Multipotent Progenitor Research Articles

Transcription Factor GATA2 Is Inevitable for the Survival and Proper Differentiation of Thymic Multipotent Progenitor Cells By Gene Expression Orchestration

Proper differentiation of T cells in the thymus is inevitable for the establishment of acquired immunity. Lymphoid-primed multipotent progenitors (LMPPs) emigrate from the bone marrow (BM) to the thymus for becoming earliest T cell progenitors, so-called early T cell progenitors (ETPs). Previous studies have shown that ETPs retain not only T cell but also innate immune cell (i.e., myeloid cells) differentiation potentials. Therefore, ETPs might be the bifurcation point of innate and adaptive immunity in thymus. However, still little is known about the mechanism by which the differentiation fates are determined in ETPs and the cellular heterogeneity in ETP subset. Since transcription factors that possess important roles in hematopoietic stem and progenitor cells (HSPCs) may play significant roles in the early differentiation of T cells as legacy stem cell genes, we sought to investigate the roles for GATA2, which is known to regulate the HSPC maintenance and myeloid differentiation, in ETPs. First, we revealed the GATA2 expression is preserved in ETPs and sharply disappear with their differentiation (GATA2 expressions were almost undetectable from double-negative 2 cells even by qPCR). To reveal the roles for GATA2 in T cell hematopoiesis, we analyzed Gata2 fl/fl; Mx1-Cre mice in which GATA2 was deletable in hematopoietic cells by polyI:C administration ( Gata2 KO). Gata2 KO mice showed a smaller thymus and a lower number of thymic T cells, including ETPs and double-positive (DP; CD4 +CD8 +) cells, than control. To rule out contributions of potential GATA2 deletions in non-hematopoietic cells, CD45.1 + wild-type unfractionated BM cells were transplanted into Gata2 KO or control mice. There were no particular differences in T cell hematopoiesis from CD45.1 + donor cells between them, supporting the notion that hematopoietic cell intrinsic GATA2 are inevitable for the proper T cell hematopoiesis. To further reveal the roles for GATA2 in ETPs, we performed global transcriptomic analysis of ETPs (RNA-seq; Gata2 KO vs control). Gene set enrichment analysis (GSEA) revealed that signatures associated with cell cycle regulation, hematopoietic progenitor cells and myeloid cells were down-regulated in Gata2 KO ETPs, suggesting that GATA2 is involved in the survival and myeloid differentiation potentials in ETPs. By taking the advantage of novel GATA2 reporter mice (GATA2-VENUS mice) in which GATA2 protein levels are detectable by flow cytometry, we revealed that the ETP fraction contained a mixture of cells with high or low GATA2 expression. To be noted, the protein expression intensity of GATA2 in GATA2-high ETPs was almost equivalent to that of LMPPs. These observations suggest that GATA2-high ETPs might be a thymic multipotent progenitors (T-MPPs), the earliest thymic progenitors in which T or myeloid cell differentiation commitment is defined. To elucidate the mechanism by which GATA2 regulates the survival and differentiation of T-MPPs, we integrated publicly available chromatin immunoprecipitation followed by sequencing data targeting GATA2 (GATA2 ChIP-seq) and our ETP RNA-seq data. We found several key genes in HSPCs maintenance (i.e. Lyl1) or myeloid differentiation (i.e. Itga2b) as potential targets of GATA2. Furthermore, we sought to reveal the effect of GATA2 deletion in progenitor cells to their progenies. To this end, we performed RNA-seq of DP cells ( Gata2 KO vs control). GSEA revealed that cell cycle related gene signature was repressed in Gata2 KO DP cells compared to control. To be noted, in CD4-Cre dependent GATA2 deletion model ( Gata2 fl/fl:CD4-Cre mice), there were almost no particular cell number and transcriptomic differences, which was also analyzed by RNA-seq, in DP cells compared to control. These results suggest that GATA2 in GATA2-high ETPs, but not in DP cells, may affect the feature of DP cells, potentially by epigenetic memory. Collectively, this study revealed that GATA2 contributes to T cell hematopoiesis. GATA2 expression was maintained in a part of the ETP subset (GATA2-high ETPs) which might be equivalent to T-MPPs. GATA2 may affect the differentiation trajectory and function from GATA2-high ETPs to their progenies. Further study of this mechanism will help us to understand how the acquired and innate immune bifurcation is defined, which might pave the way to overcome T cell development related disorders, such as T-cell/myeloid mixed-phenotype acute leukemia.

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

Mesenchymal Stromal Cells from Individuals with DNMT3A mut Clonal Hematopoiesis Instruct Young Healthy Hematopoietic Stem/Progenitor Cells Towards a Myeloid-Biased Aging Phenotype Resembling That of Patients with Clonal Cytopenia of Undetermined Significance (CCUS)

