Lymphoid-primed Multipotent Progenitors Research Articles

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Distinct roles of hematopoietic cytokines in the regulation of leukemia stem cells in murine MLL-AF9 leukemia

Lymphoid-primed multipotent progenitor (LMPP)-like and granulocyte-monocyte progenitor (GMP)-like leukemia stem cells (LSCs) co-exist in the blood of most patients with acute myeloid leukemia (AML). Complete elimination of both types of LSCs is required to cure AML. Using an MLL-AF9-induced murine AML model, we studied the role of hematopoietic cytokines in the survival of LMPP- and GMP-like LSCs. We found that SCF or FLT3L promotes the survival of LMPP-like LSCs by stimulating Stat5-mediated Mcl1 expression, whereas interleukin-3 (IL-3) or IL-6 induces the survival of GMP-like LSCs by stimulating Stat3/nuclear factor κB (NF-κB)-mediated Bcl2 expression. Functional study demonstrated that, compared to AML cells cultured in IL-3 and IL-6 medium, AML cells in SCF- or Flt3L-only culture are highly clonogenic in invitro culture and are highly leukemogenic invivo. Our study suggests that co-inhibition of both STAT5-MCL1 and STAT3/NF-κB-BCL2 signaling might represent an improved treatment strategy against AML, specifically AML cases with a monocytic phenotype and/or FLT3 mutations.

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.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

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.

Identification of Cell Type-Specific Effects of DNMT3A Mutations on Relapse in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by distinctive mutations in individual patients; therefore, each patient may display different cell-type compositions. Although most patients with AML achieve complete remission (CR) through intensive chemotherapy, the likelihood of relapse remains high. Several studies have attempted to characterize the genetic and cellular heterogeneity of AML; however, our understanding of the cellular heterogeneity of AML remains limited. In this study, we performed single-cell RNA sequencing (scRNAseq) of bone marrow-derived mononuclear cells obtained from same patients at different AML stages (diagnosis, CR, and relapse). We found that hematopoietic stem cells (HSCs) at diagnosis were abnormal compared to normal HSCs. By improving the detection of the DNMT3A R882 mutation with targeted scRNAseq, we identified that DNMT3A-mutant cells that mainly remained were granulocyte-monocyte progenitors (GMPs) or lymphoid-primed multipotential progenitors (LMPPs) from CR to relapse and that DNMT3A-mutant cells have gene signatures related to AML and leukemic cells. Copy number variation analysis at the single-cell level indicated that the cell type that possesses DNMT3A mutations is an important factor in AML relapse and that GMP and LMPP cells can affect relapse in patients with AML. This study advances our understanding of the role of DNMT3A in AML relapse and our approach can be applied to predict treatment outcomes.

