Defective Hematopoietic Stem Cells Research Articles

In Vitro Erythropoietic Response to Enzyme Replacement Therapy in Gaucher Type 1 Patients

Introduction. Gaucher disease type 1 (GD, OMIM #230800) is the most common inherited lysosomal storage disorder resulting from lysosomal glucocerebrosidase (GBA, acid-β-glucosidase) deficiency and subsequent accumulation of glucosylceramide in macrophages. This leads to anemia, thrombocytopenia and coagulopathy, visceral (hepatosplenomegaly, lungs) and skeletal manifestations (deformities, fractures, avascular osteonecrosis). Anemia is attributed to bone marrow infiltration by Gaucher cells, to inflammation and to the presence of splenomegaly; however the underlying pathophysiological mechanism hasn't been fully elucidated.Aim. The aim of this study is to evaluate the in vitro erythroid status at baseline (T0) and the response to Enzyme Replacement Therapy (ERT) after 6 months (T1) and 1 year (T2). We evaluated the expansion and differentiation in erythroid cultures of CD34+ cells obtained from peripheral blood of GD type 1 patients at diagnosis before starting ERT (T0), at 6 months (T1) and at 1 year (T2) of ERT.Methods. CD34+ cells were obtained by immunomagnetic separation from peripheral blood mononuclear cells of GD patients (n=3) and controls (n=5). Isolated CD34+ were evaluated by in vitro clonogenic capacity assays using the methylcellulose semisolid culture medium after 14-16 days of culture. Hematopoietic colonies (burst-forming unit-erythroid (BFU-E) and colony-forming unit-macrophage (CFU-M)) were scored with an inverted microscope. CD34+ cells were expanded in StemSpan medium, 1% StemSpan CC100 cytokine cocktail, 2 U/ml Erythropoietin (EPO) and 1 μM dexamethasone. Differentiation was induced in StemSpan medium with 10 ng/ml SCF and 10 U/ml EPO. Proliferation and surface marker expression (CD34, CD45, Glycophorine A, CD71) were examined by flow cytometry. Cell morphology was analyzed on cytocentrifuged smears stained with May-Grunwald-Giemsa.Results. At T0 we observed a higher number of BFU-E in GD samples (126±23 BFU-E/105 cells) compared to controls (49±12 BFU-E/105 cells) (Tab.1). Similarly, higher macrophage CFU-M were observed in GD samples (29±12 CFU-M/105 cells) compared to controls (10±1 CFU-M/105 cells) (Tab.1). The BFU-E and CFU-M morphology showed no difference between GD patients and controls. The increased proliferation of erythroid precursors of GD patients was associated to impaired ability to differentiate in liquid cultures after 14 days (45,9% proerythroblasts and only 4% polichromatophilic erythroblasts) compared to controls (27% and 19%, respectively) (Tab.1). Flow cytometry confirmed an impaired differentiation of GD erythroblasts compared to controls at day 14 of the erythroid liquid cultures, as shown by high percentage of CD71high/GlyAlow early erythroid precursor cells and low percentage of CD71high/GlyAhigh mature erythroblasts (GD: 73,9 and 4,9%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1). In samples from GD patients, after 1-year treatment with ERT (T2), we observed a reduction in the number of BFU-E compared to T0, reaching values similar to controls (GD T0: 126±23 BFU-E/105 cells, T2: 41±11 BFU-E/105 cells; controls: 49±12 BFU-E/105 cells) and an increased number of mature erythroid cells generated in GD liquid cultures (Tab. 1). Also CFU-M were reduced at T2 compared to T0 (GD T0: 29±12 CFU-M/105 cells, T2: 8±5 CFU-M/105 cells; controls: 10±1 CFU-M/105 cells) (Tab.1). An increased percentage of more differentiated erythroid cells at T2 compared to T0 was confirmed by flow cytometry in cultures from GD patients, as shown by low percentage of the CD71high/GlyAlow early erythroid precursor cells and high percentage of CD71high/GlyAhigh mature erythroblasts on day 14 (GD: 44,7 and 45,6%, respectively; controls: 67,2 and 22.4%, respectively) (Fig.1).Conclusions. These preliminary data suggest that anemia in GD might partly be related to a delay in erythroid differentiation. ERT treatment seems to correct the dyserythropoiesis by improving erythroid differentiation. Since glucosylceramide accumulates in macrophages, that are key elements in the erythroid niche, impaired erythroid differentiation might be related to the pathological micro-environment, rather than intrinsic hematopoietic stem cell defect. More extensive studies are needed to better understand the pathophysiology of anemia and pancytopenia of this rare disease.This study was partially supported by Sanofi Genzyme. [Display omitted] DisclosuresCappellini:Celgene: Honoraria; Novartis: Speakers Bureau; Vifor: Honoraria; Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau.

