Competitive Repopulation Assay Research Articles

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

Septin11 and Septin 8 Play Critical and Overlapping Roles Murine Stem Cell Function

Septins are highly conserved GTP binding proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis and are now considered the fourth component of the cytoskeleton. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previous data in our laboratory led to the discovery of a small molecular inhibitor, DW0254, which inhibits RAS function and its downstream target RhoGTPase, Rac, including the hematopoietic-specific Rac2 GTPase, also implicated in MLL leukemias. The inhibitor leads to apoptosis of leukemia cell lines. Using biophysical methods, we discovered that DW0254 binds both the RAS chaperone, PDE6D, and Septin11, which is a member of the Septin6 family (Sara CN, et al., Blood Cancer 2022). Although Septins have been reported to be involved in function of the RhoGTPase Cdc42 and hematopoietic stem progenitor cell (HSPC) polarity, the role of Septin11 in hematopoiesis has not been reported. Septin11 is a member of the Septin6 family of proteins which consists of Septin6, 8, 10, 11 and 14 with significant homology between family members. Previous data in yeast has hypothesized functional overlap within each family of Septin proteins. To better understand the functional role of Septin11, we created lentivirus vectors tagged with a GFP reporter that contained shmiRs against Septin11. Culture of Septin11 knockdown (Septin11 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 13-fold decrease in cell counts compared to controls (p=0.02) and a decrease in total clonogenic activity (Figure 1) with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies. Reduced clonogenic numbers were accompanied by a significant increase in early apoptotic cells suggesting that Septin11 is important in regulating apoptosis in HSPC. To determine the potential role of Septin11 in more primitive hematopoietic cells, we utilized competitive repopulation assays. Transduced BoyJ HSPC (GFP+ Septin11 KD and BFP+ control, mixed 1:1) were injected into lethally irradiated C57Bl/6 mice. We observed a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary transplant recipients after knockdown of Septin11 (Table 1). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin11 most clearly in LT-HSC and ST-HSC (Table 1) with no statistically significant change in MPP. Therefore, to better study the hematopoietic stem cell compartment, we performed secondary transplants utilizing the cells obtained from primary competitive repopulation assays. We observed an unexpected recovery in engraftment in secondary transplant recipients that was accompanied by an increase in the mRNA expression of Septin8. Since Septin8 and Septin11 are closely related we hypothesized that induction of Septin8 was subserving key functions of Septin11. To test this hypothesis, we created a lentivirus vector that expressed both a shmiR against Septin11 and the Septin8 cDNA. Concurrent overexpression of Septin8 with Septin11 knockdown HSPC caused a significant increase in the total number of colonies compared to controls (Figure 1). These data indicate an important role of Septin11 in murine hematopoiesis and a potential redundant role for Septin8.

Compound Loss of Dnmt3a and Kmt2c in Myeloid Malignancies

Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine 4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Individually, loss in DNMT3A activity in hematopoietic stem cells (HSCs) has shown bias towards self-renewal due to a failure to shut off stem-cell-related genes, whereas a loss of KMT2C leads to aberrant myelopoiesis. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that may accelerate development of MDS and progression to AML. To address this, we generated mice that lacked one copy of both Kmt2c and Dnmt3a in the bone marrow by conditional deletion with a hematopoietic-specific inducible Mx1-driven Cre recombinase. After inducing Cre-mediated recombination by poly(I:C) treatment creating Kmt2c(+/del):Dnmt3a(+/del) animals, the hematopoietic stem and progenitor compartment was subjected to comprehensive immunophenotypic and functional characterization. In serial competitive bone marrow repopulation assays, cells with combined heterozygous loss of both Dnmt3a and Kmt2c demonstrated increased competitive advantage in secondary transplants and retained ability to reconstitute tri-lineage hematopoiesis in tertiary transplants, unlike single gene deletion and wild-type controls. Consistently, immunophenotypic analysis of the bone marrow compartment 16 weeks after the third transplant demonstrated persistence of the primitive long-term hematopoietic stem cells (LT-HSCs) defined as Lineage -Sca1 +cKit + (LSK) CD150 +CD48 -, which were absent in other genotypes. Further, we found expansion of the LT-HSCs in aged 80-week old Kmt2c(+/del):Dnmt3a(+/del) animals that were also characterized by a gain in self-renewal as seen by their ability to serially replate in semisolid media (MethoCult GM3434). To test if combined loss of Kmt2c and Dnmt3a led to disease development, next we established a large cohort of animals non-competitively transplanted with Kmt2c(+/del):Dnmt3a(+/del), single gene deletion, and wild-type control bone marrow, and initiated a tumor watch by monthly monitoring the composition of their peripheral blood by CBCs and flow cytometry. Sixty weeks after transplantation, over a half of double knock-out animals developed myeloid disease characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly most consistent with myelodysplasia. Immunophenotypically, we found expanded LSK populations (LT-HSCs specifically) in the bone marrow and evidence of extramedullary hematopoiesis in the spleen. At the same time, the majority of mice with single gene deletion and all wild-type controls remained healthy. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in the development of myelodysplastic disease with long latency through endowing the HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles and gene expression patterns resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

Methyl-CpG Binding Domain Protein MBD2 Is a Targetable Vulnerability in TET2 Mutant Myeloid Neoplasia