Abstract A23: Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes

Abstract Malignant hematopoietic stem cells (HSCs) initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to all non-transplant therapies. As a result, allogeneic stem cell transplantation (allo-SCT) is the only curative therapy for MDS. However, relapse remains the leading cause of treatment failure after allo-SCT. Improved tools to predict relapse in this setting are necessary to allow for pre-emptive therapeutic interventions, which may be more effective when the burden of disease is still low. Given prior evidence that HSCs serve as the functional unit of resistance to non-transplant therapies, we asked whether relapse after transplant originates from MDS HSCs, and whether their persistence can predict relapse. To overcome the limiting number of HSCs available in clinical bone marrow (BM) samples, we adapted a workflow developed for assessment of cell-free DNA and utilized a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy-associated genes. Unique dual index primers (UDIs) were utilized to tag input DNA, improving PCR duplicate identification, which overcome issues with amplification bias typically faced by low-input sequencing. This ultimately allowed us to consistently capture evaluable read depths from as few as 10 sorted cells. We assembled an initial cohort of 21 patients who underwent allo-SCT for MDS or secondary acute myeloid leukemia. Inclusion criteria were follow-up time greater than 1.5 years (or relapse in this timeframe), BM sampling prior to Day+120 post-transplant and relapse, and biospecimens allowing for sorting of greater than 10 HSCs. We sequenced sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38- CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors, as well as bulk BM. With a median follow-up of two years, 9 of the 21 patients relapsed, with an average time to relapse of 362 days. Detection of disease mutations in HSCs/MPPs in any BM at D+30-D+120 post-transplant was 100% specific and 88% sensitive for relapse, showing improvements in both metrics over bulk BM assessment (22% sensitive and 66% specific), despite far lower cell input and read depth. Average time from detection to relapse was 6.9 months. In patients destined to relapse, disease mutations are often present in HSCs at a variant allele frequency of greater than 45%, while simultaneously undetectable on high-depth sequencing of bulk cells (>400x coverage). While even higher depth error-corrected sequencing bulk BM could likely detect such mutations, it would require >20x the sequencing depth, and could not identify the involved cell populations. In conclusion, we show for the first time that relapse of MDS after allo-SCT is driven by failure to eradicate MDS HSCs. Detection of MDS HSCs early post-transplant is highly predictive for relapse and opens new doors for pre-emptive interventions to prevent relapse. These findings are being validated in an independent cohort of 46 patients and updated data will be presented at the meeting. Citation Format: Benjamin Kroger, Yi Huang, Prapti Patel, Aimaz Afrough, Gurbakhash Kaur, Larry Anderson, Farrukh Awan, Praveen Ramakrishnan, Kunal Verma, Russell Vittrup, Robert H Collins, Madhuri Vusirikala, Yazan F Madanat, David Sallman, Carmelo Gurnari, Jaroslaw Maciejewski, Roni Tamari, Stephen Chung. Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A23.

Epigenetic programming defines haematopoietic stem cell fate restriction.

Haematopoietic stem cells (HSCs) are multipotent, but individual HSCs can show restricted lineage output in vivo. Currently, the molecular mechanisms and physiological role of HSC fate restriction remain unknown. Here we show that lymphoid fate is epigenetically but not transcriptionally primed in HSCs. In multi-lineage HSCs that produce lymphocytes, lymphoid-specific upstream regulatory elements (LymUREs) but not promoters are preferentially accessible compared with platelet-biased HSCs that do not produce lymphoid cell types, providing transcriptionally silent lymphoid lineage priming. Runx3 is preferentially expressed in multi-lineage HSCs, and reinstating Runx3 expression increases LymURE accessibility and lymphoid-primed multipotent progenitor 4 (MPP4) output in old, platelet-biased HSCs. In contrast, platelet-biased HSCs show elevated levels of epigenetic platelet-lineage priming and give rise to MPP2 progenitors with molecular platelet bias. These MPP2 progenitors generate platelets with faster kinetics and through a more direct cellular pathway compared with MPP2s derived from multi-lineage HSCs. Epigenetic programming therefore predicts both fate restriction and differentiation kinetics in HSCs.

Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

Single-Cell Multiomics Reveal Hypomethylating Agent Partially Restores Hematopoiesis in a LR-MDS Patient at the Cost of Leukemic Transformation