A CRISPR-Cas9 Generated Etv6 mutant Mouse Exhibits Decreased Platelet Counts and Functional Platelet Defects

We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

A Review of Molecular and Genetic Diagnostics of Myeloid Malignancies with Emphasis on Diagnostics in Bosnia and Herzegovina.

Here we describe the major genetic and genomic aberrations found in myeloid malignancies and how those markers are used in patients' diagnosis, prognosis, and targeted treatment. In Bosnia and Herzegovina, cytogenetic and molecular diagnostics for myeloid malignancies have been established and continually improved since 2005. We report the current state of available diagnostic tools for myeloid malignancies in Bosnia and Herzegovina. Myeloid malignancies are a heterogeneous group of clonal blood diseases characterized by defects in hematopoietic stem cells and myeloid progenitors that lead to abnormal proliferation, differentiation, localization, and self-renewal. Most common myeloid malignancies include myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). Molecular diagnostics of myeloid malignancies have significantly expanded in the last decade with new genetic and genomic markers for diagnosis, prognosis, and treatment. CONCLUSION: In the last decade, several new genomic markers important for patient diagnosis, prognosis, and therapy have been discovered that need to be implemented in routine molecular diagnostics not only in developed nations but also in developing nations such as Bosnia and Herzegovina.

JAK-STAT in Early Hematopoiesis and Leukemia

Hematopoietic stem cells (HSCs) produce all the terminally differentiated blood cells and are controlled by extracellular signals from the microenvironment, the bone marrow (BM) niche, as well as intrinsic cell signals. Intrinsic signals include the tightly controlled action of signaling pathways, as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Activation of JAK-STAT leads to phosphorylation of members of the STAT family to regulate proliferation, survival, and self-renewal of HSCs. Mutations in components of the JAK-STAT pathway are linked with defects in HSCs and hematologic malignancies. Accumulating mutations in HSCs and aging contribute to leukemia transformation. Here an overview of hematopoiesis, and the role of the JAK-STAT pathway in HSCs and in the promotion of leukemic transformation is presented. Therapeutic targeting of JAK-STAT and clinical implications of the existing research findings are also discussed.

TWIST1 preserves hematopoietic stem cell function via the CACNA1B/Ca2+/mitochondria axis

AbstractMitochondria of hematopoietic stem cells (HSCs) play crucial roles in regulating cell fate and preserving HSC functionality and survival. However, the mechanism underlying HSC regulation remains poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Twist1 deletion results in significantly decreased lymphoid-biased HSC frequency, markedly reduced HSC dormancy and self-renewal capacity, and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil–induced stresses and exhibit typical phenotypes of senescence. Mechanistically, Twist1 deletion induces transactivation of voltage-gated calcium channel (VGCC) Cacna1b, which exhausts lymphoid-biased HSCs, impairs genotoxic hematopoietic recovery, and enhances mitochondrial calcium levels, metabolic activity, and reactive oxygen species production. Suppression of VGCC by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through the CACNA1B/Ca2+/mitochondria axis and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate.

