Expansion Of Hematopoietic Progenitor Cells Research Articles

Ex-Vivo Human Whole Blood Assay for Evaluating the Pharmacodynamics of HT-6184: Reduction in NLRP3 Inflammasome Mediated Cytokine Production

The NLRP3 inflammasome is an intracellular multiprotein complex that plays a crucial role in the innate immune response by mediating the maturation and release of pro-inflammatory cytokines IL-1 beta, and IL-18. The release of these cytokines leads to the downstream release of TNF-alpha, IL-6, which propagate a robust inflammatory response. Dysregulation of the NLRP3 inflammasome pathway has been linked to various inflammatory diseases, suggesting the possibility for novel therapeutic strategies targeting NLRP3 activation. NLRP3 has been linked to regulating hematopoietic stem progenitor cell (HSPCs) development, expansion, release, and mobility. In addition, various hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, acute and chronic leukemias, and graft-versus-host disease (GvHD) have all been shown to involve NLRP3 inflammasome activation. Given its significant role in these conditions, inhibiting the NLRP3 inflammasome may be a novel therapeutic strategy in hematology and hemato-oncology This study utilized an ex-vivo human whole blood assay to investigate the pharmacodynamics of HT-6184, an allosteric Nek7 inhibitor. We used LPS and ATP to prime and activate the NLRP3 inflammasome and examined the effects of pre-treatment with various concentrations of HT-6184 (starting at 1 mM) on the production of critical pro-inflammatory cytokines, such as IL-1b, IL-18, IL-6 and TNF-a. Notably, preincubation with HT-6184 at concentrations as low as 4.1 nM led to a significant reduction in cytokine production (p<0.0001). Specifically, TNF-α and IL-6 were reduced by 38% and 58%, respectively, while IL-1β and IL-18 were decreased by 44% and 53% respectively. These results provide further evidence of the ability of HT-6184 to modulate the activity of the NLRP3 inflammasome in a complex biological milieu. Our study suggests that HT-6184 has significant therapeutic potential in treating a broad spectrum of inflammasome-mediated diseases. In addition, this assay has the potential to be used to measure the pharmacodynamic properties and the efficacy of HT-6184 and other inflammasome inhibitors in future healthy volunteer clinical trials. In conclusion, this study highlights the importance of innovative and physiologically relevant assay systems in identifying and evaluating potential therapeutic agents for inflammatory diseases.

Mesenchymal Stromal Cells from Perinatal Tissues as an Alternative for Ex Vivo Expansion of Hematopoietic Progenitor and Stem Cells from Umbilical Cord Blood.

Umbilical cord blood (UCB) serves as a source of hematopoietic stem and progenitor cells (HSPCs) utilized in the regeneration of hematopoietic and immune systems, forming a crucial part of the treatment for various benign and malignant hematological diseases. UCB has been utilized as an alternative HSPC source to bone marrow (BM). Although the use of UCB has extended transplantation access to many individuals, it still encounters significant challenges in selecting a histocompatible UCB unit with an adequate cell dose for a substantial proportion of adults with malignant hematological diseases. Consequently, recent research has focused on developing ex vivo expansion strategies for UCB HSPCs. Our results demonstrate that co-cultures with the investigated mesenchymal stromal cells (MSCs) enable a 10- to 15-fold increase in the cellular dose of UCB HSPCs while partially regulating the proliferation capacity when compared to HSPCs expanded with early acting cytokines. Furthermore, the secretory profile of UCB-derived MSCs closely resembles that of BM-derived MSCs. Moreover, both co-cultures exhibit alterations in cytokine secretion, which could potentially impact HSPC proliferation during the expansion process. This study underscores the fact that UCB-derived MSCs possess a remarkably similar supportive capacity to BM-derived MSCs, implying their potential use as feeder layers in the ex vivo expansion process of HSPCs.

Abstract 26 The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment

Abstract Introduction Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.

Investigation of the impact of clonal hematopoiesis on severity and pathophysiology of COVID-19 in rhesus macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection.