Background Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases characterized by ineffective hematopoiesis. Hypomethylating agents (HMA) such as decitabine and azacytidine (AZA) induce clinical responses in both lower-risk (LR) and higher-risk groups; however, the treatment eventually fails and can be followed by leukemic transformation. The mechanism underlying transient clinical benefit from low-dose HMA (restoration of hematopoiesis) is not fully understood. Here we used cutting-edge single-cell multi-omic approaches to describe the abnormal clonal architecture and transcriptome in LR-MDS and how they are altered following HMA therapy. Results First, we defined genomic differences between aged and young healthy hematopoietic stem and progenitor cells (HSCPs) by building a single-cell atlas of human HSCP with molecularly-defined concentrations of relevant CITE seq antibodies. 350k high-quality bone marrow nucleated cells were obtained from four young donors; varying race and gender. A 6-fold molecular titration of 278 CITE-seq antibodies determined those which are functional and which contribute to clustering. Next, we repeated the captures on samples from aged healthy people (n=3), MDS patients (n=4) and conducted differential analysis on cell states and transcriptomes. Compared to young marrow, healthy marrow from elderly donors revealed global upregulation of inflammatory signals and innate immunity. In erythroid progenitors (ERP) we identified abnormally high expression of the transcription factors KLF1 and GATA1, but lower than normal expression of their hemoglobin target genes. At the same time altered erythropoiesis in the elderly appeared to be compensated by increased frequency of multilineage progenitors and more mature erythroblasts mitigating clinical anemia. Comparing MDS to aged marrow, while most cell populations from MDS showed significant gene expression changes, the amplitude was dwarfed by abnormal mRNA processing gene expression in ERP and erythroblasts. Moreover, in de novo LR-MDS samples and an anemia sample (without known mutations) further downregulation of hemoglobin genes in the erythroid lineage was accompanied by decreased erythroblast numbers leading to anemia. To study the impact of AZA on LR-MDS, we longitudinally profiled a single MDS patient (pre-HMA, post-HMA, sAML transformation, cycle 2 VEN+AZA) (Panel 1). We found that 6 cycles of AZA treatment depleted HSC; whereas lymphoid-primed multipotential progenitors (LMPP), megakaryocyte-erythroid progenitors (MEP), and less mature erythroblasts were enriched after AZA therapy (Panel 2 left). Using genotyping of the transcriptome (GoT) (Panel 2 right), we defined cells with mutations to find that not only SRSF2-relevant pathways (eg. mRNA processing, and mRNA splicing) are molecularly corrected (downregulated) but also there are fewer mutant cells among primitive stem cells and lineage-committed progenitors. However, mutant ERP and erythroblasts accumulated and pathways were more active suggesting the blockade of differentiation at terminal erythropoiesis is not rescued, consistent with the clinical observation that the patient was still RBC transfusion-dependent despite platelet recovery. Notably, these mutant erythroid cells, while defective, never transformed to leukemia. The expansion of mutant multilineage progenitors and LMPP preceded AML transformation and were eliminated (as evidenced by flow and CITE-seq) following two cycles of VEN+AZA. Conclusions: Age-matched references are critical in differential analysis of hematological diseases like AML or MDS. With matched elderly donor data sets, we were able to molecularly isolate functional disruption of erythropoiesis and megakaryopoiesis underlying anemia and thrombocytopenia, respectively, observed in an LR-MDS patient. AZA rescued the dysregulated signals in SRSF2-mutated LR-MDS patient HSPCs, but not in ERP and erythroblasts, and therefore only partially restored hematopoiesis. However, as AZA drove differentiation of both wildtype and mutant HSCP, accumulation of mutant multilineage progenitors and LMPPs contributed to progression to AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Leukaemia and the CNS Niche: Insights on Bidirectional Communication from a Model of Infant Leukaemia