Advances in research for pathogenesis of paroxysmal nocturnal hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria, PNH is usually caused by the somatic mutation of X-linked PIGA gene followed by the clonal expansion of the GPI (glycosylphosphatidylinositol) anchor defective hematopoietic stem cell clone. There are two hypotheses for the mechanism of clonal expansion, one is selection theory, in which GPI deficient cells escape from attacks of cytotoxic cells, and another is benign tumor theory in which GPI deficient cells get the additional mutations and acquire proliferative nature. Recently, we identified two types of PNH patients caused by the biallelic mutation of PIGT on chromosome 20 and PIGB on chromosome 15. Both PNH clones had the germ-line mutation in one allele and another allele was somatically mutated in a hematopoietic stem cell. Both somatic mutations were loss of heterozygosity (LOH), deletion in PIGT-PNH and copy neutral LOH (CN-LOH) in PIGB-PNH. These PNH patients had typical PNH symptoms, but they have in addition auto-inflammatory features. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. Similarly, in PIGB-PNH cells, GPI intermediates are accumulated and expressed on the cell surface. Those GPIs together with complement activation cause the inflammasome activation.

New Insights on the Role of the Mesenchymal-Hematopoietic Stem Cell Axis in Autologous and Allogeneic Hematopoiesis.

Cytoreductive protocols are integral both as conditioning regimens for bone marrow (BM) transplantation and as part of therapies for malignancies, but their associated comorbidities represent a long-standing clinical problem. In particular, they cause myeloablation that debilitates the physiological role of mesenchymal stem and precursor cells (MSPCs) in sustaining hematopoiesis. This review addresses the damaging impact of cytoreductive regimens on MSPCs. In addition, it discusses prospects for alleviating the resulting iatrogenic comorbidities. New insights into the structural and functional dynamics of hematopoietic stem cell (HSC) niches reveal the existence of "empty" niches and the ability of the donor-derived healthy HSCs to outcompete the defective HSCs in occupying these niches. These findings support the notion that conditioning regimens, conventionally used to ablate the recipient hematopoiesis to create space for engraftment of the donor-derived HSCs, may not be a necessity for allogeneic BM transplantation. In addition, the capacity of the MSPCs to cross-talk with HSCs, despite major histocompatibility complex disparity, and suppress graft versus host disease indicates the possibility for development of a conditioning-free, MSPCs-enhanced protocol for BM transplantation. The clinical advantage of supplementing cytoreductive protocols with MSPCs to improve autologous hematopoiesis reconstitution and alleviate cytopenia associated with chemo and radiation therapies for cancer is also discussed.

Role of Mir-29b in T-Cell Development and in Cutaneous T-Cell Lymphoma Pathogenesis

