis. Key words: Megakaryocytes; Cell differentiation; Thrombopoiesis; Transcription factors; Apoptosis; Megakaryopoiesis

Relationship between molecular mechanism of autophagy and chronic myeloid leukemia

Autophagy is a common physiogical phenomenon in cells, which is closely related to cell growth, differentiation, apoptosis and carcinogenesis. Chronic myeloid leukemia (CML) is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells. Autophagy may play an important role in occurrence, treatment and drug resistance of CML. Therefore, the study of the molecular mechanism of autophagy and its relationship with CML may provide a theoretical guide for the development of new drugs targeting autophagy in the treatment of CML. This paper reviews the molecular mechanism of autophagy and the relationship between autophagy and CML. Key words: Autophagy; Leukemia, myeloid, chronic; Therapeutics; Drug resistance

Pathogenesis of Acquired Aplastic Anemia and the Role of the Bone Marrow Microenvironment.

Aplastic anemia (AA) is characterized by bone marrow (BM) hypocellularity, resulting in peripheral cytopenias. An antigen-driven and likely auto-immune dysregulated T-cell homeostasis results in hematopoietic stem cell injury, which ultimately leads to the pathogenesis of the acquired form of this disease. Auto-immune and inflammatory processes further influence the disease course as well as response rate to therapy, mainly consisting of intensive immunosuppressive therapy and allogeneic hematopoietic cell transplantation. Bone marrow hematopoietic stem and progenitor cells are strongly regulated by the crosstalk with the surrounding microenvironment and its components like mesenchymal stromal cells, also consistently altered in AA. Whether latter is a contributing cause or rather consequence of the disease remains an open question. Overall, niche disruption may contribute to disease progression, sustain pancytopenia and promote clonal evolution. Here we review the existing knowledge on BM microenvironmental changes in acquired AA and discuss their relevance for the pathogenesis and therapy.

Antibody Drug Conjugates Targeted to CD45 or CD117 Enable Allogeneic Hematopoietic Stem Cell Transplantation in Animal Models

IntroductionTargeted antibody drug conjugates (ADCs) to mouse CD45 or mouse CD117 have recently been shown to effectively prepare immunocompetent mice for whole bone marrow transplants (Palchaudhuri et al. Nature Biotech 2016 34:738-745; and Czechowicz et al. Blood 2016 128:493). This new targeted approach to conditioning using ADCs has the potential to expand the utility of transplantation if it can be successfully translated to humans. The anti-CD45 or anti-CD117 antibodies used previously were coupled to saporin (SAP), a ribosome-inhibiting protein, which once internalized elicits cytotoxicity in a cell cycle-independent manner. Both anti-CD45-saporin (CD45-SAP) and anti-CD117-saporin (CD117-SAP) ADCs have been shown to effectively deplete bone marrow hematopoietic stem cells (HSCs) as single dosed agents, creating vacancies that enable efficient autologous HSC engraftment (>95% long-term donor chimerism).ResultsTo further investigate and develop the utility of these tool ADCs in murine transplant models, we explored CD45-SAP (1.9 mg/kg, iv) and CD117-SAP (1 mg/kg, iv) in an allogeneic minor mismatch transplant model (Balb/c donor into DBA/2 recipients). The ADCs were used alone or in combination with an additional immune depleting agent, clone 30F11 (25 mg/kg, IP), a naked anti-CD45 antibody that mimics ATG by relying on effector function to enable potent peripheral B- and T -cell depletion. In addition to the lymphodepleting antibody, we included post-transplant Cytoxan (200 mg/kg, IP) to prevent GvHD. To compare the CD45-SAP and CD117-SAP to conventional conditioning methods, we investigated sub-lethal total body irradiation (TBI, 2Gy) or pre-transplant Cytoxan (200 mg/kg, IP) in combination with the immunosuppressants. Conditioned mice were transplanted with 2x107 whole bone marrow cells, and chimerism assessed over 12 weeks.CD45-SAP or CD117-SAP in combination with immunosuppressants (30F11 and post-transplant Cytoxan) enabled >85% peripheral donor chimerism at 12 weeks post-transplantation. Multilineage reconstitution was observed in the T-, B- and myeloid cell compartments with >80%, >90% and >90% donor chimerism respectively in both CD45-SAP and CD117-SAP groups. In contrast, 2Gy TBI in combination with immunosuppressants (30F11 and post-transplant Cytoxan) resulted in only 5% donor engraftment. Multi-dosing with 30F11 (QDx3) plus 2Gy TBI and post-transplant Cytoxan increased the peripheral donor chimerism to 40%. Pre-transplant Cytoxan plus 30F11 (QDx3) and post-transplant Cytoxan yielded 20% donor chimerism. For all groups, stem cell chimerism in the bone marrow matched the peripheral chimerism.ConclusionThese results indicate anti-CD45 or anti-CD117 ADCs may be used in combination with immunosuppression to enable highly efficient allogeneic transplants in a minor mismatch model (>85% donor chimerism). CD45-SAP and CD117-SAP were more effective at conditioning versus 2Gy TBI or pre-transplant Cytoxan. Future experiments will investigate anti-CD45 and anti-CD117 ADCs in additional allogeneic models. DisclosuresPalchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Harvard University: Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Pearse:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Sarma:Magenta Therapeutics: Employment, Equity Ownership. Aslanian:Magenta Therapeutics: Employment, Equity Ownership. Gillard:Magenta Therapeutics: Employment, Equity Ownership. Lamothe:Magenta Therapeutics: Employment, Equity Ownership. Burenkova:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Gabros:Magenta Therapeutics: Employment, Equity Ownership. McDonagh:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Inhibition of Semaphorin 3A Signaling Promotes Regeneration of Hematopoietic Stem Cells and Their Bone Marrow Vascular Niche