Infant leukaemia, an acute leukaemia within the first year of life, is a rare, aggressive entity with a poor response to intensive therapy. MLL-AF4+ infant leukaemia, the most common genetic subtype, has been shown to have: an in-utero origin, a lack of co-driver mutations, and lineage plasticity. Central nervous system (CNS) disease, typically a leukaemic infiltrate of the leptomeninges, is particularly common in these patients compared with non-infant paediatric ALL, and presents a serious clinical problem. With increasing understanding of the diverse immune cell landscape in the meninges, we sought to use our recently developed fully murine infant leukaemia model to explore the interactions between leukaemia and this microenvironment. We previously identified two microRNAs (miR128a and miR130b) that are upregulated in MLL-AF4+ infant and paediatric leukaemia patient samples. Overexpression of these microRNAs individually in mouse foetal liver (FL) Lin-Sca1+cKit+ (LSK) cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll-AF4 with prominent CNS infiltration (Malouf et al. Blood 2021). To identify potential niche-specific leukaemia-propagating cells in our Mll-AF4+ mir128a+ infant proB-ALL model, we performed serial transplants using sorted leukaemia progenitor populations (LSK IL7R+ and Lin-Sca1-cKit+ (LK/CLP)) from mice that had previously succumbed to leukaemia. We observed that these leukaemia progenitors were functionally identical with indistinguishable output. This suggests the leukaemia-initiating cells in this model can emerge from different cell populations, consistent with our current understanding of ALL-initiating cells in human disease. CNS leukaemia developed in these mice with features that also mirror human disease: an immunophenotypically polyclonal infiltrate of the leptomeninges, sparing the brain parenchyma. To complement the functional data, we transcriptionally validated the infant proB-ALL model for two key features: foetal cell-of-origin and infant-specific expression signatures. Published RNA sequencing of murine FL and adult bone marrow (BM) lymphoid-primed multipotent progenitors (LMPP) - a putative cell of origin for infant leukaemia - were reanalysed (Symeonidou et al. Cell Reports 2021 data set). BM-derived leukaemia cells from our model are more similar to FL than adult BM LMPPs. Using a published MLL-AF4+ patient leukaemia data set we showed that cells from our model are also transcriptionally closer to infant than non-infant paediatric MLL-AF4+ ALL patient samples (Figure 1)(Andersson et al. Nature Genetics 2015 data set). Transcriptomic differences were identified by RNA sequencing of leukaemia progenitors from the BM and CNS niche. Statistically significant Gene Ontology terms of particular interest based on these differences are summarised in Figure 2. Genes upregulated in CNS-derived cells included cell adhesion molecules, immune modulators and cell growth factor receptors. These suggest a CNS-specific adaptive response to the microenvironment in a bidirectional manner. These novel observations may represent infant leukaemia-specific adaptations or more generic CNS leukaemia interactions uncovered through the use of an immunocompetent fully murine disease model. In either scenario, the implications of bidirectional communication between leukaemia and the CNS niche are significant for the development of much-needed novel biomarkers and therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluating Stem Cells to Predict Post-Transplant Relapse in the Myelodysplastic Syndromes

Malignant hematopoietic stem cells (HSCs) both initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to frontline non-transplant therapies. Consequently, allogeneic transplant is the only curative therapy, but relapse can still occur. While post-transplant relapse is hypothesized to be driven by persistent MDS HSCs, their detection has proven challenging due to their limiting frequency in biospecimens. The accuracy of sequencing low-input specimens is limited by allelic drop-out and amplification bias. We adapted a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy associated genes that incorporates unique molecular identifiers capable of retaining read depths of >12x from as few as 19 sorted HSCs. We aimed to characterize residual disease after transplant with greater resolution than previously possible by sequencing sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38-CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors. We applied our technique to an MDS case characterized by bi-allelic TP53 mutations (Y220C and P75fs), assessing a bone marrow (BM) specimen D+168 after transplant, which was five months prior to clinical relapse. Both mutations were present at variant allelic frequencies (VAFs) of 8% and 4% in 258 sorted CD34+CD38- cells with 105x coverage (xc), but simultaneously undetectable in bulk BM (481xc) and CD34+CD38+ progenitor cells (239xc) despite greater cell number and read depth. We next evaluated a D+179 post-transplant BM from a case of MDS harboring ASXL1 (R713G), DNMT3A (R882H), and IDH2 (R140Q) mutations. All three mutations were detectable in 102 sorted HSCs/MPPs at VAFs of 20% (92xc), 13% (68xc), and 35% (89xc), respectively, while absent in bulk BM (369xc) and CD34+CD38+ progenitors (506xc). These findings preceded clinical relapse by nearly two years. We additionally investigated pre- and post-transplant BM samples of a patient with a germline DDX41 M1I mutation treated for MDS. In such cases, a second somatic DDX41 mutation is often acquired, impairing ribosomal biogenesis. This mutation is always present at a low VAF, and it is unclear how it promotes disease. Prior to transplant, we identified a somatic DDX41 R525H mutation at a VAF of 2.6% in bulk BM. However, this mutation was present at a VAF of 45% in sorted HSCs/MPPs, revealing the pervasiveness of this "minor clone” at the apex of the hematopoietic hierarchy (Fig. 1). Additionally, two previously unknown TP53 clones were discovered at VAFs of 22% (K132R) and 1.8% (C275F) in HSCs/MPPs, and these were undetectable in bulk BM. At D+102 post-transplant, we detected persistence of both M1I (VAF 43%) and R52H (VAF 27%) DDX41 mutations in 19 sorted HSCs/MPPs (12xc) 78 days prior to relapse. These data suggest that while impaired ribosomal biogenesis in HSCs with bi-allelic DDX41 mutations prevents effective hematopoiesis, it also increases self-renewal and accumulation of HSCs. Next, we sought to test if early identification of residual MDS HSCs post-transplant is predictive of disease relapse. We evaluated a cohort of patients who underwent myeloablative allogeneic transplant for MDS at UTSW and MSKCC (n=21). We evaluated BMs available from post-transplant dates ranging from D+30 to D+120, including any specimen from which we were able to sort at least 15 HSCs/MPPs and achieve an effective read depth of >10. We censored specimens with follow-up of less than one year. With a median follow-up of two years, detection of disease mutations in HSCs/MPPs was 100% specific and 88% sensitive for relapse (Fig. 2). The average time from detection of disease in HSCs to clinical relapse was 6.9 months. Detection in CD38+CD34+ progenitors was also 100% specific but only 67% sensitive for relapse. Assessment of mutations in bulk BM (mean 529xc) was only 22% sensitive and 50% specific for relapse. In conclusion, our research shows for the first time that relapse of MDS after allogeneic transplant is driven by failure to eradicate MDS HSCs, and that detection of MDS HSCs early after transplant is highly predictive for relapse. Validation of this assay in larger cohorts promises to yield a tool to identify patients who may benefit from early post-transplant interventions to forestall relapse. We also demonstrate how this assay can reveal novel insights into human disease HSC biology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BPDCN MYB Fusions Bind Cell Cycle Gene Promoters, Reactivate Self-Renewal in Progenitor Cells and Induce Myeloid/Dendritic Leukemia