The role of microRNA (miR) is rapidly advancing with numerous functions discovered in cancer pathogenesis. We had previously shown a significant decrease in miR-29b levels in malignant cells isolated from peripheral blood of cutaneous T-cell lymphoma (CTCL) (Kohnken et al. Blood, 2018). Replenishing miR-29b levels in vitro induced apoptosis in leukemic cells suggesting its role as tumor suppressor miR in CTCL patients. In this study, we focused on the role of mir-29b on early T-cell development and homeostasis using the miR-29b-/- mouse model. Using knockout mouse model, we show that the homozygous deletion of miR-29b locus results in an overall decrease in T-cell numbers and density in primary and secondary lymphoid organs. We observed an early thymic involution in miR-29b-/- mice, with a 5-fold decrease in the total number of thymocytes and altered T-cell development. Using the surface expression of CD25 and CD44 on double-negative (DN) cells, we observed significant decrease of the absolute counts in the four early differentiation stages (DN1-4) in miR-29b-/- mice compared to age-matched wild-type (WT) mice: DN1 (miR-29b-/- vs. WT: 4.5 x 105 ± 1.04 vs. 1.12 x 105 ± 0.201, N=4 and 6 respectively, p < 0.05); DN2 (miR-29b-/- vs. WT: 0.83 x 105 ± 0.07 vs. 0.051 x 105 ± 0.021, N=4 and 6 respectively, p < 0.001); DN3 (miR-29b-/- vs. WT: 4.05 x 105 ± 0.38 vs. 0.54 x 105 ± 0.16, N=4 and 6 respectively, p < 0.001) and DN4 (miR-29b-/- vs. WT: 2.871 x 105 ± 0.578 x 105 vs. 0.569 ± 0.14, N=4 and 6 respectively, p < 0.01). Furthermore, miR-29b-/- mice show a significant increase in regulatory T cells in comparison to WT mice in the spleen (miR-29b-/- vs. WT: 18.48 ± 0.89 vs. 10.89 ± 0.41, N=4 and 6 respectively, p < 0.001) thymus (miR-29b-/- vs. WT: 5.80 ± 0.44 vs. 3.65 ± 0.004, N=4 and 6 respectively, p < 0.01) and bone marrow (miR-29b-/- vs. WT: 50.04 ± 3.14 vs. 37.52 ± 3.29, N=4 and 6 respectively, p < 0.05). Using single-cell RNA sequencing (scRNA-seq), we found a framework of putative genes in miR-29b deficient T-cells that overlap with CTCL pathogenesis: Ccr7, Cd69, Cd74, and Lef-1. Among thymic T cells, Ccr7 affects physiologic homing of T-cells to lymph nodes and facilitate nodal metastasis in CTCL patients. As T-cells progenitors originate from the bone marrow, we observed a severe impairment in the progenitor cell population in miR-29b-/- mice and over 4-fold reduction in the absolute number of Lin-Sca1+ckit+ cell (miR-29b-/- vs. WT: 28.42 x 103 ± 4.13 vs. 5.98 x 103 ± 1.01, N=4 and 3 respectively, p < 0.01). Using the surface expression of CD48 and CD150 on Lin-Sca1+ckit+, we observed a significant decrease in hematopoietic stem cells and multipotent progenitor cells in miR-29b-/- mice. Since impairment in T-cells development can be linked to hematopoietic stem cell defects, we interrogated the engraftment capacity of Lin-Sca1+ckit+ cells into committed cell lineages in vivo. To evaluate whether the defect in T-cells development is due to reduced ability of precursor cells, we performed transplantation of Lin-Sca1+ckit+ cells from the bone marrow of miR-29b and WT (CD45.1) mice in lethally irradiated WT (CD45.2) mice. Our results show a significant reduction in T-cells reconstitution in mice transplanted with miR-29b-/- Lin-Sca1+ckit+ cells versus the bone marrow counterpart. These deficits in T-cells populations were observed in peripheral blood, thymus, spleen, and bone marrow in miR-29b-/- transplanted mice. ScRNA-seq profiling of Lin- bone marrow cells show significant changes in miR-29b-/- and WT mice. Among the upregulated genes in the mir-29b-/- mice, several genes showed greater than a 10-fold increase in the thymus (Ifna2, Zmynd10, Plekha4, Etl4, and Gm38004). In conclusion, our results highlight the importance of mir-29b in early defects in T-cells development and help us understand the complex miR-29b regulated cellular transformation machinery in CTCL pathogenesis, thus paving path to develop novel therapeutic approaches targeting miR-29b in CTCL therapy. Disclosures No relevant conflicts of interest to declare.

Cytokine Rescue and Targeting of Inflammation-Sensitive RUNX1 Deficient Human CD34+ Hematopoietic Stem and Progenitor Cells