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion and variable risk of progression to myeloid malignancy. DNMT3A is the most commonly mutated gene and while single mutations do not raise the likelihood of progression, high variant allele frequencies (VAF) and presence of co-mutations do. In myeloid diseases, the bone marrow (BM) microenvironment exhibits alterations promoting malignant transformation. However, it is unknown whether similar alterations exist in the pre-malignant niche. We set out to analyze whether human mesenchymal stromal cells (MSC) from CHIP individuals with a DNMT3A mutation ( DNMT3A mut) are altered and whether they are able to remodel the HSPC compartment in healthy hematopoiesis. From our published cohort of previously sequenced BM samples from hematologically healthy individuals undergoing hip replacement surgery, we selected samples with either an isolated DNMT3A mut at a low VAF (2.47-6.8%, n=4, median age 73.5y, termed low-risk CHIP), or DNMT3A mut samples with a high VAF (17.5-28.5%) and at least one co-mutation (n=5, median age 73y, termed high-risk CHIP) as well as 12 age-matched samples without any mutation (n=12, median age 72y). HSPC were isolated from 3 younger healthy donors (median age 50y). MSC were expanded by plastic adherence and subjected to multilevel characterization. Compared to age-matched healthy controls, MSC from DNMT3A mut BM samples (CHIP-MSC) exhibited higher proliferation rates, lower colony-forming-unit (CFU) fibroblast capacity, as well as reduced adipogenic and increased osteogenic differentiation potential. To understand how altered CHIP-MSC influence the hematopoietic compartment, younger healthy CD34+ HSPC were co-cultured with the different MSC subgroups for 42 hours in a low cytokine-containing medium, followed by bulk RNAseq on subsequently sorted cell populations (MSC: CD45-/CD90+; HSPC: CD45+/CD34+). We found 31 differentially expressed genes (DEG) (logFC±0.5, FDR<0.05) in CHIP-MSC compared to their age-matched counterparts. Functional analyses suggested an activation of communication towards HSPC via extracellular vesicles. Strikingly, differential gene expression of sorted young healthy HSPC after co-culture revealed 375 DEG (322 up-regulated, 53 down-regulated; logFC±0.5, FDR<0.05) in HSPC cultured with CHIP compared to non-CHIP age-matched MSC (Figure panel A). We then used gene set variation analysis (GSVA) to analyze HSPC compartment composition after co-culture. Based on enrichment scores, unsupervised clustering identified two distinctive profiles that correlated with HSPC cultured with either non-CHIP or high-risk CHIP-MSC, while those cultured with low-risk CHIP-MSC showed an intermediate phenotype (Figure panel B). Co-culture of HSPC with high-risk CHIP-MSC resulted in enrichment of hematopoietic stem cells (HSC) and multipotent progenitors (MPP), as well as myeloid skewing represented by higher scores for common-myeloid (CMP) and granulocyte-monocyte (GMP) progenitors. Conversely, in contrast to the control group, megakaryocyte-erythroid progenitors (MEP) were underrepresented. CFU capacity was increased in healthy HSPC cultured with CHIP-MSC and multiparameter flow cytometry analysis showed a trend towards increased CMP/GMP and decreased MEP numbers, consistent with the RNAseq data. Finally, we evaluated the BM HSPC compartment composition of a cohort of 49 CCUS patients with DNMT3A mut (VAF 1-49%, median age 76y) compared to 56 control BM samples using whole transcriptome sequencing data. Intriguingly, we observed the same profile with a myeloid shift in progenitor populations as in our healthy young HSPC exposed to high-risk CHIP-MSC. Taken together, our results indicate that MSC from individuals harboring a DNMT3Amutation are able to instruct young healthy HSPC and influence their fate, thereby promoting a differentiation bias that shifts the hematopoietic compartment toward that associated with an aged pre-malignant state, even in the absence of DNMT3A mut HSPC. These findings suggest that focusing on the interaction between MSC and HSPC is a promising therapeutic strategy to intercept disease progression. Functional analysis of identified deregulated pathways in DNMT3Amut CHIP is ongoing.

Cellular and Molecular Analysis of Hematopoietic Stem and Progenitor Cells in Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an inherited multi-organ disorder with bone marrow failure (BMF), which may include neutropenia, anemia, and thrombocytopenia, as well as a high risk of myelodysplasia and leukemia. Hematological complications are common causes of morbidity and mortality in SDS. Around 90% of SDS patients have biallelic mutations in the Shwachman-Bodian-Diamond Syndrome ( SBDS) gene, which plays an important role in ribosome biogenesis and hematopoietic cell survival. In a collaborative study led by Dr. John Dick (Science 2016) novel hematopoietic progenitor subsets were identified (noted as F1, F2 and F3), of which the abundance, clonal capacity, and function in disease states remain unknown. We hypothesized that cellular and molecular analysis of hematopoietic stem and progenitor cells (HSPC) including these novel progenitors would provide valuable insights into the pathogenesis of BMF in SDS. Using multiparametric flow cytometry we quantified the following HSPC populations: hematopoietic stem cells (HSC), multipotent progenitors (MPP F1-F3), common myeloid progenitors (CMP F1-F3), megakaryocyte-erythroid progenitors (MEP F1-F3) and granulocyte-monocyte progenitor (GMP) in 13 SDS patients with SBDS mutations and 10 normal control donors. We found a significant reduction in CD34+/CD38- and CD34+/CD38+ cells in SDS patients. It is particularly noteworthy that the sub-populations of CMPs (F1 and F2), and MEPs (F2 and F3) were prominently decreased. These cells contribute to the production of myeloid, erythroid, and megakaryocytic cells with different proportions. Using single-cell colony-forming assays, we assessed the cloning efficiency of each HSPC, revealing that HSCs, MPPs and GMPs from SDS patients exhibited a significantly lower cloning efficiency compared to those from control subjects. Notably, the typical erythroid-committing progenitor cells (CMP F2, CMP F3, MEP F2, and MEP F3) in SDS collectively produced approximately 46% fewer erythroid cells per colony compared to control progenitors, while myeloid-committing progenitor cells (CMP F1, MEP F1) from these patients produced approximately 86% fewer myeloid cells, including neutrophils, per colony compared to the corresponding control cells. Further, HSCs and MPPs-F1 cells from SDS patients had a reduced cloning efficiency of approximately 22% and 27%, respectively, compared to control HSCs and MPP-F1 progenitors. These data suggest that multiple HSPC populations from SDS patients are characterized by reduced cell numbers, functional impairments, and dysregulation compared to normal subjects. Next, we performed transcriptome analysis of each HSPC by RNA sequencing. We revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS. Using KEGG pathway analysis, we found that downregulation of the ribosome pathway reached statistical significance in HSC, CMP F1 and MEP F3. Downregulation of the hematopoietic cell lineage pathway reached statistical significance in MEP F1-2 and MPP F1-2. Additionally, the TGF-beta signaling pathway was suppressed in CMP F1-3, MPP F1, and GMP, while the NF-kappa B signaling pathway, crucial for regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells, was also found to be decreased in GMP. Interrogation of specific dysregulate genes, we found that the hematopoietic transcription factor, ZFP36 (ZFP36 ring finger protein) which involved in cellular response to cytokines and growth factors, was significantly downregulated in five subpopulations of SDS HSPCs, particularly noticeable in the four progenitors (CMP F1-3, MEP F2, and GMP) which produced substantially fewer colonies in SDS patients compared to controls. Additionally, we observed downregulation of TGFB2 and upregulation of NFKBIA and NFKBIB may inhibit the NF-kappa B signaling pathway in SDS patients. These alterations have the potential to influence the hematopoietic cell lineage pathway, leading to BMF. Functional assays are ongoing and aim to assess the impact of these dysregulated genes on formation and behavior of HSPCs. In summary, our data suggest that multiple SDS HSPC populations are characterized by reduced cell numbers and functional impairments. As well, we revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS.