Abstract 1732: Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment

Abstract CD36 has been identified as a potential therapeutic target both in the leukemic cells and in the tumor immune microenvironment. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. To establish CD36 as a viable therapeutic target in AML, we recently investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. We compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (Cd36-KO) with C57BL/6J control (WT) mice (N=6 per group). Cd36-KO mice have normal blood counts except for lower red blood cells count (5.85 vs 6.93 (x 106/μL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) than WT mice, yet these values remained within the normal range. Cd36-KO mice exhibited similar spleen T cell phenotypes compared with the WT mice and expanded similarly in vitro in the presence IL2 and PHA. The in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSPC expansion media containing SCF and TPO cytokines was also similar between Cd36-KO mice and WT mice. However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of Cd36-KO mice resulted in 40% fewer colonies than WT mouse HSPC cells (P = 0.0003). However, when HSPC obtained from Cd36-KO and WT mice were transplanted in CD45.1 mice, their percentage of engraftment were not significantly different in the bone marrow (BM: KO vs WT: 64.92% vs 63.45%, P > 0.9999), spleen (40.88% vs 40.05%, P = 0.8413), and blood (83.47% vs 82.88%, P > 0.9999). To assess whether CD36 in the leukemia microenvironment has an impact on leukemia growth and progression, we injected 5 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells via tail vein in either Cd36-KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36-KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Cd36-KO and WT mice developed similar leukemia burden when less aggressive models are used with the engraftment of 1 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells. Altogether, our preliminary data suggest that loss of Cd36 may influence the hematopoietic stem cell and erythropoiesis yet have limited detrimental overall impact on normal hematopoiesis and leukemia microenvironment. Therefore, targeting CD36 in leukemic cells presents limited toxicity on normal hematopoiesis and therapeutic approaches to target this receptor present safe strategies to treat AML. Citation Format: Yiting Meng, Mateusz Pospiech, Atham Ali, Ritu Chandwani, Sandra Onyemaechi, George Yaghmour, Houda Alachkar. Functional characterization of the impact of CD36 deletion on normal hematopoiesis and the leukemia microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1732.

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR-Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now under way. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their invitro expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology-directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during exvivo culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both invitro and invivo, with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized exvivo HSPC culture protocol can improve the efficacy, feasibility, and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology.

From bone marrow failure syndromes to VEXAS: Disentangling clonal hematopoiesis, immune system, and molecular drivers

Clonal hematopoiesis (CH) is a result of the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations originating from a primary HSC. The advent of modern genomic technologies has helped recognizing that CH is common in elderly healthy subjects as a result of the aging bone marrow (BM). CH in healthy subjects without abnormalities in blood counts is known as CH of indeterminate potential. CH is also seen in BM failure (BMF) disorders. Whether CH alarms for the risk to develop malignant evolution in BMF or creates an adaptation to selective pressure is a matter of controversy. As such, a continuum might exist from pre-malignant to malignant hematopoietic diseases. This review summarizes how somatic mutations and immune derangement in HSCs shape disease evolution and describes the complexity of disorders such as VEXAS as the prototypic tetrad of somatic mutations, morphologic features, inflammatory pathways and immune overshooting. In such a view, we interconnect the axis aging and immune-hematopoietic system, which all convey important clues for the risk to develop malignancies.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Identification of SALL4 Expressing Islet-1+ Cardiovascular Progenitor Cell Clones.

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

Simulated microgravity affects stroma-dependent ex vivo myelopoiesis

Microgravity is known negatively affect physiology of living beings, including hematopoiesis. Dysregulation of hematopoietic cells and supporting stroma relationships in bone marrow niche may be in charge. We compared the efficacy of ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) in presence of native or osteocommitted MSCs under simulated microgravity (Smg) using Random Positioning Machine (RPM). In comparison with 1 g, a decrease of MSC-associated HSPCs and an increase of floating HSPCs was observed after 7 days of Smg exposure. Among floating HSPCs, primitive progenitors were presented by late CD34+/133–. Total CFUs as well as erythroid (BFU-E) and granulocytic (CFU-G) numbers were lower. MSC-associated primitive HSPCs demonstrated increased proportion of late CD34+/133- in expense of early CD34-/133+. Osteo-MSCs preferentially supported late primitive CD34+ and more committed HSPCs as followed from increase of CFUs, and CD235a+ erythroid progenitors. Under Smg, an increased VEGF, eotaxin, and GRO-a levels, and a decrease in RANTES were found in the osteo-MSC-HSPC co-cultures. IL-6,-8, -13, G-CSF, GRO-a, MCP-3, MIP-1b, VEGF increased in co-culture with osteo-MSCs vs intact MSCs. Based on the findings, the misbalance between primitive/committed HSPCs and a decrease in hematopoiesis-supportive activity of osteocommitted cells are supposed to underline hematopoietic disorders during space flights.

Chronic Inflammation Drives p53 Mutant Clonal Hematopoiesis Via Activating the NLRP1 Inflammasome