Background & Significance. Epigenetic dysregulation leading to acquired disorganization of CpG methylation (mCpG) is a hallmark of cancer in general and myeloid neoplasia (MN) in particular. TET dioxygenases (TET1, TET2 and TET3) progressively oxidize 5-methyl cytosine (mC) to facilitate CpG demethylation, a process critical to efficient transcription, lineage differentiation, and proliferation. Loss of function TET2 mutation ( TET2 MT), the predominant TET in HSPCs, isone of the most common genetic lesions in MN and often occurs early in MDS/AML as a founder lesion. It is frequently observed in clonal hematopoiesis of indeterminate potential (CHIP) 1,2, a prodromal condition linked with increased risk of MN, coronary heart disease 3, and pulmonary arterial hypertension 4. TET2 MT results in abnormal accumulation of mC in the genome resulting in loss of lineage plasticity due to transcriptional repression of differentiation-promoting genes and tumor suppressor genes (TSGs), contributing to a volatile pre-neoplastic state. Indeed, the accumulation of mC at otherwise unmethylated promoter- and enhancer-associated CpGs is a hallmark cancer 2,5. The epigenetic reader mCpG binding domain protein 2 (MBD2) specifically recognizes mCpG, thereby modulating regional gene transcription 6. This modulation occurs through the recruitment of co-repressor complexes which deliver enzymatic machinery to reorganize chromatin into compact, inaccessible, and stably silenced regions. This tightly regulated process, required for normal development and cell lineage differentiation, is hijacked by malignant cells to maintain a pro-oncogenic transcriptional program. Therefore, MBD2 inhibition may restore the normal hematopoiesis in TET2 MT HSPCs and may represent a selective therapeutic target. Methods. Genetic and epigenetic in vitro and in vivo model systems of clonal evolution including competitive repopulation assays and CRISPR-engineered cells coupled with NGS and biochemical analysis were used to establish the functional relevance of MBD2 in the clonal evolution of TET2 MT HSPCs. In addition, gene knockout or knockdown (ko/kd) were used to establish MBD2 as a novel therapeutic vulnerability in TET2 MT MN. Results. Here we report that loss of TET activity, either by TET2 MT or by neomorphic IDH1/2 MT, selectively enriches promoter/enhancer mCpG contributing to malignant evolution of TET2 MT HSPCs that creates sensitivity to MBD2 inhibition. TET2 MT creates regional accumulation of mC that recruits the MBD2-NuRd complex, resulting in silencing of TSGs as demonstrated via MBD2 chromatin immunoprecipitation assay. MBD2 ko/kd in TET2 MT cells (SIG-M5, OCIAML-5) or isogenic CRISPR-engineered leukemic cells (THP1, K562) resulted in the termination of the malignant program as reflected in a complete loss of colony forming ability in vitro. Orthotropic transplant of SIG-M5 cells (10 5 cells/mice) created leukemia in SGM3-NSGS mice with a maximum survival of 45 days, however, MBD2 ko in transplanted cells resulted in 100% disease-free survival of these mice. To further test if Mbd2 loss is sufficient to restore normal hematopoiesis and if it can abrogate the proliferative advantage of Tet2 -/- HSPCs, we developed a Tet2 -/-;Mbd2 -/- double ko (DKO) murine model by crossing Tet2 -/-with Mbd2 -/- mice. The DKO mice developed normally with restored hematopoiesis within the myeloid lineage compared to Tet2 -/-. Most importantly, the proliferative advantage of Tet2 -/-HSPCs was ablated, as reflected in the mutant fraction of total WBC and the restoration of CD11b + myeloid cells following Mbd2 loss in competitive bone marrow repopulation assays ( Fig. A).This restoration was mainly driven by the transcriptional rescue of differentiation-promoting genes which were silenced by promoter methylation in Tet2 -/- HSPCs ( Fig. B). Discussion & Conclusion. Here, for the first time, we demonstrate that MBD2 is required for maintaining the undifferentiated state of TET2 MT HSPCs with myeloid skewing. We provide several lines of evidence in human cells as well as in murine models that MBD2 is a targetable vulnerability in TET2 MT-associated leukemia. Our current findings provide a rationale for development of novel and potent non-cytotoxic therapeutic agents targeting MBD2 and their application in a biomarker-driven fashion for a large proportion of leukemias.

Prostaglandin E2 as transduction enhancer affects competitive engraftment of human hematopoietic stem and progenitor cells

Exvivo gene therapy (GT) is a promising treatment for inherited genetic diseases. An ideal transduction protocol should determine high gene marking in long-term self-renewing hematopoietic stem cells (HSCs), preserving their repopulation potential during invitro manipulation. In the context of the improvement of a clinically applicable transduction protocol, we tested prostaglandin E2 (PGE2) as a transduction enhancer (TE). The addition of PGE2 shortly before transduction of human CD34+ cells determined a significant transduction increase in the invitro cell progeny paralleled by a significant reduction of their clonogenic potential. This effect increased with the duration of PGE2 exposure and correlated with an increase of CXCR4 expression. Blockage of CXCR4 with AMD3100 (plerixafor, Mozobil) did not affect transduction efficiency but partially rescued CD34+ clonogenic impairment invitro. Once transplanted invivo in a competitive repopulation assay, human CD34+ cells transduced with PGE2 contributed significantly less than cells transduced with a standard protocol to the repopulation of recipient mice, indicating a relative repopulation disadvantage of the PGE2-treated CD34+ cells and a counter-selection for the PGE2-treated cell progeny invivo. In conclusion, our data indicate the need for risk/benefit evaluations in the use of PGE2 as a TE for clinical protocols of GT.

Impaired Repopulating Ability of Uhrf2-/- Hematopoietic Progenitor Cells in Mice.

UHRF proteins catalyze the ubiquitination of target proteins and are involved in regulating gene expression. Some studies reported a reduced expression of UHRF2 in acute leukemia cells, but the role of UHRF2 in hematopoiesis remains unknown. Here, we generated Uhrf2-/- mice to clarify the role of UHRF2 deletion in hematopoiesis. Compared to Uhrf2+/+ mice, Uhrf2-/- mice showed no differences in complete blood counts, as well as bone marrow (BM) findings and spleen weights. Proportions of cells in progenitor fractions in BM were comparable between Uhrf2+/+ mice and Uhrf2-/- mice. However, in competitive repopulation assays with BM transplants (BMT), the proportions of Uhrf2-/- cells were decreased relative to Uhrf2+/+ cells in all lineages. After the second BMT, Uhrf2-/- neutrophils were few, while 20-30% of Uhrf2-/- T cells and B cells were still detected. RNA sequencing showed downregulation of some genes associated with stem-cell function in Uhrf2-/- hematopoietic stem/progenitor cells (HSPCs). Interestingly, trimethylated histone H3 lysine 9 was increased in Uhrf2-/- HSPCs in a cleavage under targets and tagmentation assay. While UHRF2 deletion did not cause hematologic malignancy or confer a growth advantage of HSPCs, our results suggest that UHRF2 may play a role in the regulation of hematopoiesis.