Acute Inflammation Redirect Bone Marrow Hematopoietic Stem Cell Cycling and Differentiation Program By Reshaping Their Chromatin Architecture at Long-Term

Background: Acute and persistent inflammation induced by microbial infections/sepsis or by therapeutic interventions, such as whole-body irradiation and CAR-T therapy, triggers a rapid overwhelming immune response in the host. While these responses are well defined in the short-term, the long-term impact of the inflammatory injury on the hematopoietic system still needs to be unveiled. Using a murine model of severe inflammation induced by bacteria P. aeruginosa ( P. aer.) or LPS, our group has previously reported that bone marrow (BM) hematopoietic stem cells (HSC) respond to infections with an aberrant expansion associated with their transitory inability to generate the downstream myeloid progenitors. Such dysfunctions correlated with the suppression of transcription factors governing stem-cell self-renewal and myeloid differentiation. Furthermore, when transplanted into healthy mice, the “inflamed” HSC showed reduced ability to contribute to the myeloid lineage and to engraft at long-term, suggesting that they retain memory of the LPS-mediated insult and maintain long-lasting changes. Thus, we hypothesized that severe inflammation alters the HSC epigenome, reshaping their transcriptional programs and affecting cell fate decisions and differentiation. Methods: We performed time-course experiments in WT mice challenged with a single dose of LPS or PBS, as control, and investigated 1) the kinetics of hematopoietic recovery by flow-cytometric immunophenotypic characterization, including cell-cycle 2) changes in the chromatin landscape of Lin- sorted BM subpopulations identified by the expression of the CD150 and CD48 markers as Long-Term (LT-HSC), Short-Term (ST-HSC), and Multipotent Progenitors (MPP1/2 and MPP3/4) using ATAC-seq. Results: Our studies revealed that the LPS challenge resulted in a significant increase in the cell-cycle of LT-HSC, ST-HSC, MPPs, common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP), which persisted up to 3 days from the inflammatory injury. Immunophenotypic analysis showed that while HSC frequencies returned to baseline at 5 days from LPS, the abundance and cell-cycle of myeloid progenitors and mature cells remained altered. Surprisingly, at 15 days post LPS, the cell-cycle of HSC markedly decreased, and such dysregulation was maintained until our last observation at 30 days from LPS. The pool of BM HSC was severely depleted and associated with exhaustion of T-cells but increased B-cells. GMP and Gr1+Mac1+ cells were also significantly decreased in the BM whereas mature lineages were increased in the peripheral blood, exhibiting high WBC, lymphocytes, and platelet count, neutrophilia, monocytosis, basophilia, and eosinophilia. ATAC-seq analysis revealed prominent differences in the chromatin architecture of the four HSC subpopulations at steady state (PBS). In all of them, LPS induced both i) a transient reorganization of the chromatin in some loci, and ii) the formation of persistently reshaped regions. Deeper investigations into the long-term changed sites revealed that chromatic accessibility was altered in proximity of CTCF-enriched regions fundamental to mediate chromatin reorganization and drive gene expression. Moreover, LPS modified the accessibility of genomic loci involved in the maintenance of HSC stemness and quiescence as well as regulation of their cell-cycle, self-renewal, and cell fate decision. Conclusions: Taken together these results suggest that LPS-mediated acute inflammation has long-lasting effects on HSC epigenetic regulation, influencing their cell-cycle and multilineage decisions. Relevance: The association between the resulting phenotype and the persistency of epigenetically remodeled HSC loci identified here may provide potential insights for future therapeutic approaches to re-direct “inflamed” HSC to a normal differentiation program, thereby enabling an efficient innate host response. Disclosures: No relevant conflicts of interest to declare.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Distinct Cellular, Transcriptional, and Mechanistic Properties Determine HSC Fate during Emergency Granulopoiesis

Emergency granulopoiesis (EG) is the process responsible for the rapid and enhanced production of granulocytes during acute infections. Traditionally, EG was understood as a cellular mechanism mostly initiated and supported by myeloid progenitors, however, recent data suggest that hematopoietic stem cells (HSCs) might also participate in the process. To investigate whether and how HSCs contribute to the initial steps of EG, we performed scRNA-seq and ATAC-seq analysis of sorted murine HSCs 4 hours after in vivo lipopolysaccharide (LPS) administration, mimicking an acute bacterial infection. Strikingly, we observed radical transcriptional changes between HSCs isolated from PBS control and LPS treated mice, mostly marked by alterations in HSC lineage bias. We identified a steady state lymphoid-biased subpopulation of HSCs, marked by expression of Procr (CD201). Following LPS stimulation, the CD201 expression was lost and this population was transcriptionally rewired to a myeloid-biased HSC population. Accordingly, ATACseq data corroborated opening of myeloid-bias loci upon LPS administration rather than steady-state lymphoid-biased loci. Further, we confirmed the loss of CD201 expression in HSCs by flow cytometry in mice after LPS and G-CSF treatment, as well as Candida albicans infection, suggesting that the loss of CD201 expression is a general event during EG induced by different stimuli. Interestingly, the CD201 downregulation in HSCs was independent of the master regulator of EG C/EBPβ, while we observed the contribution of the TLR4-MyD88 signaling axis. Next, we functionally validated our scRNA-seq results in vitro and in vivo. When cultivated under myeloid differentiation conditions, the CD201- HSCs gave rise to mature granulocytes, whereas CD201+ HSCs remained rather immature. In transplantation settings, CD201+ HSCs showed increased engraftment and preferential bias to produce lymphoid cells, while CD201- HSC showed decreased engraftment ability and a bias towards myeloid production. Surprisingly, LPS challenge of BM chimeras transplanted with lymphoid-biased CD201+ HSCs led to EG response marked by rapid loss of lymphoid-biased multipotent progenitors (MPPs) and the expansion of myeloid-biased MPPs, thus confirming the lympho-myeloid switch observed in the scRNA-seq data. Interestingly, the treatment of WT mice with CD201 blocking antibody was able to partially impair the lympho-myeloid switch, suggesting that CD201 plays an active role in HSCs during EG. Mechanistically, we observed that both CD201+ and CD201- HSCs rely on different signaling pathways under EG. While CD201+ HSCs express higher levels of TLR4 and have higher activation of NF-κB signaling upon in vitro LPS stimulation, the CD201- HSCs express higher levels of G-CSF-R and have higher activation of pSTAT3 signaling upon G-CSF-stimulation. Moreover, while the LIP isoform of C/EBPβ, important for cell proliferation, is present in both CD201+ and CD201- HSCs, the LAP/LAP* isoform, important for myeloid differentiation, is present only in the CD201- HSCs fraction. Altogether, our data suggest that EG is supported by a population of HSCs which upon pathogen sensing undergo a radical transcriptional rewiring that promotes their myeloid output. Initially, the pathogen is directly sensed by TLR4 on the surface of a steady state lymphoid-biased CD201+ HSCs, causing a rapid activation of the downstream NF-κB signaling pathway. Subsequently, the lymphoid-myeloid transcriptional switch, marked by the loss of CD201 expression, occurs and EG is then supported by myeloid-biased CD201- HSCs. CD201- HSCs respond to the infection in an indirect manner through G-CSF-R on their surface and exhibit enhanced pSTAT3 activation and elevated LAP/LAP* C/EBPβ isoform, cellular mechanisms known to promote myeloid differentiation and granulocytic production. In conclusion, the switch from CD201+ to CD201- HSCs facilitates both fast and sustained EG leading to the supply of new granulocytes to fight the infection. This detailed understanding of the distinct cellular, transcriptional, and mechanistic properties that determine HSC fate during emergency granulopoiesis opens new venues to potentially modulate granulocytic production in certain clinical conditions such as sepsis. This work was partially supported by a GACR 22-18300S, GAUK 327722, and IMG institutional funding RVO 68378050.