Individuals with somatic TP53 mutations manifest clonal hematopoiesis and are at high risk of developing hematological malignancies. However, how environmental factors influence the expansion of p53 mutant hematopoietic stem and progenitor cells (HSPCs) is unclear. We found that pI:pC or LPS treatment promotes the clonal expansion of p53 mutant HSPCs in transplantation assays. To determine the impact of LPS on HSPC function in vivo, we treated p53+/+ and p53R248W/+ mice with LPS or PBS and then performed competitive transplantation assays. LPS-treated WT BM cells show decreased engraftment in both primary and secondary transplantation assays, whereas LPS-treated p53 mutant BM cells continued to show increased engraftment. To understand how LPS-induced chronic inflammation promotes p53 mutant HSPC expansion, we examined the impact of mutant p53 on cytokine secretion from BM derived macrophages. We found that p53 mutant macrophages show increased secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-12 (p70), KC, VEGF, IP-10, CXCL-9, LIX, and MCP-1. To further investigate how chronic inflammation promotes p53 mutant HSPC expansion, we performed ATAC-seq assays in LSKs to identify differential regions of chromatin accessibility. Notably, we found that mutant p53 increases the chromatin accessibility to inflammasome genes, including Nlrp1a and Nlrp1b. Inflammasomes are multiprotein complexes that activate Caspase-1 and increase the release of pro-inflammatory cytokines such as IL-1β, leading to caspase-1-dependent death, known as pyroptosis. We found that loss of NLRP1 significantly decreased the secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-6, MCP-1, KC, M-CSF, RANTES, LIX, GM-CSF G-CSF, TNF-α and IL-12 (p40), from macrophages expressing mutant p53. Loss of NLRP1 significantly decreases the repopulating potential of HSPCs expressing mutant p53 in vivo. To further understand how chronic inflammation may confer a competitive advantage to p53 mutant HSPCs, we performed in vitro cell competition assays. We found that WT CD45.1+ cells co-cultured with p53 mutant cells show decreased replating potential compared to WT CD45.1+ cells co-cultured with WT CD45.2+ cells. We measured the levels of cytokines in the condition media utilizing cytokine arrays and found increased levels of IL-1β, IL-6, CXCL-9, and IL-4 in mutant cell cultures compared to that of the WT cells. We found that WT CD45.1+ cells co-cultured with mutant cells show increased pyroptosis. Importantly, we found that the enhanced pyroptosis of WT CD45.1+ cells seen in the mutant group is IL-1β dependent. We found that IL-1β treatment decreases the replating potential of WT BM cells, but not that of p53 mutant cells. To determine the mechanism by which p53 mutant HSPCs are resistant to IL-1β treatment, we performed RNA sequencing in p53+/+ and p53R248W/+ LSKs treated with IL-1β (10 ng/ml) or PBS in vitro for 90 minutes. GSEA analysis revealed that hematopoietic stem and progenitor cell differentiation genes are significantly upregulated in p53 mutant LSKs compared to WT LSKs following IL-1β stimulation. Notably, we found that p53 mutant LSKs treated with IL-1β are enriched with MYC target genes. Furthermore, blocking of IL-1β using IL-1β neutralizing antibody decreases the fitness of p53 mutant HSPCs. Our studies will likely establish both the NLRP1 inflammasome and the pro-inflammatory cytokine IL-1β as novel potential therapeutic targets for preventing clonal hematopoiesis development and treating hematological malignancies with TP53 mutations.

GAS6 Promotes Robust Expansion of Human Hematopoietic Stem and Progenitor Cells with High Serial Engraftment Activity

Hematopoietic stem cell transplantation is a curative therapy for over 30 hematopoietic illnesses. The inability to sufficiently expand functional HSCs in cultures has partly hampered their therapeutic potential and limited molecular understanding of HSCs. Stem cell agonists (SCA) such as UM171 and StemReginin1 (SR1) have been shown to promote HSC expansion. Recently, we discovered that Ascorbic acid-2-phosphate (AA2P) can act as a novel SCA. Furthermore, using mathematical modeling we formulated a series of stem cell agonist cocktails (SCACs) composed of varying concentrations of 4 SCAs. SCACs promote robust expansion of HSPCs and limited dilution transplant assay revealed that the lead cocktail X2A promoted 15- and 4-fold expansions of scid repopulating cells, when compared to non-cultured cells or cells expanded with the combination of UM171 and SR1 (Manesia et al., 2019). The objective of the present study was to characterize the molecular programs supporting the strong expansion of HSCs in X2A cultures. RNA-Seq data from SCACs expanded CD34+CD45RA- cells revealed the upregulation of several receptor tyrosine kinases (RTKs) including AXL, but also its ligand GAS6 preferentially with X2A. AXL was recently shown to be essential for the self-renewal of leukemia initiating cells. Single cell RNA-seq analyses of CD34+ cells was done to characterize the graft composition of SCACs expanded HSPCs. These analyses revealed that AXL expression is enriched within CD34+CD45RA-CD90+CD49f+ HSC-enriched clusters. Image-flowcytometry analyses confirmed that HSC-enriched cell populations respond to GAS6-activation, resulting in the activation of ERK1/2 downstream of AXL activation. Epigenetic analysis revealed that GAS6/AXL upregulation is regulated by AA2P through DNA-demethylation in a TET dependent manner. Using genetic knockdown, chemical inhibition and GAS6 neutralizing antibody approaches we confirmed that GAS6 activation of AXL drives human HSPC expansion within X2A. In addition, supplementation of SCAC or single SCA-based cultures with GAS6 significantly enhanced the expansion of HSPC subsets with little induction of differentiation (e.g., 640- Vs. 1975-fold for CD34+CD45RA- HSPC in X2A and X2A+GAS6 cultures, respectively (n=3, p<0.05)). Transplant assays with day-14 expanded HSPC (progeny of 850 CD34+ starting cells/mouse) revealed that GAS6 supplementation in X2A cultures increased ensuing engraftment by 10-fold in both primary and secondary NSG mice (n=2, p<0.0001). For instance, the % of human CD45+ BM cells in secondary mice were of 2.3±0.1% and 25.7±1.3% in X2A and X2A+GAS6 groups. Remarkably, the net numbers of human colony-forming unit and CD34+ BM cells were increased by 7- and 15-fold (p<0.0001) in primary, and by 20- and 26-fold (p<0.0001) in secondary transplants by GAS6, respectively. In contrast, GAS6 neutralization in cultures impaired ensuing serial engraftment by 8- and 30-fold respectively Vs. X2A control (p<0.01). Lastly, SCAC and GAS6 were also shown to improve the expansion of adult G-CSF mobilized peripheral blood stem cells. In conclusion, we demonstrate that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and its ligand GAS6. We also show for the first time that GAS6 is a potent growth factor that promote robust expansion of human HSPCs including serial engrafting HSCs resulting in unexpectedly high engraftment.