Introduction: Loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) are commonly found in both germline and somatic hematopoietic malignancies and confer particularly poor prognosis in AML. However, it remains unclear how RUNX1 functions during hematopoietic and leukemic development, particularly because RUNX1 mutations alone are not sufficient to cause myeloid malignancy and some models show that RUNX1 mutations confer hematopoietic stem cell defects. Recently, mouse models have shown that RUNX1-deficient neutrophils upregulate NFκB activity, and hematopoietic stem and progenitor cells (HSPCs) with overactive inflammatory pathways gain competitive advantage under chronic inflammation. Thus, we hypothesized that while RUNX1 mutations impair normal HSPC function, inflammation may select for or rescue RUNX1 mutant HSPCs. Methods: To interrogate the effect of RUNX1 loss in human CD34+ HSPCs, we disrupted the RUNX1 locus using CRISPR/Cas9 and AAV6-mediated homology directed repair. Importantly, by using an AAV6 vector that carries arms of homology flanking a fluorescent reporter expression cassette, we are able to track and isolate cells edited at the RUNX1 locus for in vitro and in vivo functional analyses and for molecular characterization using RNA-seq and ATAC-seq. Results: First, we used this system to evaluate the functional consequences of RUNX1 knockout (KO) in human CD34+ HSPCs. Loss of RUNX1 caused early erythroid-megakaryocytic differentiation arrest and skewing toward monocytic differentiation. RUNX1 KO cells demonstrated decreased proliferation, cell cycle arrest, and reduction in serial replating potential in vitro. In competitive transplantation experiments in NSG mice, RUNX1 KO engraftment decreased over time in both primary and secondary transplant, revealing a competitive disadvantage. Second, ATAC-seq peak motif analysis showed that PU.1 and NFκB motifs are more accessible upon RUNX1 KO whereas GATA, TAL1, and RUNX motifs were less accessible. Similarly, gene set enrichment analysis of transcriptional data confirmed the broad upregulation of NFκB-mediated inflammatory programs; downregulation of GATA1-dependent heme metabolism and platelet development pathways; and downregulation of MYC- and E2F-dependent cell cycle programs. These observations imply that RUNX1 directs cell fate decisions by recruiting and activating lineage-specific hematopoietic transcription factors and augmenting stem cell proliferation programs. We next sought to determine which cytokines are sufficient to drive RUNX1 KO cell expansion. RUNX1 KO cells not only expanded preferentially in NSG mice expressing human SCF, GM-CSF, and IL-3 (NSGS mice), but also were no longer defective in competitive transplants in these mice. Further, treatment with IL-3 was sufficient to significantly expand RUNX1 KO cells in vitro. Flow cytometry revealed that the IL-3 receptor CD123 is upregulated in RUNX1 KO cells compared to control. Similarly, RUNX1-mutant AML patient samples express higher levels of CD123 than RUNX1-wildtype AML patient samples. Finally, evaluation of publicly available RUNX1 ChIP-seq of bone marrow CD34+ HSPCs revealed that RUNX1 directly binds the promoter of CD123. Ongoing efforts are aimed at determining whether targeting CD123 and IL-3 signaling may be a viable therapeutic approach for the prevention or treatment of RUNX1-mutant myeloid malignancies. Conclusion: In summary, we established a RUNX1-deficient human HSPC model not only to evaluate the role of RUNX1 in hematopoiesis, but also to characterize intrinsic and extrinsic factors involved in RUNX1-deficient clonal expansion and leukemic transformation. We show that RUNX1 KO causes monocytic skew at the expense of erythro-megakaryocytic potential and severely limits HSC engraftment and expansion in vivo. Molecular profiling reveals that these effects are associated with dysregulation of both transcription factor activity and cytokine signaling. However, exposure to IL-3 rescues RUNX1-deficient cell proliferative defects in vitro and competitive engraftment defects in vivo. This hypersensitivity to IL-3 signaling is mediated in part by increased expression of the IL-3 receptor CD123. These findings reveal how RUNX1 mutations may initially behave in a deleterious manner but can ultimately confer an advantage to HSPCs under certain environmental conditions. Disclosures Majeti: CD47 Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Kodikaz Therapeutic Solutions Inc: Membership on an entity's Board of Directors or advisory committees.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-β pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation

Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy.

3097 – MULTILEVEL REGULATION OF THE MICROTUBULE PLUS-END TRACKING PROTEIN CLASP2 DURING MOUSE AND ZEBRAFISH HEMATOPOIETIC STEM CELL DEVELOPMENT.