A Mouse Model of Zhu-Tokita-Takenouchi-Kim Syndrome Reveals Indispensable Son Functions in Myeloid-Lymphoid Lineage Balance and Terminal Differentiation

SON is a DNA- and RNA-binding protein mainly localized in nuclear speckles. Compromised SON function leads to aberrant and alternative RNA splicing, particularly for transcripts bearing weak splice sites. SON also binds to DNA and suppresses H3K4me3 modifications at transcription start sites by inhibiting the MLL1/2 methyltransferase complex. More recent studies suggest that SON serves as the core of nuclear speckles, and SON is required for the association between nuclear speckles and p53 target genes to boost RNA expression. Therefore, SON governs the expression of a myriad of target genes by regulating transcription and RNA splicing as well as nuclear speckle assembly, yet exerts its effects on specific sets of genes. However, the role of SON in organ development and hematopoiesis remains poorly understood. Recently, we identified that heterozygous loss-of-function (LoF) mutations in the SON gene cause a neurodevelopmental disorder with multi-organ anomalies in human patients. This syndrome has been designated as a novel rare disease named Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome. The ZTTK SON-Shine Foundation, a family-initiated non-profit organization, was launched, and through this foundation, we work with families to characterize clinical symptoms associated with SON LoF. Among the clinical symptoms observed in individuals with ZTTK syndrome, a noticeable feature is a wide range of hematological abnormalities, including bone marrow failure, high mean corpuscular volume (MCV), polycythemia, severe anemia, and thrombosis. Furthermore, immunoglobulin deficiency, poor responses to vaccines, and frequent infections were reported, implicating SON LoF in various hematopoietic abnormalities. To study how heterozygous LoF of SON affects organ development and causes clinical features associated with ZTTK syndrome, we created a mouse model of Son haploinsufficiency ( Son +/-). While complete knockout of Son ( Son -/-) led to embryonic lethality, Son +/- mice were viable and recapitulated many clinical features of human ZTTK syndrome patients. The Son +/- mice showed severe growth retardation, signs of cognitive impairment, skeletal abnormalities, kidney agenesis/hypoplasia, and, importantly, hematopoietic abnormalities, such as high MCV, low lymphocyte counts, and immunoglobulin deficiency. To examine the hematopoietic features found in ZTTK syndrome patients are due to SON LoF, we further examined hematopoiesis of our Son +/- mice. We found that Son +/- fetal liver as well as adult mouse bone marrow contain a reduced size of the early-stage hematopoietic stem and progenitor cell (HSPC) pool (marked as Lin-Sca1+cKit+: LSK). Analysis of LSK subpopulations revealed that Son +/- mice have myeloid-lymphoid lineage imbalance at the stage of multipotent progenitor (MPP), with increased megakaryocyte/erythroid lineage-biased MPPs (MPP2) while lymphoid lineage-biased MPPs (MPP4) are decreased. In addition, erythroid terminal differentiation and B cell maturation were impaired in Son +/- mice. Single-cell RNA-sequencing of mouse bone marrow Lin-cKit+ cells and transcriptome-based clustering also revealed that Son haploinsufficiency leads to a reduction in lymphoid lineage-committed progenitor cells and decreased expression of lymphoid lineage/B cell development genes, while erythroid lineage-promoting genes were aberrantly activated in myeloid lineage progenitors. Deep bulk RNA-sequencing and alternative splicing analysis identified that that Son haploinsufficiency caused alternative splicing of chromatin modifiers genes and epigenetic regulator genes. Furthermore, from our ATAC-sequencing analysis, we found that Son haploinsufficiency causes decreased chromatin accessibility in the gene loci critical for lymphoid lineage specification. Taken together, our Son +/- mouse model revealed that the normal level of Son is required to achieve the sufficient expression levels of the genes directing lymphoid lineage specification, B cell lineage development, and erythroid terminal differentiation. Our study demonstrates the indispensable roles of SON in multiple stages of hematopoiesis and provides valuable insight into the clinical significance of abnormal SON levels in hematopoietic diseases.