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare acute leukemia with poor prognosis arising from the plasmacytoid dendritic cell (pDC) lineage. MYB is a hematopoietic transcription factor overexpressed in many leukemias. Genomic rearrangements of MYB were identified in a significant fraction of BPDCNs, including in cases without any other clear driver mutations, and result in its fusion to one of several partner genes (Suzuki et al., Leukemia 2017). These fusions are rare or absent in other acute leukemias and the mechanisms by which they contribute to BPDCN oncogenesis are not understood. To examine the impact of Myb fusions on hematopoietic differentiation and leukemogenesis, we used mouse bone marrow progenitors immortalized by estradiol (E2)-dependent activation of HoxB8 and culture with Flt3 ligand (HoxB8-FL cells). We used HoxB8-FL cells deficient in Cdkn2a, since CDKN2A deletion is present in up to ~67% of BPDCNs (Lucioni et al., Blood 2011) but is rare in other myeloid leukemias. HoxB8-FL cells differentiate into myeloid, lymphoid and dendritic cells in vitro upon withdrawal of E2, or in vivo upon transplantation. They therefore represent lymphoid-primed multipotent progenitor (LMPP)-like cells that are unable to self-renew in the absence of E2. We co-expressed a dTom reporter with V5-tagged Myb constructs in HoxB8-FL cells: wild-type full-length Myb (Myb-FL), truncated Myb (Myb-TR) or Myb-PLEKHO1. MYB-PLEKHO1 is the most frequent fusion in BPDCN patients, while truncated Myb is analogous to recurrent BPDCN MYB fusions in which the partner is out-of-frame. Empty vector was used as a control. HoxB8-FL cells expressing Myb-FL, Myb-TR or Myb-PLEKHO1 displayed arrested differentiation upon withdrawal of E2 in vitro (Figure 1). While all empty vector-transduced cells had upregulated CD11b and/or CD11c at day 7 post-E2 withdrawal, Myb overexpression caused an accumulation of CD11b-CD11c- undifferentiated cells. In vivo, a similar effect was observed at day 7 post-transplantation: dTom+ CD11b-CD11c-CD19-B220- undifferentiated cells were still present in the bone marrow of Myb-FL, Myb-TR and Myb-PLEKHO1 recipient mice, while in empty vector recipients all dTom+ cells expressed at least one of these differentiation markers. Surprisingly, we observed long-term persistence of dTom+ cells in the peripheral blood of Myb-TR and Myb-PLEKHO1, but not Myb-FL, recipients. This developed into lethal malignancy in 100% of Myb-TR and Myb-PLEKHO1 recipients by 28 weeks (Figure 2). Malignant cells showed blast-like morphology of acute leukemia and expressed markers of a myeloid/dendritic progenitor (Kit+CD11b+Cx3cr1+). dTom+ disease was never observed in Myb-FL or empty vector recipients (log-rank P<0.001). Together, while expression of either full-length Myb or Myb fusions impaired myeloid/dendritic differentiation, only Myb rearrangements associated with BPDCN generated a fully penetrant leukemia. To investigate chromatin-level differences between physiologically expressed wild-type MYB and MYB fusions, we performed Cut&Run in K562 cells with V5-tagged MYB-FL, MYB-TR or MYB-PLEKHO1 knocked in to the endogenous MYB locus using CRISPR/Cas9 and homology-directed repair. We observed increased binding of MYB-TR and MYB-PLEKHO1 relative to MYB-FL at promoters of cell cycle genes that function at the G2/M checkpoint, including CDC20, CDCA3 and CKS2. These binding sites contained canonical MYB binding motifs, indicating that MYB fusions increase direct DNA binding at chromatin involved in control of specific cell cycle genes. Cut&Run for V5-Myb-PLEKHO1 in mouse leukemias revealed similar direct DNA binding at cell cycle gene promoters. Moreover, expression of G2/M cell cycle genes was enriched in patient BPDCNs relative to normal pDCs. Our data suggest that BPDCN MYB fusions exert distinct oncogenic effects to overexpression of wild-type MYB. In addition to arresting differentiation, BPDCN MYB fusions can reactivate self-renewal in progenitor cells, which may occur through increased direct DNA binding at cell cycle gene promoters. This may explain the simple genetics of MYB-rearranged BPDCN, which lack other recurrent myeloid-type mutations, if the fusions are sufficient to generate leukemia via these dual roles. These data also indicate that therapeutic strategies targeting MYB and/or specific cell cycle checkpoints may be active in BPDCN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Decoding lymphomyeloid divergence and immune hyporesponsiveness in G-CSF-primed human bone marrow by single-cell RNA-seq