Lin- PU.1dim GATA-1- defines haematopoietic stem cells with long-term multilineage reconstitution activity.

Despite extensive characterization of the state and function of haematopoietic stem cells (HSCs), the use of transcription factors to define the HSC population is still limited. We show here that the HSC population in mouse bone marrow can be defined by the distinct expression levels of Spi1 and Gata1. By using a double fluorescence knock-in mouse model, PGdKI, in which the expression levels of PU.1 and GATA-1 are indicated by the expression of GFP and mCherry, respectively, we uncover that the HSCs with lymphoid and myeloid repopulating activity are specifically enriched in a Lin- PU.1dim GATA-1- (LPG) cell subset. In vivo competitive repopulation assays demonstrate that bone marrow cells gated by LPG exhibit haematopoietic reconstitution activity which is comparable to that of classical Lin- Sca1+ c-kit+ (LSK). The integrated analysis of single-cell RNA sequence data from LPG and LSK-gated cells reveals that a transcriptional network governed by core TFs contributes to regulation of HSC multipotency. These discoveries provide new clues for HSC characterization and functional study.

Dysfunctional Clusterin-Positive Hematopoietic Stem Cells Expand with Aging

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes dysregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old), and aged (20-month-old) mice to gain insight into the dynamics of HSC aging. scRNA-seq data revealed that transcriptomic profiles of HSCs change with aging, although middle-aged and aged HSCs showed very similar profiles to each other. scRNA-seq data divided young and aged HSCs into 2 and 3 clusters, respectively. While both young and aged HSCs had clusters with myeloid or lymphoid gene signature, the cluster with myeloid signature dominated in aged HSCs, in which aged gene signature (Nupr1, Mt1, Mt2, Selp) was highly enriched. Clusterin (Clu) was also significantly up-regulated in this cluster. Clu encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis (Itakura et al., JCB 2020). We hypothesized that Clu would be useful as a marker of aged HSCs, and conducted further analysis using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory elements of the Clu gene. Bone marrow (BM) analysis of Clu-reporter mice revealed that Clu expression is confined to a part of HSCs in BM and significantly up-regulated within the HSC compartment with aging (10 and 70 % of HSCs are Clu-positive in young and middle-aged mice, respectively). In competitive repopulation assays, Clu-positive HSCs showed severely impaired repopulation capacity and preferentially differentiated into myeloid cells while Clu-negative HSCs largely retained repopulation capacity and showed balanced differentiation. We analyzed the transcriptomic profile of Clu-positive HSCs with aging. RNA-sequence analysis revealed that young and middle-aged Clu-positive HSCs were significantly enriched for aged HSC gene signature and myeloid-biased HSC signature compared to Clu-negative HSCs. We evaluated the transcriptional regulation mechanism of Clu by Assay for Transposase-Accessible Chromatin sequence (ATAC-seq) analysis. Chromatin accessibility at the Clu promoter was already open in young HSCs, and largely enhanced in Clu-positive HSCs but not in Clu-negative HSCs among middle-aged HSCs. The open chromatin region at the Clu promoter had binding motifs of AP-1 and STAT family transcription factors, suggesting that external signals or stresses activate Clu expression. These results suggest that Clu-positive cells represent myeloid-biased HSCs which expand with aging and that Clu serves as a faithful marker to trace alterations in HSC heterogeneity with aging.

Septin-6 Regulates Murine and Human Hematopoiesis, and Its Dysregulation Is Associated with Pediatric Myelodysplasia

Septin family proteins are key regulators of cytoskeletal organization and cytokinesis although little is known about their function in normal hematopoiesis. Septins are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we identified a non-syndromic newborn with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022). Next-generation sequencing revealed a novel X-linked germline mutation in the SEPTIN-6 gene associated with dysmyelopoiesis (SEPTIN6 c.1282T>C); and a second stop-gain somatic mutation hypothesized to rescue hematopoietic function, but the mechanism of MDS was not defined. Based on our initial publication, a second patient with neutropenia/MDS has since been identified in Seattle with a mutation in the same codon (SEPTIN6 c.1282T>A) suggesting an important new molecular mechanism for pediatric MDS. Both identified patients had a high degree of myeloid tetraploidy in the marrow and were refractory to G-CSF. To assess the pathobiology of the published patient's mutation, we replicated the germline T>C mutation using single base-pair gene editing in normal donor primary CD34+ cells. Editing rates were ~50-70%. Morphological studies demonstrated that SEPTIN-6 edited human CD34+ cells demonstrated enlarged, multi-nucleated and dysplastic nuclei. These findings were similar to the appearance of the patient's bone marrow myeloid precursor cells. These edited cells also demonstrated a decline in clonogenic activity notably with a selective reduction in granulocyte-macrophage (GM) colonies (Table 1). Both myeloid and erythroid colonies showed a reduction in colony size. These data directly confirm an important role for this mutated SEPTIN-6 in abnormal hematopoietic function. The septin-6 family of proteins consist of septin-6, 8, 10, 11 and 14 which may have overlapping functions in cells. Murine single cell mRNA expression data demonstrate that hematopoietic stem and progenitor cells (HSPC) express significant levels of Septin-6, Septin-11 and 8, with little to no expression of Septin-10 and 14. To better understand the functional role of Septin-6, we created lentivirus vectors tagged with a GFP reporter that contained shRNAs against Septin-6. Culture of Septin-6 knockdown (Septin-6 KD) murine HSPC in IMDM (10ng/ml IL-3, 25ng/ml of SCF, TPO, Flt-3 and IL-6) resulted in an average of 18-fold decrease in cell counts compared to controls (p=0.02). This was accompanied by an increase in cell cycle: S-phase: Septin-6 KD 55.7%; controls 42.5%, p=0.05 and a cumulative decrease in the G0/G1 and G2/M phase suggesting that Septin-6 is important in regulating cell cycle in HSPC. Controls were a non-targeting shRNA and an empty vector for all experiments described. Furthermore, knockdown of Septin-6 caused a decrease in total clonogenic activity with defects seen in both myeloid (GM) and erythroid (BFU-E) colonies (Table 1). Competitive repopulation assays, wherein transduced BoyJ HSPC (GFP+ Septin KD and BFP+ control were mixed 1:1) were injected into lethally irradiated C57Bl/6 mice, demonstrated a reduction in multilineage chimerism in the peripheral blood, bone marrow, spleen and thymus in primary and secondary transplant recipients after knockdown of Septin-6 (Table 2). To better define function in specific hematopoietic populations of HSPC, we performed knockdown in FACS-sorted purified cells. Engraftment studies demonstrated a significant reduction in bone marrow chimerism in primary transplant recipients after knockdown of Septin-6 most clearly in ST-HSC (Table 2) with a trend towards a decrease in LT-HSC and MPP. These data support a key role of Septin-6 in murine and human hematopoiesis via alterations in cell cycle and validate the pathogenic nature of a specific mutation in human SEPTIN-6. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Loss of DHX15 Function Impairs Hematopoiesis in Vav-Dhx15-KO Mice and Cause DNA Damage-Induced Cell Cycle Arrest and Apoptosis in Leukemia Cell Lines