Metformin Treatment Abrogates the Competitive Advantage of Dnmt3a R878H HSPCs By Enhancing DNA Methylation Activity

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying leukemogenic mutations in individuals without evidence of a blood disorder. CHIP carriers are at an increased risk of blood cancers and cardiovascular diseases. Targeting the clonal expansion of mutant HSPCs has the potential to mitigate CHIP-related illnesses. The most commonly mutated gene in CHIP is DNMT3A, which encodes an enzyme that catalyzes the methyl transfer from S-adenosylmethionine (SAM) to cytosines in DNA. The heterozygous DNMT3A hotspot mutation at R882 exerts a dominant negative effect on the wild-type (WT) allele, resulting in decreased DNA methylation activity. Prior studies have shown that murine HSPCs carrying mutant Dnmt3a R878H (equivalent to R882 in humans) have an advantage over WT cells in competition assays. Here, we aimed to first identify differences in the functional properties between Dnmt3a-mutated and WT HSPCs. Given the crosstalk between mitochondrial metabolism and DNA methylation, we investigated potential differences in mitochondrial activity and found that the level of mitochondrial respiration was higher in Dnmt3aR878H/+ HSPCs than in WT cells. To determine if this translated to differential dependencies, we silenced the expression of critical subunits in the each of five electron transport chain (ETC) complexes. Knockdown of NDUFV1 in Complex I was the most effective in reducing the competitive advantage of Dnmt3aR878H/+ cells in vitro. To explore translational relevance of this finding, we tested the impact of metformin, a pharmacologic inhibitor of Complex I, on Dnmt3a-mutated HSPCs. Treatment with metformin at clinically relevant concentrations suppressed the competitive advantage of mutant cells in vitro. Importantly, this effect was rescued by expression of NDI1, a metformin-resistant yeast analog of Complex I. To extend these findings, we conducted a competitive repopulation assay between CD45.2+ Dnmt3aR878H/+ and CD45.1+ Dnmt3a+/+ HSPCs. Metformin treatment abrogated the in vivo competitive advantage of mutant cells over an 8-month period based on peripheral blood chimerism. To decipher the mechanism of action, we performed transcriptome and metabolome profiling of mutant HSPCs treated with or without metformin for 1 month. GSEA analysis of the transcriptome data revealed a downregulation of genes associated with stemness in metformin-treated cells. Intriguingly, we found that metformin increased the expression of genes involved in one-carbon metabolism, which generates SAM. Consistent with these findings, metabolomics analysis showed that the levels of several key metabolites in one-carbon metabolism, including SAM, were higher in metformin-treated cells compared with controls. Genetic and pharmacologic inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism, as well as the addition of exogeneous S-adenosylhomocysteine (SAH), an inhibitor of SAM-dependent methyltransferases, effectively rescued the effect of metformin on mutant cells in vitro. Based on the above findings, we hypothesized that metformin treatment might enhance the residual DNA methylation activity found in Dnmt3aR878H/+ mutated cells by increasing SAM concentration. In support of this hypothesis, we found that exogeneous SAM reproduced the effect of metformin, and HSPCs with homozygous Dnmt3aR878H mutations were insensitive to metformin. Furthermore, we performed DNA methylome profiling of untreated and metformin-treated mutant and WT HSPCs using RRBS. This analysis revealed that the hypomethylated regions (HMRs) in untreated Dnmt3aR878H/+ HSPCs relative to WT controls were enriched for regions of repressed chromatin with H3K27 methylation. Importantly, metformin treatment increased the level of DNA methylation at these regions in mutated HSPCs. In summary, our findings demonstrate that Dnmt3aR878H/+ HSPCs depend on ETC activity and particularly complex I activity to maintain their competitive advantage over WT cells. We propose a model in which inhibition of complex I activity with metformin increases one-carbon metabolism and SAM level, thereby restoring DNA methylation at HMRs and the differentiation of mutant HSPCs. Our findings have important implications for the development of preventive interventions against DNMT3A-mutated CHIP.