Radiation injury damages both the bone marrow hematopoietic stem cell and its supportive endothelial niche. In response to radiation stress, bone marrow endothelial cells (BM ECs) upregulate expression of a gene, Sema3A, which encodes for the secreted protein, Semaphorin 3A (SEMA3A), and its receptor, Neuropilin 1 (Nrp1). Commensurate with this, we observed a substantial increase in SEMA3A protein levels in the BM of mice and in NRP1 expression on VE-cadherin+ BM ECs following 500 cGy total body irradiation (TBI) (p<0.001, p<0.0001). We also found that treatment of irradiated primary murine BM ECs with SEMA3A significantly increased BM EC apoptosis (p<0.01). Based on these results, we hypothesized that SEMA3A may be a negative regulator of BM vascular recovery following injury and that inhibition of SEMA3A/NRP1 signaling might accelerate BM vascular niche recovery following irradiation. In keeping with this hypothesis, we found that a single systemic administration of 2 µg SEMA3A (IV) to C57BL/6 mice following 500 cGy TBI significantly increased BM EC apoptosis in vivo at 24 hours and also markedly decreased BM vascular integrity at 24 hours as measured by Evans Blue Dye extravasation in the BM (p<0.01, p<0.01). Interestingly, SEMA3A administration (IV, every other day) also significantly suppressed BM hematopoietic regeneration with decreased BM cell counts and BM ckit+sca-1+lin- (KSL) progenitor cell recovery at day +10 compared to irradiated, control mice (p=0.04, p=0.001).In contrast, systemic administration of anti-NRP1 antibody (10 µg, IV every other day), which blocks SEMA3A binding to NRP1 on BM ECs, accelerated regeneration of the sinusoidal BM vasculature at day +10 following 500 cGy TBI compared to irradiated controls. Concordant with these findings, anti-NRP1 treatment increased the recovery of peripheral blood WBCs, neutrophils, and BM KSL cells, compared to irradiated controls (p=0.002, p=0.04, p=0.04). Competitive repopulation assays confirmed that anti-NRP1 treatment increased the recovery of long-term HSCs capable of 20-week multilineage repopulation in congenic mice (p=0.03 for % donor CD45.1+ cell engraftment at 20 weeks). Importantly, anti-NRP1 treatment also markedly improved 60-day survival of irradiated mice from 17% (2/12 controls) to 83% (10/12 anti-NRP1) following 800 cGy TBI.In order to confirm the role of SEMA3A/NRP1 signaling in BM ECs in regulating the BM vascular and hematopoietic response to injury, we generated mice with tamoxifen-inducible, EC - specific deletion of Nrp1 (VE-Cad-Cre-ERT2;Nrp1 fl/fl mice). Adult VE-Cad-Cre-ERT2;Nrp1 fl/fl mice were viable and had no baseline hematologic phenotype. However, following 500 cGy TBI, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice displayed accelerated BM vascular regeneration at day +10 compared to irradiated control mice. Furthermore, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice demonstrated significantly increased peripheral blood WBCs, neutrophils, and BM KSL cells at day +10 following radiation injury (p<0.001, p=0.002, p=0.002). These data suggest that the SEMA3A-NRP1 pathway is an autocrine signaling mechanism that negatively regulates BM vascular niche recovery following myelosuppressive irradiation. Targeted inhibition of SEMA3A-NRP1 signaling in BM ECs has therapeutic potential to accelerate both BM vascular niche regeneration and hematopoietic reconstitution following myelosuppression. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Targeting of Osteoblast-Induced MDS and AML