DHX15, a member of the DEAD-box RNA helicase family, acts as splicing factor in pre-messenger RNA splicing, pre-ribosomal RNA, and RNA metabolism. Our previous studies showed that DHX15 overexpression is associated with poor prognosis in acute myeloid leukemia (AML), and DHX15 regulates cell apoptosis via the NF-kB signaling pathway(Pan et al., 2017). However, its molecular function remains obscure in the blood system. First, with the Cre-loxp knockout system, Dhx15fl/fl male mice were crossed with vav-icre female mice (specifically expressed in blood system), we found that Dhx15fl/fl; vav-icre homozygous knockout mice (vav-Dhx15-KO) are lethal by E17.5-E18.5. Then, to explore the inner difference in blood system, the hematopoietic stem cells (HSC) were analyzed by flow cytometry in fetal liver (FL) (E13.5, E15.5). Compared with Dhx15fl/fl (wild-type, WT) mice, the lineage-negative cells (Lin- cells; Lin+ Cocktail, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4 and Ter-119) significantly increased in vav-Dhx15-KO FL cells (P<0.0001) and the short-term HSC(ST-HSC) proportion compensatively increased in LSK (Lin-c-Kit+Sca1+cells) population (P<0.01), while the proportion of LK(Lin-c-Kit+Sca1-cells) (P<0.0001 in E13.5, P<0.001 in E15.5), LSK(Lin-c-Kit+Sca1+cells)(P<0.1 in E15.5), and multipotent progenitor (MPP) (P<0.0001 in E15.5) all witnessed decrease in the Lin-/LSK population respectively. Meanwhile, vav-Dhx15-KO mice exhibited impaired multi-lineage hematopoiesis especially in erythroid, where the precursors and immature erythrocytes increased, mature erythrocytes decreased (P<0.001 in E13.5). Next, in vivo competitive repopulation assay were performed to further examine any potential cell-intrinsic defect of HSC function caused by Dhx15-inactivation. Thus, 1,000,000 E13.5/E15.5 total FL donor cells (CD45.2, vav-Dhx15-KO or WT) together with 1,000,000 CD45.1 unfractionated BM competitor cells were intra-venous transplanted into lethally irradiated (9Gy) B6.SJL (CD45.1) recipient mice. Recipient mice were sacrificed 8-weeks after transplantation, flow cytometry was conducted to analysis the ratio of CD45.2 in mice, the results showed that vav-Dhx15-KO FL was severely deficient compared to WT FL(0.1±0.04%vs 62.2±2.8% in BM, 0.2±0.05% vs 64.5±6.1% in SP, 0.1±0.05% vs 45.1±5.5% in PB). These results indicate that Dhx15 is crucial in mice blood system, loss of its function may block cell differentiation, leading to the reduction of mature cells. At the same time, to further explore the molecular mechanism of DHX15 in blood system, MOLM-13 and NB4 leukemia cell lines were selected for shRNA-KD (knockdown) experiments due to the lethality of vav-Dhx15-KO mice. We found that Dhx15-KD significantly impaired the proliferation ability of these cell lines in vitro and in vivo, by experiments of survival competition, clone formation, subcutaneous and intra-venous tumorigenesis. Besides, our data demonstrate DHX15-KD-mediated G0/G1 cell cycle arrest and increase in early-late apoptotic cells by Hoechest 33342-EdU cell cycle and DAPI-Annexin V cell apoptosis assays. Mechanistically, we found that UV_RESPONSE signaling pathway which is mainly related to DNA damage was up-regulated by RNAseq and bioinformatics analysis (DHX15-KD cell line vs WT cell line). Correspondingly, the DNA damage indicators γH2AX and 53BP1 were also increased in DHX15-KD cell lines detected by WB and immunofluorescence experiments, suggesting that the deletion of DHX15 could lead to DNA damage-mediated cell cycle arrest and apoptosis in leukemia cell lines. In summary, these results show that DHX15 deficiency leads to DNA damage-induced cell cycle arrest and apoptosis which further impairs the proliferative capacity of leukemia cell lines and lost function of Dhx15 impairs multi-lineage hematopoiesis in mouse embryos, which may lead to its perinatal death.