During embryonic development, the first definitive hematopoietic stem cells (HSCs) are generated from hemogenic endothelial cells through an endothelial to hematopoietic transition (EHT) occurring in the main arteries of vertebrate embryos. HSCs then mature and expand during development prior to colonizing the adult hematopoietic niche. The complex and highly regulated process of HSC production is not fully understood yet. The journey of HSCs through their subsequent niches requires cell shape remodelling, attachment, division, migration and homing, all steps involving extensive cytoskeletal rearrangements. CLASP2 is a microtubule-associated protein that regulates microtubule dynamics by stabilizing the plus-end of microtubules. We have previously shown that Clasp2 knockout mice (Clasp2-/-) have an HSC defect in the bone marrow, resulting in severe pancytopenia. Our goal is now to investigate the role of CLASP2 in hematopoietic stem and progenitor cells (HSPCs) during embryonic development. We studied the role of CLASP2 on hematopoiesis by performing loss-of-function experiments in mouse and zebrafish embryos. Here, we show that CLASP2 ensures the proper dynamics of EHT and is involved in the maturation of HSC precursors into fully functional HSCs in the aorta. At later time points during embryogenesis, CLASP2 prevents the exhaustion of the blood cell pool by ensuring self-renewal of HSCs and by precluding their premature differentiation. Our data indicate that CLASP2 is involved in both, cellular fidelity during mitosis and protein formation at the post-translational level in the Golgi and/or their trafficking to the cell membrane, as exemplified for KIT. In conclusion, our data emphasize an earlier role of CLASP2 as an important and conserved regulator of HSC generation and expansion at multiple steps of hematopoiesis during mouse and zebrafish development.

2007 – DIFFERENTIAL ALTERNATIVE POLYADENYLATION LANDSCAPES MEDIATE HEMATOPOIETIC STEM CELL ACTIVATION AND REGULATE GLUTAMINE METABOLISM

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3'-untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies in vivo remains elusive. Using extensive in vitro and in vivo analysis approaches we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Further, we performed low input 3'-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors determining the genome-wide APA landscape (APAome). This revealed transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation (Sommerkamp et al., Cell Stem Cell, 2020). Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching. This process is mediated by Nudt21 and is required for the proper stress response of HSCs. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Inhibition of this metabolic adjustment leads to impaired HSC function and a partial block in differentiation. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal and commitment.

1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Dysregulation of ribosome biogenesis is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. As prominently illustrated by one type of ribosomopathies, Shwachman-Diamond Syndrome (SDS), germline biallelic mutations in three assembly factors essential for the maturation of the 60S ribosome subunit result in progressive hematopoietic stem and progenitor cell (HSPC) failure, suggesting that HSPCs are sensitive to perturbations in ribosome assembly. However, how ribosome assembly is regulated in HSPCs remains poorly understood, as are approaches to restore in hematopoietic stem cell (HSC) function in the context of defective ribosome biogenesis. Here we uncover a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. HSCs from hematopoietic-specific <i>Hectd1</i> knockout mice exhibit a striking defect in transplantation ability and self-renewal. <i>Hectd1</i>-deficient HSCs fail to be maintained ex vivo, concomitant with a reduction in global protein synthesis and growth rate. The mechanism underlying HSC dysfunction upon <i>Hectd1</i> deficiency is directly linked to aberrant ribosome assembly. We demonstrate that HectD1 ubiquitinates and degrades an assembly factor for the polypeptide exit tunnel of the 60S ribosome in the cytoplasm. Depletion of <i>HectD1</i> led to an accumulation of the anti-association factor eIF6 on the 60S and decreased 80S to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, we found that restoration of ribosome assembly in <i>HectD1</i>-deficient cells promoted protein synthesis and enhanced HSC reconstitution capacity. These findings highlight the importance of ribosome assembly in HSC regeneration and represent a rare <i>in vivo</i> example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis.

Aging-Related Reduced Expression of CXCR4 on Bone Marrow Mesenchymal Stromal Cells Contributes to Hematopoietic Stem and Progenitor Cell Defects.

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function.

The Fanconi Anemia-Associated Protein NIPA Is Essential for the Nuclear Abundance of FANCD2

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (&gt;26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents &amp; Royalties: royalties for the invention of NUP98-HOXD13.