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of >1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR <0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p <0.05 was considered statistically significant. Results Cluster annotation revealed HPC, early and committed erythroid and MK, and myeloid cells (Figure 1). D7 T21/G1S HPCs surprisingly showed an erythroid bias, with upregulation of genes such as GYPA and AHSP, while euploid/G1S HPCs showed a MK bias, with upregulation of genes including PF4V1 and PF4. With wtGATA1, neither euploid nor T21 D7 HPCs showed a significant lineage skew. On D9 and D11, the euploid/WT, T21/WT, and T21/G1S cells developed subpopulations committing to the erythroid lineage, while the euploid/G1S cells demonstrated minimal erythroid potential (Figure 1). Across all 3 timepoints, early and committed erythrocytes from T21 and euploid cells with G1S had a greater number of combined DEGs than their wtGATA1 counterparts (G1S vs. WT, T21: 692 vs. 378; euploid: 1235 vs. 599). On D9, T21 was associated with a greater erythroid skew (38.6% of T21/WT and 32.2% of T21/G1S cells vs. 27.8% of euploid/WT cells; p <0.00001). By D11, this erythroid drive was no longer apparent, and the T21/G1S erythrocytes were significantly less mature (Table 1; p <0.00001). These data suggest that T21 initially enhances erythropoietic drive, while G1S results in a near-complete erythroid block in euploid cells but an incomplete developmental block with T21. All 4 genotypes yielded MK subpopulations on D9 and D11 (Figure 1). With wtGATA1, euploid and T21 MKs were predominantly early MKs by D11 (Table 1). Interestingly, G1S was associated with enhanced MK commitment and maturation in the euploid context but arrest at the early stage with T21 (Table 1; p <0.00001). G1S was again associated with increased DEGs compared to wtGATA1 (G1S vs. WT, T21: 594 vs. 454; euploid: 1689 vs. 1310). These data suggest that G1S in a euploid background promotes MK differentiation, but T21 and G1S cooperate to generate immature, abnormal MKs. Conclusions Although the hematopoietic abnormalities and leukemic risk of T21 have been well-described, the underlying developmental events remain unclear. This scRNA-seq timecourse of HPCs differentiated from isogenic iPSCs reveals the individual and synergistic effects of T21 and G1S. Overall, T21 enhances erythropoietic drive while G1S alone suppresses erythropoiesis. G1S in euploid cells enhances megakaryopoiesis, but with T21, megakaryopoietic commitment and maturation are uncoupled with a strong bias to early MKs. Together, the competing effects of T21 and G1S appear to result in abnormal, immature erythrocytes and MKs with impaired lineage commitment, consistent with a pro-leukemic phenotype.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

Reduction in Lymphoid-Supporting Factors By Mesenchymal Stromal Cells Drives Myeloid-Biased Hematopoiesis at Middle Age

The bone marrow “niche” for hematopoietic stem cells (HSCs) encompasses various cell types, extracellular matrices and secreted factors that maintain the specialized functions of HSCs such as quiescence, self-renewal, and production of multipotent progenitor cells. In old age, changes in this niche contribute to dysfunction of HSCs and the hematopoietic system including increased inflammation and myeloid-biased molecular programs. We have observed that HSC and hematopoietic aging phenotypes become apparent by mid-age (Young et al. Cell Stem Cell 2021, Young et al. J Exp Med 2016), leading to the hypothesis that evaluating alterations in the niche at this stage would reveal the earliest changes initiating HSC aging. To test this hypothesis, we examined both the hematopoietic and non-hematopoietic compartments in individual middle-aged (12-14 months of age, n = 9) and compared them to young C57BL/6 mice (2-4 months of age, n = 5). Immunostaining of femurs showed an increase in sinusoid volume, vessel dysmorphia, and an increase in megakaryocyte abundance in the bone marrow of middle-aged mice. No alterations were observed in arterioles nor adipocyte volume. This data provides supporting evidence that a subset of HSC niche remodeling phenotypes observed in old age are present by mid-age. We then performed single-cell RNA-seq to comprehensively assess changes in abundance and transcriptomes of hematopoietic and non-hematopoietic cells at mid-age. Analysis of this data revealed significant expansion of HSCs in middle-aged mice. In the expanded HSC population, a reduction in B lymphoid differentiation signatures was observed without alterations in inflammation or myeloid differentiation signatures. Computational predictions suggested that the reduced B lymphopoiesis program in middle-aged HSCs resulted from decreased interaction with soluble factors produced by subsets of mesenchymal stromal cells (MSCs), rather than endothelial cells or other hematopoietic cell compartments. Specifically, reduced MSC-to-HSC interactions were linked to a decline in signaling mediated by adiponectin (ADIPOQ; Adipoq), stem cell factor (SCF; Kitl), midkine (MDK; Mdk), and insulin-like growth factor 1 (IGF1; Igf1). Among these, reductions in Kitl and Igf1 in MSCs were correlated with the loss of B lymphopoiesis programs in HSCs comparing individual middle-aged mice. To test the functional impact of the decline in Igf1 in MSCs on lymphopoiesis, we created three MSC-selective Igf1 conditional knockout mouse models using distinct Cre recombinases (Lepr-Cre, nestin-CreER and Prx1-CreER). In all three of these models, MSC-selective knockout of Igf1 reduced B lymphopoiesis resulting in myeloid-biased hematopoiesis, replicating hematopoietic aging phenotypes observed at middle age. These findings indicate that the decline in B lymphopoiesis from HSCs during aging is initiated by a reduction in lymphoid-supporting factors produced by MSCs in the bone marrow microenvironment, and this occurs independently of inflammation or activation of myeloid-promoting transcriptional programs in HSCs.

Single-Cell Whole-Genome Sequencing after Chemotherapeutic Treatment Reveals Cell Type-Specific Mutational Burdens