Tumor Necrosis Factor from Leukemic Microenvironment Provides Non-Leukemic Hematopoietic Stem Cells with Megakaryocyte Lineage Bias

Background: Acute myeloid leukemia (AML) is characterized by proliferation of malignant cells and impairment of normal hematopoiesis, leading to the severe bone marrow failure. Although genetic abnormalities and pathogenesis of AML are now investigated in detail, the precise mechanism impairing normal hematopoiesis in AML is not fully elucidated yet. Hematopoietic stem cells (HSC) retain the self-renewal capacity together with multipotent differentiation potential into all mature blood cells, playing a major role in maintaining the normal hematopoiesis. The ability of HSC is maintained by complex network of intrinsic factors and external stimulation from microenvironment called as HSC niche. According to these backgrounds, we hypothesized that the impairment of normal hematopoiesis in AML is caused by abnormal function of non-leukemic (nl) HSC. To verify this hypothesis, we isolated nl-HSC and HSC niche cells from MLL-AF9-induced AML mice and further analyzed their function and gene expression profiles. Results: The frequencies of nl-HSC/hematopoietic progenitor fraction (Lin-cKit+Sca1+ cells, HSPC) were significantly increased at the initiation phase of leukemic proliferation in AML mice (leukemic cells : 10-30% in BM)（normal: 0.76+/-0.34%, AML: 2.07 +/-0.93 %, p<0.001）. Surface expression level of CD41 was also upregulated (normal: 15.1+/- 0.526%, AML: 25.4+/- 9.28%, p<0.05). Interestingly, the results of in vitro culture of nl-HSPC demonstrated that they proliferated more efficiently than HSPC from normal mouse BM, and that they tended to differentiate into megakaryocyte (MgK) lineage. Gene set enrichment analysis revealed that MgK-related gene sets and several inflammatory cytokines signaling pathways, including tumor necrosis factor (TNF) pathway, were statistically enriched in nl-HSC. These data suggested that nl-HSC/HSPC retain the MgK-biased character. On the other hands, the gene expression profiles of perivascular stromal cells (ICAM1+CD31-CD45-TER119-cells) in AML mice demonstrated the significantly decreased expression of HSC-supporting factors (e.g., lpl, ptn, rspo2, angptl4, kitl, cxcl12), when compared with those in normal mice. These data suggested that the changes of multiple external humoral factors from leukemic environment induce functional alternation of nl-HSC. To further investigate the role of inflammatory humoral factors in HSC self-renew and differentiation, we next utilized polyvinyl alcohol (PVA)-based ex vivo HSC expansion system and focused on the role of TNF signaling. In the analysis of highly purified normal HSC (CD150+CD48-CD34-cKit+Sca1+Lin-cells) with PVA, TNF alpha increased both proliferation and MgK differentiation. UMAP analysis of single cell RNA sequence data for ex vivo-expanded HSCs with TNF alpha showed the increased frequencies of the clusters expressing MgK-related genes (e.g., itga2b and gp1ba) and relatively decreased immature HSC related genes (e.g., hlf and procr). The results of competitive repopulation assay using ex-vivo expanded normal HSC with TNF alpha stimulation in HSC promoted myeloid differentiation of HSC, rather than self-renew and expansion of HSC. Conclusion: Our data suggested that TNF alpha from leukemic microenvironment may play a key role for the driver signaling of HSCs to MgK, eventually resulting in the impairment of normal HSC maintenance.

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

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

Elongation of Long-Chain Fatty Acids Is Crucial for Hematopoietic Stem Cell Engraftment and Leukemia Propagation

Lipid metabolism plays a crucial role in regulating physiological stem cells and cancer cells. Variations in length or degree of desaturation of fatty acid (FA) chains greatly contribute to lipid diversity, but whether it significantly regulates hematopoietic cell biology remains unknown. The enzyme ELOVL6 converts C16 saturated and monounsaturated FAs to C18 species. Elovl6 gene disruption (Elo6KO) in mice decreases the proportions of stearate (C18:0) and oleate (C18:1n-9) and increases those of palmitate (C16:0) and palmitoleate (C16:1n-7). Here, we report that ELOVL6 is essential for hematopoietic stem cells (HSC) engraftment after bone marrow (BM) transplantation and acute myeloid leukemia (AML) development in a mouse model. To determine whether hematopoietic cells of Elo6KO mice exhibit changes in FAs composition, we analyzed FAs in BM cell lysates. We found that the ratio of stearate to palmitate (C18:0/C16:0 ratio) was significantly decreased in Elo6KO cells compared to WT cells. To assess the effects of Elovl6 loss on the mouse HSC, we performed competitive repopulation assays. Using control or Elo6KO donor BM cells mixed in a 1:1 ratio with competitor BM cells, the chimerism of peripheral blood (PB) was about 50% in mice transplanted with control cells but remained at < 0.2% in those with Elo6KO cells at 12 weeks after transplantation. Even when transplanted Elo6KO donor cells with the competitor in a 10:1 ratio, the chimerism of PB was similarly low. Next, we investigated the roles of Elovl6 in leukemogenesis. We infected lineage-negative and c-Kit+ cells from WT or Elo6KO BM with MLL-AF9(MA9) retrovirus and transplanted lethally irradiated mice with either WT or Elo6KO MA9 cells. All mice receiving WT MA9 cells developed AML; however, AML propagation in Elo6KO MA9 cell-transplanted recipients was blocked. Elo6KO MA9 cell-transplanted mice survived until 6 months after transplantation. To assess the mechanisms underlying these phenotypes, we performed RNA-seq and gene enrichment analysis (GSEA) using CD34-, lineage-negative, Sca-1+ and c-Kit+ (CD34-LSK) cells and MA9 cells from Elo6KO or control mice. Chemotaxis, CXCR4 pathways, and their related target gene sets were enriched in Elo6KO cells, relative to control in both CD34-LSK cells and MA9 cells. We tested the chemotaxis of Elo6KO CD34-LSK and MA9 cells toward CXCL12 in a migration assay. A greater fraction of WT CD34-LSK and MA9 cells than that of these Elo6KO cells migrated to the CXCL12-containing chamber. We then examined the pharmacologic effects of inhibiting the PI3K pathway on chemotaxis. In a migration assay, the presence of PI3K inhibitor, Copanlisib, decreased the migration rate in WT MA9 cells, but not significantly in Elo6KO MA9 cells, which was already compromised. By contrast, AKT inhibitor, MK2206, did not alter WT or Elo6KO MA9 cell migration. In GSEA, Rac1-related pathways were enriched in Elo6KO cells. We thus investigated whether Rac1 activation was blocked by Elovl6 loss. The levels of the GTP-bound Rac1 were markedly increased in WT MA9 cells following CXCL12 stimulation compared to control, while those were not significantly different with/wo CXCL12 stimulation in Elo6KO MA9 cells. Given impaired chemotaxis and CXCR4-PI3K-Rac1 signaling in Elo6KO cells, we then investigated whether these cells exhibited altered actin remodeling in response to CXCL12. As expected, CXCL12 treatment induced lamellipodia formation in WT CD34-LSK cells, but not in Elo6KO CD34-LSK cells. Thus, ELOVL6 may regulate a CXCL12-CXCR4 pathway, resulting in PI3K-Rac1-mediated cytoskeletal remodeling and chemotaxis. Finally, to determine whether exogenously expressed Elovl6 could rescue Elo6KO MA9 cell phenotypes, we generated Elo6KO MA9 cells expressing Flag-tagged ELOVL6 (Elo6KO MA9Elo6) and mock controls (Elo6KO MA9mock). To evaluate the rescue of AML propagation, we transplanted lethally irradiated syngeneic mice with either Elo6KO MA9mock or Elo6KO MA9Elo6 cells. Among Elo6KO MA9mock transplanted mice, few recipients developed AML, while 10 out of 14 recipients had died due to AML after Elo6KO MA9Elo6 cell transplant. In summary, this study demonstrates novel and unexpected outcomes in normal HSCs and AML driver-transformed cells following lipid changes induced by Elovl6 deficiency. Altering lipid diversity could be a potential strategy to treat AML.