The bone marrow hematopoietic stem cell niche has emerged as a regulator and instigator of hematological myeloid malignancies. Activation of b-catenin signaling in osteoblasts, by deletion of exon3 or by inactivating mutations in APC, induces myelodysplasia (MDS), rapidly progressing to acute myeloid leukemia (AML) in mice. Activated b-catenin signaling is present in osteoblasts of one third of MDS and AML patients and it is the most active pathway in stromal cells of MDS patients suggesting that it may sustain dysplastic hematopoiesis and progression to MDS and AML in humans. Therefore, targeting this pathway may provide a new therapeutic approach for this subgroup of MDS/AML patients. To examine this hypothesis we searched for an FDA-approved compound with the ability to inhibit b-catenin signaling and identified all-trans-retinoic acid (ATRA) as a potential candidate. We show here that treatment of osteoblastic cells with ATRA inhibits the induction of b-catenin target genes by Wnt3a and abrogates nuclear localization of b-catenin, as shown by immunohistochemistry and western blot analysis. These effects are at least in part independent of its phosphorylation by GSK3b on exon3 and are mediated by the retinoic acid receptor a (RARa) since silencing RARa, but not RARb or RARg, in osteoblasts abolishes the inhibitory effect of ATRA on Wnt3a-induced b-catenin signaling. Similarly, treatment of leukemic mice expressing constitutively active b-catenin in their osteoblasts with ATRA, inhibited b-catenin signaling as measured by expression of its target genes Axin2 and Lef1 in bone. At the same time, ATRA improved anemia and thrombocytopenia, decreased the percentage of blasts in bone marrow and blood and prolonged overall survival in these leukemic mice as compared to vehicle-treated mice. The response to ATRA treatment of two MDS patients with active b-catenin in their osteoblasts, and not responding to other treatments, was monitored. The hematologic phenotype of the patients improved and disease status stabilized so that patients remain transfusion independent and disease-related symptom-free with a good quality of life thereafter. The improvement in disease status coincided with absence of b-catenin in the nucleus as flow cytometry analysis showed a high percentage of patient osteoblasts expressing activated b-catenin in pre-therapy samples but not in on-therapy samples. These results suggest that ATRA may find a new use in the treatment of the specific portion of MDS/AML patients with activated b-catenin in their osteoblasts. DisclosuresRaza:Geoptix: Speakers Bureau; Onconova: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Kura Oncology: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Syros: Research Funding.