Introduction The hematopoietic system is especially vulnerable to chemotherapeutic treatment-related long-term consequences, including clonal and malignant hematopoiesis. These diseases are caused by the acquisition of driver mutations in hematopoietic stem and progenitor cells (HSPCs). An increased rate of mutation accumulation, as identified in HSPCs of patients who receive chemotherapeutic treatment, can therefore result in disrupted blood production. Accordingly, childhood cancer survivors have a five-fold increased risk of secondary cancers, amongst which therapy-related myeloid neoplasms (t-MN) are one of the most prevalent. Here, we aim to increase the understanding of the origin of t-MN through the characterization of the short- and long-term mutational consequences of chemotherapy in different hematopoietic stem and progenitor cell types. Methods We studied the genome-wide mutation burden in the HSPC compartment at the time of an initial cancer diagnosis, following chemotherapeutic treatment, and upon a second cancer diagnosis. We performed whole-genome sequencing (WGS) of single HSPCs, including hematopoietic stem cells (HSCs, CD34+CD38-CD45RA-CD90+), multipotent progenitor cells (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitor cells (CMPs, CD34+CD38+CD45RA-), and granulocyte-monocyte progenitor cells (GMPs, CD34+CD38+CD45RA+). In order to obtain enough DNA for WGS, we performed clonal expansion of single cells or primary template-directed amplification of the DNA of one cell. Next, we normalized the mutation burden in each cell to the expected number of mutations (Osorio et al. Cell Reports 2018). In addition, we performed both whole genome and transcriptome sequencing of individual HSCs/MPPs. Results First, we investigated pediatric ten t-MN patients with a latency between the initial and second cancer diagnosis spanning 1-12 years. We applied WGS to clonally expanded HSPCs (CD34+CD38-CD45RA-) and found that the relative increase in mutation burden in HSPCs is restricted to the first five years after initial treatment. To gain more insight into the heterogeneous HSPC compartment, we subsequently investigated cell-type specific mutational loads based on immunophenotypic markers. So far, we have included three pediatric cancer patients with comparable treatment regimens and diagnoses, namely medium risk acute lymphoblastic leukemia. Here, we found that the mutation burden is lower in HSCs when compared to the progenitor cell types (Figure 1A). When comparing the mutation load in the HSCs to the linear mutation accumulation observed in HSCs and MPPs of healthy donors, we identified a fraction of cells overlapping with the expected mutation load based on the age of the patient (Figure 1B). These data suggest that HSCs are more protected against chemotherapy-induced mutagenesis than progenitor cells, although this protective effect varies between individual cells. Therefore, we also performed both whole genome and transcriptome sequencing from a single cell, to characterize the transcriptional state of HSCs/MPPs showing a relatively high or low mutation load within their respective cell type. Conclusions Our results indicate that primitive hematopoietic stem cells are more protected against chemotherapy-induced mutations than the more proliferative progenitor cells. In addition, we find that in t-MN patients with a long latency (>5 years), the mutation load in HSPCs returns to the expected baseline. Based on our data, we hypothesize that the more damaged progenitor cells may be replaced over time, fueled by the undamaged primitive stem cell pool. Thus, our results may explain why the risk of t-MN drops 5-7 years after treatment end.

Acquired microRNA-142 Deficit Drives Escape Mechanisms of Anti-Leukemic Surveillance during Blast Crisis Transformation of Chronic Myeloid Leukemia (CML)

CML may evolve from a chronic phase (CP) into blast crisis (BC), but the underlying mechanisms of the transformation remain to be fully elucidated. We reported that microRNA (miR)-142 is downregulated in BC compared with CP patients; and in a murine model of CP CML (i.e., BCR-ABL mouse), miR-142 knock-out (KO) induced a BC-like phenotype, substantiating a mechanistic role of miR-142 deficit in BC transformation. Mechanistically, miR-142 KO induced mitochondrial fusion and increased oxidative phosphorylation (OxPhos) in leukemic stem cells (LSCs), causing a shift of leukemic phenotype from CP to BC ( Nat Commun, in press). Herein, we report that miR-142 deficit also occurs in T lymphocytes of BC patients due to inflammatory cytokines that are aberrantly produced by the proliferating leukemic cells. Using the Mir142 −/−BCR-ABL mouse, we observed that miR-142 deficit resulted in loss of T cell number and activity, suppressed the antileukemic immune surveillance, and contributed to BC transformation. In fact, miR-142 KO hampered thymic lymphoid-primed multipotent progenitor (LMPP) differentiation into T cells and rendered mature T cells dysfunctional and exhausted, with increase of PD-1 levels, and decrease of the apoptotic threshold, cell cycling and cytokine production, via blockade of OxPhos/glycolysis switch that regulates the metabolism of otherwise activated T cells. These changes translated into a decrease of T-cell antileukemic surveillance as demonstrated by increased numbers of BC murine Lin -Sca-1 +c-Kit + (LSKs) or human CD34+ blasts cocultured with Mir142 −/−T-cells vs those cocultured with Mir142 +/+ T-cells. Furthermore, congenic B6 (lethally irradiated to eradicate host T cells) or immunodeficient NSG (no T cells) recipient mice transplanted with Mir142 −/−BCR-ABL LSK and Mir142 −/− T cells had reduced T cells (both: p<0.0001), increased blasts (B6: p=0.01; NSG: p<0.0001) and a shorter survival (median survival for B6: 58 days vs unreached, p=0.008; for NSG: 28 days vs unreached, p<0.0001) compared with the respective controls transplanted with Mir142 −/−BCR-ABL LSK and Mir142 +/+ T cells. Transplantation of Mir142 −/−BCR-ABL LSKs into Mir142 −/− or Mir142 flox(f)/fLck-cre+ (i.e., miR-142 KO only in T cells; Mir142T Δ/Δ) recipients also resulted in a shorter survival than Mir142 +/+ recipients (median survival for Mir142 −/− vs Mir142 +/+ recipients: 44 vs 53 days, p=0.001; for Mir142T Δ/Δ vs Mir142 +/+ recipients: 42 vs 53 days, p=0.0002). To correct miR-142 deficit, we produced a synthetic miR-142 mimic oligonucleotide (CpG-M-miR-142). We treated a cohort of NSG mice transplanted with Mir142 −/−BCR-ABL LSKs and Mir142 −/− T cells, with CpG-M-miR-142 (30mg/kg/day, IV) or CpG-scramble RNA (SCR) for 3 weeks. We observed increased T cells (p=0.04), decreased blasts (p=0.0029) and longer survival (median survival: unreached vs 37 days, p=0.0005) in CpG-M-miR-142-treated recipients vs SCR-treated controls. Similar results were observed in CpG-M-miR-142-treated Mir142 −/−BCR-ABL mice vs SCR-treated controls. To test CpG-M-miR-142 activity in human T cells, a cohort of BC CML patient-derived xenograft (PDX) mice were given patient's autologous T cells and CpG-M-miR-142. Mice receiving T+CpG-M-miR-142 had increased T cell expansion (p=0.04), reduced leukemic cells (p=0.0078), and longer survival (median survival: 53 vs 38 days, p=0.01) than controls receiving T+SCR. In secondary transplants, the recipients of BM from T+CpG-M-miR-142-treated donors survived longer (median survival: 73 vs 53 days, p<0.0001) than the recipients of BM from T+SCR-treated donors, suggesting a decrease in LSC burden. Finally, we tested the efficacy of tyrosine kinase inhibitors (TKIs), which represent the primary targeting approach for CML, in combination with CpG-M-miR-142 and/or PD-1 inhibitor in Mir142 −/−BCR-ABL mice. The mice treated with TKI+CpG-M-miR-142 (median survival: 107 days, p=0.001) or TKI+PD-1 inhibitor (104 days, p=0.002) survived longer than the controls treated with TKI alone (66 days); the triplet combination (TKI+CpG-M-miR-142+PD-1 inhibitor) is expected to improve survival further (ongoing experiment). In summary, CpG-M-miR-142 alone and in combination with TKI or PD-1 inhibitor rescued T-cell antileukemic activity and prolonged survival in BC CML murine and PDX models, thereby providing a potentially new therapeutic approach for BC CML.