Functional Role of Histone Variant macroH2A1 (H2AFY) in Acute Myeloid Leukemia (AML)

Histone variants can replace replication-coupled histones in the nucleosome. Among core histones, the H2A family exhibits highest sequence divergence resulting in the largest number of variants. Dysregulated expression of macrohistone (macroH2A) variants has been reported in different cancers. Their role in normal hematopoiesis and hematopoietic cancers remained so far poorly understood. We found high expression of macrohistone H2AFY (macroH2A1) in hematopoietic stem and progenitor cells (HSPCs) and primary AML as compared to differentiated hematopoietic cells. To assess for the functional dependency of human AML cells on macroH2A1, we performed in vitro CRISPR-Cas9 negative selection competition assays. Genetic inactivation of H2AFY in 4/9 AML cell lines (Molm-13, MV4;11, THP-1, OCI-AML3) resulted in outcompetition of H2AFY-deleted cells against non-deleted competitors. To confirm these findings in vivo, we performed xenotransplantation experiments. Genetic inactivation of H2AFY in human AML cells resulted in delayed leukemia development in recipient mice (median survival for NT-control: 35.5 days vs. H2AFY-KO: 44.0 days; p=0.002). To validate the functional impact of macroH2A1 on leukemic stem cells (LSCs), we generated a novel conditional knockout mouse model for H2AFY [B6(Cg)-H2afytm1Flh/J (H2afy flox/flox)]. Here, isolated lineage-depleted H2afy+/+, Mx1-Cre+ and H2afyfl/fl, Mx1-Cre+ bone marrow cells (Lin-BMCs) were retrovirally infected with MLL-AF9-GFP (MA9) and transplanted into sub-lethally irradiated recipient mice. Following establishment of leukemia, we transplanted 1x105 leukemic cells and inactivated H2afy in the leukemic cells by pIpC injection on day 7, 9 and 11 post-transplantation. Genetic deletion of H2afy resulted in delayed disease progression (median survival +/+: 83.5 days vs. -/-: 162.0 days, p=0.0013) and reduction of disease penetrance to 61%. Organ infiltration by leukemic cells led to disease development in a limited number of H2afy-/- recipient mice. Isolated H2afy-/- leukemic cells showed a decreased in proliferation and an increased in differentiation. Importantly, 37% of H2afy-/- recipient mice that developed AML showed only partial excision of H2afy, indicating selection of partially excised clones. Long-term survivors without leukemia development showed no molecular evidence for counterselection. Irrespective of the time of disease development and survival, immunophenotypic analysis of (Lin-cKit+Sca-1-FcγR+CD34+GFP+) LGMPs revealed decreased numbers of LSCs following H2afy-deletion (+/+: 2631/106 BMC; -/-: 727/106 BMC; p=0.0002). To assess for the functional consequences of H2afy-deletion on the LSC pool, we performed limiting dilution assays. When transplanting 1x105, 1x104, 1x103 and 1x102 H2afy+/+ or -/- leukemic cells into sub-lethally irradiated recipients, LGMP frequency in H2afy-deficient cells was significantly reduced (1/1 vs. 1/29928; p=1.25e-19). The reduction of LGMP frequency correlated with reduced plating efficiency and increased differentiation. Consistently, primary human AML cells showed impaired colony forming capacity following H2afy deletion (mean colony number NT-control: 60.0±17.1 vs. H2afy-KD: 30.1±8.9; p=0.0023). To assess for the impact of H2afy deletion on normal HSPCs, we performed competitive repopulation assays. Immunophenotypic abundance of HSPC in H2afy+/+ vs. -/- animals revealed comparable numbers in all relevant subpopulations. Serial competitive transplantation of H2afy+/+ and -/- BMC into primary and secondary recipient animals showed no competitive disadvantage or lack of self-renewal capacity of H2afy-/- cells. Likewise, colony formation of healthy human donor CD34+ cells was not impaired following depletion of H2AFY by RNAi. Transcriptome analysis of LGMP populations indicated increased of myeloid cell development and loss of LSC signatures in H2afy-deficient LGMPs. Likewise, ATAC-seq of H2afy+/+ and -/- LGMP population revealed profound changes in the chromatin structure and a strong correlation between chromatin accessibility and mRNA expression. Together, our results provide first evidence for a critical function of H2afy (macroH2A1) in models of AML while being dispensable for normal HSPC function. Experiments to assess for the mechanistic consequences of H2afy deletion on chromatin are currently under way.