Loss of Cd36 Expression Has Limited Impact on Mouse Normal Hematopoiesis

CD36 has been identified as a potential therapeutic target both in the leukemic cells and in tumor immune microenvironment. In acute myeloid leukemia, we recently found that APOC2 acts with CD36 to promote acute myeloid leukemia (AML) growth by activating the LYN-ERK signaling. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. CD36 is also present on various types of normal hematopoietic cells, including monocytes, macrophages, endothelial cells, and platelets and contribute to their normal functions. Whether targeting CD36 presents unwanted consequences on normal hematopoiesis remains unknown. Here we analyzed mouse RNA-Seq data (BloodSpot, GSE60101) and found that Cd36 expression is low in the hematopoietic stem cells (HSC) and increases significantly as they differentiate to mature cells (115.4-fold in erythrocytes, 61.8-fold in NK cells, 32.9-fold in B cells, P < 0.001). We also observed differential expression patterns of CD36 RNA between human and mouse. The haemopedia human RNAseq data revealed high level of CD36 expression in dendritic cell lineage, macrophage/monocytes, and NK cells. While haemopedia mouse RNAseq data showed higher Cd36 expression in progenitor cells, erythrocytes, mast cells, basophils, and B cells. To investigate the effect of loss of Cd36 in normal hematopoiesis, we compared B6.129S1-Cd36tm1Mfe/J Cd36 knockout (KO) with C57BL/6J control (WT) mice (N=6 per group). The mice strains were confirmed by genotyping. In bone marrow (BM), spleen, and liver cells, the KO mice had reduced Cd36 mRNA level (~46-72%, P < 0.05), and decreased Cd36 surface expression (~20-40% Cd36+ population, P < 0.05) compared with WT mice. Differential blood count and hematological analysis revealed lower red blood cells count (5.85 vs 6.93 (x 106/uL), P = 0.0332), hemoglobin (10.03 vs 11.35 (gr/dL), P = 0.0109), and hematocrit (31.67 vs 35.48 (%), P = 0.02) in KO mice than WT mice, yet these values remained within the normal range. On the other hand, the count of white blood cells (1.47 vs 2.06 (x 103/uL), P = 0.2461), neutrophil (5.77 vs 6.10 (%), P = 0.8750), lymphocytes (81.55 vs 86.48 (%), P = 0.1509), monocytes (5.27 vs 3.23 (%), P = 0.0848), eosinophils (7.40 vs 4.08 (%), P = 0.3223), and platelets (230.67 vs 280.17 (x 1000/uL), P = 0.6779) were not significantly different between Cd36 KO and WT mice. Cd36 KO mice also exhibited similar T cell phenotype in the spleen compared with the WT mice: (% lymphocytes: 72.13% vs 67.97%, P = 0.7857; % CD3+: 29.80% vs 29.10%, P = 0.6982; % CD4+: 57.02% vs 54.52%, P = 0.0901; % CD8+: 32.12% vs 33.70%, P = 0.1246; and % CD25+: 12.03% vs 21.03%, P = 0.9920). We also assessed the in vitro expansion of hematopoietic stem and progenitor cells (HSPC) in HSC expansion media containing SCF and TPO cytokines. The expansion of HSPC was not significantly different (P = 0.2461) between WT and KO mice (Day 10 to Day 5 fold change: 1.65-fold, P = 0.0187 in WT and 1.92-fold, P = 0.0049 in KO). Consistently, the MFI of CFSE labeled HSPC was decreased by 35% (P = 0.0047) in WT and 32% (P = 0.0006) in KO on day 9 relative to day 1 (P = 0.8292 between KO and WT). However, mouse colony forming cell assay using methylcellulose-based media showed that the HSPC of KO mice resulted in less colonies compared with WT mice (WT vs KO = 44 vs 26, P = 0.0108). We also assessed T cells proliferation in CD36 KO mice by in vitro culturing of mouse splenocytes with T cell expansion media containing IL2 and PHA. The number of viable T cells was 1.94-fold (P < 0.0001) in WT mice and 2.24-fold (P < 0.0001) in KO mice on day 10 relative to day 5 (P = 0.2519 comparing WT vs KO). In addition, the cell trace dye MFI was decreased by 67% (P = 0.0002) in WT splenocytes and 59% (P = 0.0030) in KO solenocytes (P = 0.2879 comparing WT vs KO). To assess the leukemia burden in KO mice, we injected 5 million FLT3-ITD/MLL-PTD mice leukemic cells via tail vein in either Cd36 KO mice (N = 5) or WT mice (N = 6) and compared the percentage of engraftment in their tissues. Cd36 KO mice exhibited similar leukemia engraftment in the BM (88.92% vs 86.38%, P = 0.0497), spleen (76.48% vs 68.10%, P = 0.1627), liver (16.12% vs 6.70%, P = 0.2521), and blood (16.33% vs 0.96%, P = 0.2504) compared with the WT group. Altogether, our preliminary data suggest that Cd36-KO has limited impact on normal hematopoietic cells.