CYP26 Activity Maintains the Bone Marrow Niche and Stem Cell Homeostasis

Vitamin A and its active compound, retinoic acid (RA), manifest pleomorphic activities in hematopoiesis. Vitamin A homeostasis is maintained systemically through hepatic CYP26. Emerging data suggests that CYP26 mediated RA metabolism also plays a role in controlling hematopoietic stem and progenitor cells (HSPCs) behavior (Ghiaur G et al. PNAS 2013, Schonberger K et al. Cell Stem Cell 2022). We generated Cyp26a1/ Cyp26b1 inducible knockout (CYP26KO) using ROSA26CreERT Cyp26a1/ Cyp26b1 conditional mice to study the impact on hematopoiesis and the bone marrow microenvironment (BME). Tamoxifen induces complete CYP26KO in these mice and subsequently higher retinoid levels and systemic inflammation (Snyder JM et al. FASEB J 2020). Here, we characterize the hematopoietic, bone and bone marrow (BM) phenotype of these mice. Compared to the CYP26 wild type controls (CTR), we observed leukocytosis and mild anemia as well as signs of extramedullary hematopoiesis in CYP26KO mice (Table). Spleen histology and flow cytometry showed an increased number of all mature elements, including megakaryocytes as well as increased absolute numbers of all hematopoietic progenitor populations (HPCs): LSK, CMP, GMP and MEP (Table). BM analysis showed decreased cellularity and myeloid biased in CYP26KO mice (Table). There was an increased frequency of colony forming unit cell (CFU-C) in the BM of CYP26KO mice but overall preserved absolute numbers when accounted for cellularity (Table). We observed increased frequency of LSK (Table) but decreased numbers of CMP, GMP and MEP in the BM of CYP26KO mice. Both CFU assay and flow cytometry showed myeloid biased HPCs found in the BM of CYP26KO mice (Table). When analyzing the stem cell compartment, short term stem cells and multipotent progenitor cells number were decreased in CYP26KO BM (p<0.01), but the long-term HSCs (LT-HSC) were relatively preserved (0.04 ± 0.01 vs. 0.17 ± 0.04 x10 6/femur, CTR vs CYP26KO, p<0.05). Both flow cytometry and CFU assay confirmed the presence of circulating HPCs in CYP26KO mice (Table). Thus, hematopoiesis of CYP26KO mice migrated away from the BM and into extramedullary tissues. The hematopoietic phenotype was most likely due to the cell extrinsic role of CYP26 since bone marrow transplantation experiments confirmed that a CYP26KO recipient (regardless of the genotype of the hematopoietic cells) and not CYP26 wild type recipient recapitulates the observed hematopoietic findings. This observation, together with necropsy findings of thin, frail bones in CYP26KO mice, prompted a closer look at the BM matrix/microenvironment of these mice. CYP26KO mice had smaller BM cavities, decreased trabecular bone and an increased fat/bone marrow volume ratio (Table). Consistent with these findings, CYP26KO mesenchymal stroma cells (MSC) had no osteoblastic differentiation potential in vitro but showed preserved adipogenic potential. The number of CFU-fibroblasts was decreased in the BM of CYP26KO mice but were found circulating in the PB and were dramatically increased in the spleen of these mice (Table). Single cell RNAseq showed quantitative and qualitative alterations in the BME populations with relative reduction of Cxcl12 abundant MSC2, which explained the qRT-PCR observation of lower levels of Cxcl12 mRNA in MSC isolated from CYP26KO mice. More so, Spp1 mRNA was downregulated in various MSC populations of CYP26KO bone marrow, consistent with their osteopenia and with potential implications for the observed HSPC phenotype. Receptor ligand analysis revealed that various components of the BME have impaired interactions with the extracellular matrix. Thus, the leading hypothesis is that absence of CYP26 leads to a dysregulated BME and to migration of a HSCs-niche unit out of the BM and into secondary hematopoietic organs. Interestingly, scRNAseq experiments uncovered upregulation of S100a8/9 in the CYP26KO neutrophil progenitors, which may be in response to systemic stress. The observed bone alterations (osteopenia and increased BM fat) coupled with noted changes in hematopoiesis (i.e. myeloid bias and inflammatory stress) may represent early signs of aging of the bone and bone marrow and suggest a central role of retinoid homeostasis in this process.

Identifying Stress Granules As Determinants of Leukemia Stem Cell Maintenance and Stress Adaptation