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image:Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

Abstract 841: Mutant Trp53 R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice

Abstract Background: Missense mutations of the TP53 gene frequently occur in patients with myelodysplastic syndrome and acute myeloid leukemia. The functional consequences of mutant p53 expression on hematopoiesis remain incompletely studied. Whether TP53 mutations confer a loss-of-function, gain-of-function, or dominant-negative effect in response to chemotherapies has not been fully explored. One of these hotspot mutations is a G-to-A substitution at nucleotide 525 of TP53, which codes for an Arg175His (R175H) substitution. Thus, we characterized hematopoiesis in Trp53R172H constitutive knock-in mice, corresponding to R175H TP53 mutation in patients, and compared the results to Trp53 knock-out and control mice. Methods: Five genotypes of mice were analyzed (Trp53+/+ [WT], Trp53R172H/+, Trp53R172H/R172H, Trp53+/-, and Trp53-/-). Frequencies of peripheral blood (PB) and bone marrow (BM) cells were measured by flow cytometry and fluorouracil (5-FU) and N-ethyl-N-nitrosourea (ENU) were used as hematopoietic stressors. Results: There was no change in the PB counts or lineage distribution of mature PB and BM cells in Trp53R172H/+ and Trp53R172H/R172H mice compared to WT or Trp53 knock-out mice at 8-15 weeks of age. We found that expression of Trp53R172H mutation in mice does not cause hematopoietic stem cell (HSC) expansion under basal conditions, in contrast to heterozygous and homozygous deletion of Trp53, but does confer a functional advantage to HSCs in a competitive repopulation assay. Trp53R172H HSCs expand further following ENU exposure, similar to Trp53-/- cells and consistent with a dominant-negative effect of Trp53R172H. Mechanistically, mutant p53R172H has a dominant-negative effect on p21-induced expression following ENU treatment. Repeated 5-FU injections drive proliferation of progenitors. To test the hematopoietic cell-intrinsic effects of mutant p53R172H on white blood cell count recovery, we exposed mice reconstituted with Trp53+/+, Trp53+/-, Trp53-/- or Trp53R172H/+ BM cells to a single dose of 5-FU and followed mice weekly for a month. Trp53R172H/+ mice were least affected by the 5-FU challenge and showed the fastest and largest recovery of WBC count compared to Trp53+/+, Trp53+/- and Trp53-/- mice. These results demonstrate that mutant p53R172H confers a gain-of-function property following 5-FU exposure that was not seen with deletion of Trp53. Conclusion: Our data suggest that in vivo expression of mutant Trp53R172H has minimal effects on basal hematopoiesis. However, mutant Trp53R172H induces either a gain-of-function or a dominant-negative effect depending on the type of hematopoietic stress and hematopoietic phenotype being observed. Overall, our results indicate that the functional consequences induced by various Trp53 mutations (e.g., deletion and point mutation) are not identical, but are influenced by the type of hematopoietic stress. Citation Format: Tuoen Liu, Tanzir Ahmed, Michael O. Alberti, Brian A. Wadugu, Jin Shao, Sarah Grieb, Grace Przybyl, Matthew Ndonwi, Matthew J. Walter. Mutant Trp53 R172H has gain-of-function or dominant-negative effects in response to hematopoietic stressors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 841.

Conditional deletion of mTOR discloses its essential role in early B-cell development.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.

JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm

IntroductionRecent studies implicated megakaryocytes (MKs) in regulating hematopoietic stem cell (HSC) function. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). In the present study, we investigated the effects of JAK2V617F-bearing MKs on HSC aging in a murine model of MPN during a 2-yr follow up.MethodsJAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) and Pf4-Cre mice (which express Cre under the promoter of platelet factor 4, a MK-specific gene) were crossed to generate MK lineage-specific human JAK2V617F transgenic mouse line (Pf4 +FF1 +).ResultsDuring aging, the Pf4 +FF1 +mice maintained an essential thrombocythemia phenotype with no evidence of transformation to leukemia or myelofibrosis (Figure 1).Myeloid-biased HSCs (Lin -cKit +Sca1 +CD150 +CD48 -CD41 +) were significantly expanded in 1yr old and 2yr old Pf4 +FF1 +mice compared to age-matched control mice, while no difference in myeloid-biased HSC numbers was detected between young (6mo) Pf4 +FF1 +mice and control mice. Competitive repopulation assays and serial transplantation assays showed that HSCs from old Pf4 +FF1 +mice had a reduced engraftment and self-renewal capacity with a skewed differentiation towards the myeloid lineage. Results from the serial transplantation experiments also indicated that the functional decline of HSCs in aged Pf4 +FF1 +mice were HSC-intrinsic and was not reversible. Both cell cycle analysis by Hoechst33342 and Pyronin Y staining and cell proliferation analysis by in vivo BrdU labeling revealed that the JAK2V617F mutant MK niche can promote hematopoietic aging in old Pf4 +FF1 +mice by increasing HSC proliferation/cycling. Taken together, the Pf4 +FF1 +mice demonstrated several hallmarks of accelerated HSC aging.(Figure 2)Next, we examined the hematopoietic microenvironment in the old Pf4 +FF1 + mice. Using whole-mount immunofluorescent staining and confocal imaging, we found that the cKit +HSPCs were located further from MKs in old Pf4 +FF1 +mice compared to old control mice. We also found that CD45 -CD31 +Sca1 - sinusoidal marrow endothelial cell (EC) number (by flow cytometry analysis) and marrow vascular area (by in vivo VE-cadherin staining) were significantly decreased in aged Pf4 +FF1 +mice compared to control mice. Tube formation assays confirmed that conditioned medium from old Pf4 +FF1 +MK culture significantly inhibited EC angiogenesis in vitro. These findings suggest that the JAK2V617F mutant MK niche not only altered its own interaction with HSCs during aging, but also suppressed the vascular niche function to promote HSC aging. (Figure 3)To further understand the mechanisms by which JAK2V617F mutant MKs promote HSC aging, we performed both RNA sequencing and targeted cytokine arrays of wild-type and JAK2V617F mutant MKs from both young (6mo) and old (2yr) Pf4-cre control and Pf4 +FF1 +mice. We found that HSC aging had a profound effect on MK transcriptomic profiles and dysregulated pathways in cell adhesion molecules, MAPK signaling, NF-kappa B signaling, hematopoietic cell lineage, and cytokine-cytokine receptor interaction were highly upregulated in old JAK2V617F mutant MKs compared to young JAK2V617F mutant MKs. FAS ligand, IL-12, tissue inhibitor of metalloproteinases-1, IL-10, IL-6, MIG, macrophage inflammatory protein 1a, GCSF, IL-5, MIP-1g were produced in increased amounts in old mutant MKs compared to old wild-type MKs. Many of these factors are involved in inflammation, angiogenesis, extracellular matrix remodeling, and hematopoiesis regulation.Sensitive PCR assays confirmed that human JAK2 gene was expressed in MKs but not in HSCs in 2yr old Pf4 +FF1 +mice. In vitro co-culture and in vivo co-transplantation assays provided further evidence that the JAK2V617F mutant MKs affected wild-type HSC function directly. Therefore, the hematopoietic aging phenotype we have observed was not caused by any direct effect of the JAK2V617F mutation on HSC function because the Pf4 promoter was ‘leaky’.ConclusionsResults from this study support that, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging in MPNs – the JAK2V617F-bearing MKs can alter the hematopoietic niche to accelerate HSC aging, and HSC aging in turn can profoundly remodel the niche e.g. by affecting MK transcriptomics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IL-10R Inhibition Selects for Jak2V617F Hematopoiesis By Augmenting Inflammation Induced Exhaustion of Wild-Type but Not Jak2V617F Hematopoietic Stem Cells

An emerging theme among clonal hematopoiesis (CH) associated mutations is that these mutations endow hematopoietic stem cells (HSC) protection from chronic inflammatory stress. Specific inflammatory stressors may select for specific mutants, such that presence of particular mutant clones could be leveraged as a biomarker to herald the presence of a specific stressor. JAK2 V617F clonal hematopoiesis is less common among the general aging population but clusters within families suggesting that a specific stressor present among members of myeloproliferative neoplasm (MPN) families selects for JAK2 V617F mutant cells. We have recently found that monocytes from MPN patients and their unaffected family members have defective negative regulation of Toll Like Receptor Ligand (TLR) signaling due to a dampened response to the anti-inflammatory cytokine Interleukin-10 (IL-10) which leads to persistent TLR signaling and increased TNF production.We hypothesized that IL-10 serves to curtail TLR signaling in HSCs just as it does in monocytes. Therefore, we assessed activation and proliferation of HSCs in WT mice treated with the TLR agonist lipopolysaccharide (LPS) versus mice treated with LPS + IL-10 receptor (IL-10R) blocking antibody (Fig 1A). As expected, LPS treatment induced proliferation of HSCs at day 1 which returned to dormancy by day 3. However, with IL-10R blockade, WT HSCs remained highly active and proliferative even at 3 days post stimulation (Fig 1A). In contrast, blockade of IL-10R in Jak2 V617F HSC did not extend the time period that HSC were actively proliferating post-LPS stimulation (Fig 1B).To assess how chronic inhibition of IL-10R signaling impacts HSC fitness, we treated WT mice with twice weekly IL-10R blocking antibody and after 8 weeks harvested bone marrow for use in competitive repopulation assays. Bone marrow from mice exposed to chronic IL-10R blockade was inferior to untreated bone marrow (Fig 2A). We next tested whether blockade of IL-10R results in a functional decline in HSC when exposed to a short course of LPS exposure which would not otherwise result in defective competitive potential. We treated mice with 2 weeks of LPS +/- IL-10R blocking antibody before harvesting bone marrow for competitive repopulation assays. Whereas bone marrow from mice treated with 2 weeks of LPS competed equivalently to unexposed bone marrow, bone marrow from mice treated with 2 weeks of LPS + IL-10R blocking antibody were inferior to bone marrow from unexposed mice (Fig 2B). This demonstrates that IL-10R blockade accelerates inflammation induced exhaustion of WT HSC.Reasoning that blockade of IL-10R signaling may lead to an inflammatory state that allows for Jak2 V617F mutant cells to gain a selective advantage, we injected bone marrow mosaic mice twice weekly with IL-10R blocking antibody starting at 50 days post-transplant. We observed an increase in the chimerism of Jak2 V617F mutant cells in mice exposed to IL-10R blocking antibody as compared to mice exposed to PBS, both in peripheral blood (Fig 3A) as well as in the stem and progenitor cell compartment of the bone marrow (Fig 3B). Moreover, IL-10R blockade exaggerated the MPN phenotype, with increased spleen size (Fig 3C) and increased megakaryocytes in the bone marrow and spleen (data not shown).Together, these data demonstrate that IL-10R blockade accelerates inflammation induced exhaustion of WT HSC, likely due to increased cycling. Jak2 V617F HSC are protected from this accelerated exhaustion of HSC from IL-10R blockade, allowing them to gain a selective advantage in the context of chronic inflammation when IL-10R is blocked. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.