Granulocyte colony-stimulating factor (G-CSF) has been widely used to mobilize bone marrow hematopoietic stem/progenitor cells for transplantation in the treatment of hematological malignancies for decades. Additionally, G-CSF is also accepted as an essential mediator in immune regulation, leading to reduced graft-versus-host disease following transplantation. Despite the important clinical roles of G-CSF, a comprehensive, unbiased, and high-resolution survey into the cellular and molecular ecosystem of the human G-CSF-primed bone marrow (G-BM) is lacking so far. Here, we employed single-cell RNA sequencing to profile hematopoietic cells in human bone marrow from two healthy donors before and after 5-day G-CSF administration. Through unbiased bioinformatics analysis, our data systematically showed the alterations in the transcriptional landscape of hematopoietic cells in G-BM, and revealed that G-CSF-induced myeloid-biased differentiation initiated from the stage of lymphoid-primed multipotent progenitors. We also illustrated the cellular and molecular basis of hyporesponsiveness of T cells and natural killer (NK) cells caused by G-CSF stimulation, including the potential direct mechanisms and indirect regulations mediated by ligand–receptor interactions. Taken together, our data extend the understanding of lymphomyeloid divergence and potential mechanisms involved in hyporesponsiveness of T and NK cells in human G-BM, which might provide basis for optimization of stem cell transplantation in hematological malignancy treatment.

Oral Trypanosoma cruzi Acute Infection in Mice Targets Primary Lymphoid Organs and Triggers Extramedullary Hematopoiesis.