A Circulating Microbial Metabolite Drives the Clonal Expansion of Pre-Leukemic Cells

Inflammatory mediators, including cytokines and microbial ligands, are implicated in the development of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A consequence of aging and various autoimmune and inflammatory conditions is loss of intestinal barrier integrity and translocation of microbial species and metabolites into circulation. More recent findings revealed that patients with inflammatory bowel disease (IBD) harbor mutant hematopoietic clones and often present with clinical manifestations of MDS/AML. Moreover, intestinal barrier dysfunction is commonly reported at diagnosis in patients with MDS and AML. These observations led us to speculate that microbial-derived signals may contribute to the expansion of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) and development of myeloid malignancies. To determine if loss of intestinal barrier integrity contributes to the expansion of pre-leukemic DNMT3a-mutant HSPCs, we generated a model of IBD by inducing gut epithelial injury using dextran sulfate sodium (DSS). Upon gut epithelial injury, we observed a significant expansion of DNMT3a-mutant HSPCs, but not of wild-type (WT) HSPCs, in vivo. Unexpectedly, DNMT3a-mutant HSPCs do not exhibit a competitive advantage over WT cells in the absence of intestinal barrier dysfunction, such as following DSS or high-dose radiation. Fecal material from DSS-treated WT mice was sufficient to induce expansion of DNMT3a-mutant HSPCs and this expansion was prevented with broad-spectrum antibiotics. These findings indicate that the competitive advantage of DNMT3a-mutant HSPCs following gut epithelial injury was due to alterations in the microbiome. In parallel sequencing studies, we identified a significant enrichment of gram-negative bacterial phyla in stool samples from the IBD mouse model, IBD patients, and treatment-naive AML patients. All gram-negative bacteria uniquely generate a microbial intermediate, ADP-heptose, as part of the lipopolysaccharide biosynthesis pathway. ADP-heptose can enter the blood circulation and freely translocate into cells and activate the ALPK1-TIFA complex ("TIFAsome"). Alpha-protein kinase 1 (ALPK1) and tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with an FHA domain (TIFA) are overexpressed in primary human MDS and AML HSPCs as compared to healthy donors and their expression is correlated with poor prognosis. Gram negative bacteria and corresponding ADP-heptose levels were also significantly increased in the plasma of individuals with intestinal barrier injury, such with IBD, MDS, or upon aging, as compared to young healthy individuals. The circulating levels of ADP-heptose from aged individuals and IBD or MDS patients were sufficient to induce the TIFAsome in leukemic cells. Moreover, administration of disease relevant levels of ADP-heptose resulted in expansion and enhanced function of mouse DNMT3a-mutant HSPCs and of human AML cell lines and primary MDS cells in vivo. Expansion of DNMT3a-mutant HSPCs and human MDS/AML cells was directly related to overexpression of ALPK1, as WT HSPCs or ALPK1-deficient AML cells did not respond to ADP-heptose. To gain deeper mechanistic insight, we examined global transcriptional and epigenetic changes in purified WT and DNTM3a-mutant HSCs treated with ADP-heptose. DNMT3a-mutant HSCs treated with ADP-heptose resulted in epigenetic changes and increased expression of gene programs related to non-canonical NF-kB, ERK/MAPK, cell cycle activation, cell survival, and stem cell function. Indeed, ADP-heptose caused increased cycling and self-renewal of DNMT3a-mutant HSPCs, but not WT HSPCs, via the TIFAsome in vitro and in vivo. Based on the enriched pathways in DNMT3a-mutant HSCs treated with ADP-heptose, we screened small molecule inhibitors of TIFAsome activation. We found that targeting UBE2N abrogated ADP-heptose-mediated signaling via the TIFAsome and expansion of patient-derived AML xenografts. In summary, we report that conditions leading to intestinal barrier dysfunction and circulating ADP-heptose from gram-negative bacteria induce expansion of pre-leukemic and MDS/AML cells via ALPK1-TIFA. Lastly, we provide a potential therapeutic strategy to prevent the competitive advantage of pre-leukemic cells prior to overt malignant transformation.