Hematopoietic Stem Cell Function in β-Thalassemia Is Impaired and Is Rescued By Targeting the Bone Marrow Niche

Hematopoietic stem cells (HSC) are regulated by signals from the bone marrow (BM) niche and little is known about their fate in altered hematological conditions associated to non-malignant diseases. In β-thalassemia ineffective erythropoiesis and secondary alterations, as abnormal regulation of bone metabolism, iron overload and hormonal factors, induce changes in the BM homeostasis with a potential impact on HSC-niche interaction. We addressed these unexplored issues in the murine disease model and in patients' cells. We investigated hematopoiesis in thalassemic Hbbth3/+ (th3) mutant mice and we found lower frequency, reduced quiescence and reconstituting potential of HSC. th3 HSC have impaired self-renewal, which is rescued upon transplantation in a normal BM, proving an active role of the niche microenvironment. Both stromal and hematopoietic components of the BM niche are altered in th3 mice. Consistently with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. Low PTH negatively affects bone deposition and expression of the Notch-ligand Jag1 by th3 mesenchymal and osteolineage cells, thus reducing the activation of Notch1 in HSC and consequently impairing their function. In vivo activation of PTH signaling through the reestablished Jag1-Notch1 pathway restores the functional pool of th3 HSC by correcting HSC-niche crosstalk. In addition to the stromal component of the BM, hematopoietic cells with a key role in regulating the fate of HSC, such as megakaryocytes (Mk), were found defective in maturation, possibly due to reduced circulating levels of thrombopoietin (TPO). We are currently investigating the molecular causes of dysmegakaryopoiesis and the Mk-HSC interaction in thalassemic mice. Strikingly, reduced HSC quiescence was confirmed in samples from patients affected by β-thalassemia, along with impaired stromal niche and megakaryopoiesis, thus highlighting the clinical relevance of our findings. Further investigation will unravel the multiple molecular mechanisms that affect in trans HSC functions in the complexity of the stressed thalassemic BM microenvironment. Our results uncover a defect of HSC in β-thalassemia, induced by an altered BM niche and provide new relevant insight for improving transplantation and gene therapy approaches. Disclosures No relevant conflicts of interest to declare.

ALTERNATIVE POLYADENYLATION REGULATES HEMATOPOIETIC STEM CELL METABOLISM

Alternative Polyadenylation (APA) is emerging as an important regulatory mechanism controlling 3’-UTRs and thereby RNA and protein isoform expression. However, the relevance of APA in stem cell hierarchies in vivo remains unexplored. Here, we show that deregulation of the APA regulator Pabpn1 results in severe hematopoietic stem cell (HSC) defects. Furthermore, we performed low input 3’-sequencing and established bioinformatic pipelines to uncover dynamic APA patterns in numerous genes of HSCs and multipotent progenitors. The 3’-UTRs of HSCs undergo shortening upon transition from quiescence to proliferation during homeostasis and in response to inflammation as well as during HSC differentiation. Strikingly, we observe APA-mediated metabolic reprogramming upon HSC activation leading to an increase in glutaminolysis. This increase is mediated by APA regulators and required for proper HSC function. Inhibition of this metabolic adjustment by targeting APA-regulators leads to impaired HSC function and a partial block in differentiation. Our study establishes APA and associated adaptations of the glutamine metabolism as novel layers of regulation that orchestrate HSC behavior and commitment in the hematopoietic cascade.

Control of Hematopoietic Stem and Progenitor Cell Function through Epigenetic Regulation of Energy Metabolism and Genome Integrity.

SummaryIt remains largely unclear how stem cells regulate bioenergetics and genome integrity to ensure tissue homeostasis. Here, our integrative gene analyses suggest that metabolic and genotoxic stresses may underlie the common functional defects of both fetal and adult hematopoietic stem and progenitor cells (HSPCs) upon loss of DPY30, an epigenetic modulator that facilitates H3K4 methylation. DPY30 directly regulates expression of several key glycolytic genes, and its loss in HSPCs critically impaired energy metabolism, including both glycolytic and mitochondrial pathways. We also found significant increase in DNA breaks as a result of impaired DNA repair upon DPY30 loss, and inhibition of DNA damage response partially rescued clonogenicity of the DPY30-deficient HSPCs. Moreover, CDK inhibitor p21 was upregulated in DPY30-deficient HSPCs, and p21 deletion alleviated their functional defect. These results demonstrate that epigenetic mechanisms by H3K4 methylation play a crucial role in HSPC function through control of energy metabolism and protecting genome integrity.