During the acute phase of Chagas disease, Trypanosoma cruzi circulation through the bloodstream leads to high tissue parasitism in the host. In primary lymphoid organs, progenitor cell reduction paralleled transient immunosuppression. Herein we showed that acute oral infection in mice promotes diffuse parasitism in bone marrow cells at 14 and 21 days post-infection (dpi), with perivascular regions, intravascular regions, and regions near the bone being target sites of parasite replication. Phenotypic analysis of hematopoietic differentiation in the bone marrow of infected mice showed that the cell number in the tissue is decreased (lineage-negative and lineage-positive cells). Interestingly, analysis of hematopoietic branching points showed that hematopoietic stem and progenitor cells (HSPCs) were significantly increased at 14 dpi. In addition, the pool of progenitors with stem plasticity (HSC-MPP3), as well as multipotent progenitors (MPPs) such as MPP4, also showed this pattern of increase. In contrast, subsequent progenitors that arise from MPPs, such as common lymphoid progenitors (CLPs), lymphoid-primed MPPs (LMPPs), and myeloid progenitors, were not enhanced; conversely, all presented numeric decline. Annexin V staining revealed that cell death increase in the initial hematopoietic branching point probably is not linked to CLPs and that myeloid progenitors decreased at 14 and 21 dpi. In parallel, our investigation provided clues that myeloid progenitor decrease could be associated with an atypical expression of Sca-1 in this population leading to a remarkable increase on LSK-like cells at 14 dpi within the HSPC compartment. Finally, these results led us to investigate HSPC presence in the spleen as a phenomenon triggered during emergency hematopoiesis due to mobilization or expansion of these cells in extramedullary sites. Splenocyte analysis showed a progressive increase in HSPCs between 14 and 21 dpi. Altogether, our study shows that the bone marrow is a target tissue in T. cruzi orally infected mice, leading to a hematopoietic disturbance with LSK-like cell bias accounting on HSPCs possibly affecting myeloid progenitor numbers. The LMPP and CLP reduction converges with defective thymocyte development. Lastly, it is tempting to speculate that the extramedullary hematopoiesis seen in the spleen is a mechanism involved in the hematological maintenance reported during the acute phase of oral T. cruzi infection.

1021 – SWI/SNF CHROMATIN REMODELING COMPLEXES GOVERN LYMPHOID LINEAGE COMMITMENT

Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis. Lymphocyte development from hemopoietic stem cells (HSCs) is accompanied by a loss of self-renewal capacity and a progressive restriction of developmental potential. Although key transcriptional pathways controlling lymphoid development have been identified, detailed molecular mechanisms explaining how multipotent HSCs initially commit to the lymphoid lineage are still lacking. Work from our laboratory indicates that combinatorial assembly of SWI/SNF chromatin remodeling complexes is a key epigenetic mechanism controlling cell fate decisions in the hemopoietic tissue. Single-cell RNA-seq analyses in bone marrow cells revealed that expression of the Smarcd1 subunit is enriched in lymphoid-primed multipotent progenitors. Using a conditional knock-out mouse model, we showed that Smarcd1 is specifically required for the development of the lymphoid lineage. Acute deletion of Smarcd1 in adult hematopoiesis causes lymphopenia with near complete absence of lymphoid-primed multipotent progenitors, indicating a role in lymphoid lineage commitment. Genome-wide studies revealed a functional collaboration between Smarcd1 and the bHLH transcription factor E2A in activating the lymphoid program of gene expression. Mechanistically, we show that Smarcd1 binds to and regulates chromatin accessibility of E2A at H3K27ac/H3K4me1-enriched enhancers that coordinate activation of the early lymphoid signature in hemopoietic stem cells. Altogether, these studies identify Smarcd1 as a chromatin remodeler that collaborates with E2A to determine the lymphoid cell fate during hemopoiesis.

Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis.

ABSTRACTThe bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin−Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.