The Long Non-Coding RNA MALAT1 Is Necessary for Hematopoietic Stem and Progenitor Cell Expansion and Preleukemic Myeloproliferation in TET2 Deficient Myeloid Neoplasms

Ten Eleven Translocation 2 (TET2) is a dioxygenase which regulates gene expression by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine. Loss of function mutations in TET2 occur in up to 58% and 30% of myeloid malignancies and clonal hematopoiesis respectively. Loss of TET2 function also occurs with isocitrate dehydrogenase 2 (IDH2) mutations in Acute Myeloid Leukemia (AML) via 2-hydroxyglutarate (2-HG). Genetically engineered murine models (GEMM) of Tet2 deficiency produce a Chronic Myelomonocytic Leukemia (CMML)-like phenotype with splenomegaly, granulomonocytic expansion and increased bone marrow hematopoietic stem and progenitors (LSK). Further, bone marrow mononuclear cell (BMNC) transplant models of Tet2-/- clonal hematopoiesis suggest that this myeloid expansion leads to enhanced inflammation and increased cardiovascular disease risk. However, the downstream effects of TET2 loss of function leading to this phenotype are unknown. Here we show that TET2-deficient granulomonocytic expansion and inflammation are mediated via MALAT1, a long non-coding RNA overexpressed in several malignancies. MALAT1 expression was increased in BMNCs from IDH2 mutated AML patients (n=152 WT n=17 mutant IDH2, p=0.04). Increased MALAT1 expression was also seen in human and murine models of TET2 deficiency including isogenic IDH2 mutated TF-1 cells (5 biological replicates p=0.005), 2-HG treated leukemia cell lines (2 biological replicates p<0.0001) and BMNCs from Tet2f/f mice (n= 7/grp p=0.005). To evaluate the impact of Malat1 on normal and Tet2 deficient hematopoiesis we utilized the Malat1tg overexpression and Tet2-/-Malat1-/-GEMM models. Similar to Tet2-/- mice, bone marrow (n=4/grp p=0.02) and spleen (n=8/grp p=0.004) LSK numbers were increased in Malat1tg mice compared to control. However, unlike Tet2-/-LSKs, MALAT1tgLSKs did not show increased self-renewal in serial in vivo competitive transplants. Further, depleting Malat1 in Tet2-/- mice did not rescue the increased self-renewal capacity seen in Tet2-/- LSKs. Collectively, these data suggest that Malat1 overexpression is sufficient for LSK expansion but does not impact LSK function. Splenomegaly, myeloproliferation and extramedullary hematopoiesis is a phenotype termed Preleukemic Myeloproliferation (PMP) and is predictive of myeloid neoplasm development in Tet2-/- models (Meisel M et al, Nature 2018). Consistent with a role of Malat1 in PMP (Fig A), Malat1 overexpression recapitulated PMP at 8 (n=10/grp p<0.0001) and 20 (n=10/grp p=0.0005) weeks. Malat1 overexpression was also associated with an increase in classical monocytes in the peripheral blood- a hallmark of CMML (8 weeks n= 7/grp p<0.0001, 20 weeks n=10/grp p=0.0007). This was recapitulated in Tet2-/-mice (n=3/grp p=0.001) and CMML patients with TET2 mutations (n=27 mutant TET2 v 109 WT, odds ratio 5.6 p= 0.001). Finally, depleting Malat1 in a Tet2-deficient GEMM rescued PMP (n=4 Tet2-/-Malat1-/-v 5 Tet2-/-p=0.01) and classical monocytosis (n=2/grp p=0.002) suggesting that Malat1 overexpression is both sufficient and necessary for PMP in Tet2 deficient leukemia. This impact was also observed in recipients in BMNC competitive transplant experiments (Fig B). Initial insights into the molecular mechanism underlying the role of Malat1 in PMP were derived from analyzing competitor and host cells during in vivo competitive transplants. Interestingly, transplant recipients with Malat1tg LSK demonstrated myeloid and granulomonocytic expansion in both donor and non-donor cells suggesting that the effects of Malat1 overexpression are, in part, cell extrinsic. Consistent with this hypothesis and the impact of NF-kB on inflammatory cytokine secretion and Tet2-/- PMP, we show that MALAT1 deletion in human leukemia attenuates NF-kB activity by enhancing the phosphatase activity of PP2A. Further, Interleukin 6 (IL-6), a major downstream target of NF-kB and potent activator of myelopoiesis in response to inflammatory stimuli, was increased in the plasma of MALAT1tg mice (n=8/grp p=0.01) suggesting a possible cell-extrinsic mechanism for Malat1 dependent PMP. Ongoing studies are focused on demonstrating that IL-6 is necessary for Malat1 dependent PMP. Our current data provides hitherto unknown insights establishing MALAT1 as a major effector of TET2 loss of function which may lead to novel therapeutic options in TET2 deficient myeloid neoplasms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A chimeric feeder comprising transforming growth factor beta 1- and basic fibroblast growth factor-primed bone marrow-derived mesenchymal stromal cells suppresses the expansion of hematopoietic stem and progenitor cells.