The development of novel leukemic stem cell (LSC)-targeted therapeutics, which spare hematopoietic stem cells (HSC), is an urgent goal towards reducing the high, LSC-driven, relapse rates in acute myeloid leukemia (AML) patients. Towards elucidating key post-transcriptional networks underlying LSC maintenance we previously performed an in vivo pooled CRISPR dropout screen targeting 128 RNA binding proteins (RBPs) highly expressed in AML LSCs while having low expression in HSCs. This screen highlighted 12 RBPs crucial for murine AML LSC-mediated leukemic growth, of which there was a significant enrichment of stress granule (SG) RBPs. Further, gene set enrichment analysis shows the SG proteome is enriched in human AML LSCs vs non-LSCs while normal HSCs have reduced SG RBP expression relative to multipotent progenitor cells. SGs are dynamic biomolecular condensates, composed of ribonucleoprotein complexes, which may form as a protective mechanism against a variety of stress conditions. SGs are gaining increasing attention as contributors to cancer metastasis and chemoresistance, but their role in LSCs is virtually unexplored. The intriguing enrichment of SG RBPs in our screen hints at a larger role for SGs in LSC maintenance and/or stress resistance. To address this, we focused on the core SG nucleation RBP, G3BP1, a molecular switch that guides SG assembly. Fluorescence microscopy was first performed to describe SG dynamics in leukemia vs primitive normal cells where interestingly, a subset of patient AML cells contained G3BP1 SGs in the absence of added stress. In contrast, primitive healthy murine stem cells had reduced heat shock-induced SG formation in comparison to downstream normal progenitors. A series of shG3BP1 knockdown experiments performed in human leukemia cell lines and patient AML specimens demonstrated that shG3BP1-transduced cells showed increased apoptosis and differentiation and reduced colony forming capacity and competition in vitro compared to control cells. The same set of assays showed minimal impact of SG impairment to healthy umbilical cord blood cells. Importantly, in vivo xenotransplantation demonstrated that LSCs with G3BP1 knocked down were significantly compromised in their capacity to generate leukemic grafts whereas normal HSCs assessed in a similar manner display much less G3BP1 dependency. Towards determination of the SG mechanisms essential for bulk AML and LSC regulation, we performed matched RNA-sequencing and proteomics in G3BP1 knocked down AML cells as well as BioID in AML cell lines, primary patient AML cell subsets, and healthy hematopoietic cells to allow for the uncovering of an AML specific SG proteome. These analyses highlighted numerous potential SG mechanisms of cancer cell stress resistance and/or stem cell regulatory processes including sequestration of apoptotic factors and oncogenic signalling pathways. Of note, RNA-seq and proteomics in SG impaired shG3BP1 leukemia cells showed both a negative enrichment of the SG proteome and LSC signature as well as a positive enrichment of the innate immune response. Interestingly, our BioID uncovered that the AML specific SG proteome is also enriched for positive regulation of RIG-I signaling pathway (GO: Biological Process). Together this data suggests SGs play a protective role through the downregulation of overactive innate immune signalling in AML. Work using optimized small input approaches to map SG RNA and protein interactomes in LSCs vs HSCs to confirm similar regulatory relationships as well as identify LSC-context specific ones is ongoing. Overall, these results elucidate SGs as key AML LSC stress adaptive regulatory networks and highlights them as novel targets for consideration in AML therapeutics.

TNFα-Driven Interplay between Hematopoietic Stem Cells and Antigen-Specific Cytotoxic T Cells

Clonal hematopoiesis (CH) is governed by aberrant hematopoietic stem cell (HSC) clones with leukemia-associated somatic mutations that arise spontaneously during organismal aging or after exposure to genotoxic insults. Cells with a somatic mutation can be eliminated through presentation of neoantigens on MHC class I (MHC-I) to antigen-specific cytotoxic T cells. Indeed, many of the CH-related mutations, such as JAK2V617F, are reported or predicted to elicit antigen-specific cytotoxic T cell response. On the other hand, accumulating evidence suggests that inflammatory environment facilitates clonal expansion of abnormal HSCs. However, much remains to be elucidated on whether HSCs expressing such non-self peptides can be eliminated by antigen-specific cytotoxic T cells and how inflammation affects the T cell-mediated HSC regulation. Our RNA-seq analyses revealed that genes involved in MHC-I-dependent antigen presentations, such as Nlrc5, H2-kb and B2m, are most abundantly expressed in HSCs (Lin -/c-Kit +/Sca-1 +/Flk2 -/CD48 -/CD150 +) among murine bone marrow immature hematopoietic cells. Using transgenic mice that ubiquitously express a model antigen ovalbumin (OVA) and a monoclonal antibody that specifically recognizes MHC-I-OVA complex, we found that while MHC-I protein is abundantly expressed on the surface of hematopoietic cells except granulocytes, HSCs and multipotent progenitor cells (MPPs) possess greater capacity to present intracellular antigens through MHC-I compared to myeloid progenitor and mature cells. Remarkably, in vitro co-culture experiments revealed that whereas OVA-expressing (OVA +) HSCs, MPPs and myeloid progenitors could directly activate OVA-specific OT-I CD8 + T cells through MHC-I-dependent antigen presentation, HSCs are the most susceptible population to the killing effect by cytotoxic T cells. Indeed, co-infused OT-I CD8 + T cells totally abolished the long-term repopulation capacity of OVA + HSCs in a serial transplantation setting, and adoptive transfer of OT-I CD8 + T cells to mixed chimeric mice that harbor both wild-type and OVA + hematopoietic cells completely and specifically eliminated hematopoiesis governed by OVA + HSCs. TNFα is not only a prototypical inflammatory cytokine but also one of the major cytokines that are secreted by CD8 + cytotoxic T cells. Notably, genes directly involved in T cell regulation including Cxcl9, Cd274, and Pdcd1lg2 were induced in HSCs, but not myeloid progenitor cells, upon exposure to TNFα (Yamashita and Passegué, Cell Stem Cell 2019). Indeed, upon co-culturing OT-I CD8 + T cells produced TNFα as well as IFNγ and Granzyme B and OVA + HSCs but not myeloid progenitor cells produced CXCL9 largely in a TNFα receptor-dependent manner. Interestingly, such HSC response to T cell-derived TNFα was critical for naïve CD8 + T cells to rapidly differentiate to effector cytotoxic T cells, as TNFα receptor-deficient ( Tnfr1 -/-Tnfr2 -/-) OVA + HSCs stimulated proliferation and cytokine production of OT-I CD8 + T cells but failed to stimulate Granzyme B production, whereas IFNγ receptor deficiency ( Ifngr -/-) did not have such effect. As a result, Tnfr1 -/-Tnfr2 -/- OVA + HSCs, but not Ifngr -/- OVA + HSCs, could escape from the killing by OT-I CD8 +T cells. In sharp contrast, prior exposure to environmental TNFα, but not to IL-1β or IL-6, before interaction with OT-I CD8 + T cells rendered OVA + HSCs resistant to the killing by OT-I CD8 + T cells largely through upregulation of PD-L1 and PD-L2. Of note, upon 24-month aging or acquisition of Jak2V617F mutation, OVA + HSCs still maintained robust MHC-I-dependent antigen presentation activity and high susceptibility to the killing by OT-I CD8 + T cells, though prior exposure to TNFα allowed for their immune escape. Consequently, adoptive transfer of OT-I CD8 + T cells completely and specifically eliminated Jak2V617F-mutant OVA + HSCs and reversed myeloproliferative neoplasms in the mixed chimeric mice. Taken together, our results reveal the robustness of HSC quality control via MHC-I-dependent antigen presentation to antigen-specific cytotoxic T cells. They also highlight a critical but complex role of TNFα in active interplay between HSCs and antigen-specific cytotoxic T cells as well as immune evasion by HSCs during inflammation. Specifically targeting abnormal HSCs by antigen-specific cytotoxic T cells may pave the way to eradicate mutant HSC clones and prevent CH-associated disease.