Single Cell Analysis of Adult Human Hematopoietic Stem and Progenitor Cells Identifies a Novel Lymphoid Primed Multipotent Progenitor That Expands in Relapsed Acute Myeloid Leukemia

Background: Hematopoietic stem and progenitor cells (HSPCs) are capable of generating all human blood cells. Although these cells have been extensively evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al. Nat. Commun. 2019), there remains significant uncertainty as to the degree of heterogeneity within HSPC subpopulations and their relevance to disease. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of numerous blood disorders including hematologic malignancies. To address this need, we extensively characterized healthy bone marrow mononuclear cells (BMMCs) with both whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and multiplexed (n=35) surface marker analysis, we could not only improve HSPC clustering but also characterize specific phenotypic states along unique hematopoietic differentiation trajectories. This analysis allowed us to identify new subpopulations within currently defined hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and lymphoid primed multipotent progenitors (LMPPs).Methods: Three healthy adult BMMC samples were stained with antibody conjugated oligonucleotides (BD Abseq) and analyzed using a nanowell cell capture system (BD Rhapsody). We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. These cells were first clustered using matrix factorization in order to identify unique HSPC states. Novel clusters within the HSC, MPP, and LMPP subpopulations were purified using flow cytometry, and functionally evaluated with both in vitro and in vivo assays.Results: The antibody-derived tags (ADTs) obtained from BD Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 versus 934 for mRNA alone for 20 clusters). Additionally, we were able to design a targeted HSPC panel (n=500 genes) with Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (Rand index = 0.88). HSPC clustering identified the putative hematopoietic stem cell (HSC), a multipotent progenitor (MPP), and a new lymphoid primed multipotent progenitor (LMPP). A new computational sorting strategy was derived to purify these primitive HSPCs, and subsequently validated with flow cytometry. The functional evaluation of these sorted populations revealed that HSC and MPP cells were capable of increased serial replating ability in vitro suggesting enhanced self-renewal capabilities compared to LMPP cells. All three HSPC subpopulations produced gradients of erythroid and myeloid colonies in methylcellulose, and T-cells in T-cell expansion assays in vitro. The HSCs were the only cells that were able to produce long term engraftment in immunodeficient mice after serial transplantations. Additionally, common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers were identified. The primitive HSPC clusters were converted into a signature matrix using CIBERSORTx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. Using multivariate Cox proportional hazard analysis, we found that high levels of MPPs at diagnosis was the strongest feature associated with worse overall survival (HR = 78.46, 95% confidence interval 7.32-828). Interestingly, after performing paired analysis of diagnostic and relapse cases, the HSC and LMPP expanded significantly in relapsed AML after chemotherapy (p < 0.05) whereas the MPP expanded considerably after stem cell transplant but did not reach significance (p = 0.09).Conclusions: In summary, we identified new cell type clusters within previously defined HSC, MPP, and LMPP subpopulations, and unique surface marker associations using combined single cell WTA and surface marker analysis. We were able to purify these primitive HSPCs using these new markers, in addition to classical markers like CD34, CD90, and CD45RA. Importantly, deconvolution analysis provided preliminary insights into their clinical relevance in AML. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc: Membership on an entity's Board of Directors or advisory committees; Circbio inc: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutics Solutions Inc: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead Sciences, Inc: Patents & Royalties.

Cdc42-Borg4-Septin7 axis regulates HSC polarity and function.

Aging of hematopoietic stem cells (HSCs) is caused by the elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (Borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal Septin network. Here, we show that Cdc42 interacts with Borg4, which in turn interacts with Septin7 to regulate the polar distribution of Cdc42, Borg4, and Septin7 within HSCs. Genetic deletion of either Borg4 or Septin7 results in a reduced frequency of HSCs polar for Cdc42 or Borg4 or Septin7, a reduced engraftment potential and decreased lymphoid‐primed multipotent progenitor (LMPP) frequency in the bone marrow. Taken together, our data identify a Cdc42‐Borg4‐Septin7 axis essential for the maintenance of polarity within HSCs and for HSC function and provide a rationale for further investigating the role of Borgs and Septins in the regulation of compartmentalization within stem cells.