Bone marrow-derivedmesenchymal stromal cells (BMSCs) physically associate with the hematopoietic stem cells (HSCs), forming a unique HSC niche. Owing to this proximity, the signaling mechanisms prevailing in the BMSCs affect the fate of the HSCs. In addition to cell-cell and cell-extracellular matrixinteractions, various cytokinesand growth factors present in the BM milieu evoke signaling mechanisms in the BMSCs. Previously, I have shown that priming of human BMSCs with transforming growth factor β1 (TGFβ1), a cytokine consistently found at active sites of hematopoiesis, boosts their hematopoiesis-supportive ability. Basic fibroblast growth factor (bFGF), another cytokine present in the marrow microenvironment, positively regulates hematopoiesis. Hence, I examined whether priming human BMSCs with bFGF improves their hematopoiesis-supportive ability. I found that bFGF-primed BMSCs stimulate hematopoiesis, as seen by a significant increase in colony formation from the bone marrow cells briefly interacted with them and the extensive proliferation of CD34+ HSCs cocultured with them. However, contrary to my expectation, I found that chimeric feeders comprising a mixture of TGF-primed and bFGF-primed BMSCs exerted a suppressive effect. These data demonstrate that though the TGF- and bFGF-primed BMSCs exert a salutary effect on hematopoiesis when used independently, they exert a suppressive effect when presented as a chimera. These findings suggest that the combinatorial effect of various priming agents and cytokines on the functionality of BMSCs towardthe target tissues needs to be critically evaluated before they are clinically applied.

Abstract 3 Generation of Myeloid Cells from Cord Blood-Derived CD34+ Cells for Therapeutic Intent

IntroductionMacrophages and monocytes traffic to and infiltrate complex solid tumor microenvironments where conditions can promote an immunosuppressive phenotype in resident tumor-associated macrophages that hinder effective anti-tumor immune responses. Ongoing advances in cell engineering are being adapted to modify monocytes and macrophages into efficacious anti-tumor cell therapies. However, a limiting factor to this promising therapy has been cell numbers, since monocytes obtained from adult peripheral blood mononuclear cells do not expand ex vivo.ObjectiveWe are developing an expanded allogeneic, cryopreserved, off-the-shelf myeloid cell product for the treatment of solid tumors.MethodsCD34+ cells isolated from pooled donor cord blood are the starting material for generation of myeloid cells. The process has three phases: (1) expansion of hematopoietic stem and progenitor cells, (2) continued expansion and differentiation into monocytes, and (3) differentiation of monocytes into mature macrophages. Cells are characterized before and after cryopreservation by immune phenotyping, morphology, and phagocytic capacity.ResultsThe current culture method produces several thousand HLA-DR+ CD11b+ myeloid cells at both the monocyte and macrophage stage per starting CD34+ cell. These cells make up ~70% of the final cell product along with a potentially beneficial heterogeneous mix of additional myeloid-derived cells. Immunophenotyping demonstrates expression of canonical monocyte/macrophage markers, including CD14, CD86, and CD163. Cell morphology after macrophage differentiation is similar to that of peripheral blood-derived mature adult macrophages. Importantly, the final cell product can be cryopreserved with excellent recovery of viable cells post-thaw that are functional, as demonstrated by maintenance of phagocytic potential when compared with pre-cryopreservation function.DiscussionThis proprietary CD34+ cell expansion and directed differentiation platform results in the generation of therapeutically relevant numbers of functional myeloid cells. Furthermore, the ability to cryopreserve these cells, with demonstrated viability and function upon thaw, facilitates their use as an off-the-shelf cell therapy, with potential for faster and less expensive routes to treatments for solid tumors that have proven resistant to other cell therapies. Additional work is being done toward engineering these myeloid cells to express proteins to enhance a patient’s anti-tumor immune responses, whether these cells are used alone or in combination with other therapeutics.

Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs

Mesenchymal stromal cells (MSCs) have been employed invitro to support hematopoietic stem and progenitor cell (HSPC) expansion and invivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential invitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.

An Interferon-γ/FLT3 Axis Positively Regulates Hematopoietic Progenitor Cell Expansion from Human Pluripotent Stem Cells.

Since it became possible to differentiate human pluripotent stem cells (hPSCs) into hematopoietic cells in vitro, great efforts have been made to obtain highly potent hematopoietic stem/progenitor cells (HSPCs) from hPSCs. Immunophenotypical HSPCs can be obtained from hPSCs, but their repopulating potential in vivo is low. Here, we developed a novel hematopoietic differentiation method for human-induced pluripotent stem cells (hiPSCs) to determine why the existing hPSC differentiation systems are inadequate. hiPSC-derived CD45+CD34+ cells in our system were mostly CD38- immunophenotypical HSPCs. The vast majority of human CD45+CD34+ cells in umbilical cord blood, fetal liver, and bone marrow are CD38+ hematopoietic progenitor cells (HPCs); therefore, the poor production of CD38+ HPCs was indicative of a systematic problem. hiPSC-derived CD45+CD34+ cells did not express FLT3, a receptor tyrosine kinase. Exogenous FLT3 activity significantly enhanced the production of CD38+ HPCs from hiPSCs. Thus, poor production of CD38+ HPCs was due to a lack of FLT3 expression. Interferon-γ upregulated expression of FLT3 and increased the number of CD38+ HPCs among hiPSC-derived CD45+CD34+ cells. These results suggest that the poor production of CD38+ HPCs with hPSC differentiation systems is due to a lack of FLT3 expression, and that the addition of interferon-γ can solve this problem.