Granulocyte-monocyte Progenitors Research Articles

FLT3 ITD and NUP98 translocations Cooperate to Induce Type I Interferon Signaling That Impedes Hematopoietic Progenitor Differentiation

FLT3-Internal Tandem Duplication (FLT3 ITD) mutations occur at similar frequencies in both childhood and adult acute myeloid leukemia (AML) patients, but they cooperate with different initiating mutations at different ages. In childhood AML, FLT3 ITD often cooperates with translocations that generate fusion proteins, such as NUP98 translocations (NUP98-t). In adult AML, FLT3 ITD often cooperates with mutations that inactivate RUNX1, DNMT3A, TET2 and other genes that regulate self-renewal and myeloid differentiation. This raises the question of whether FLT3 ITD induces similar, or distinct, changes in gene expression and cell fate when it arises in the setting of pediatric and adult driver mutations. To answer this question, we evaluated pre-leukemic changes in hematopoiesis and gene expression in neonatal and juvenile mice that carried pediatric-like FLT3 ITD/NUP98-HOXD13 and adult-like FLT3 ITD/Runx1 DEL/+ mutation profiles. Both NUP98-HOXD13 (NHD13) and Runx1 DEL/+ severely depleted phenotypic hematopoietic stem cells (HSCs) when coupled with FLT3 ITD. However, FLT3 ITD/NHD13 cooperated to enhancer ectopic self-renewal capacity in lineage committed hematopoietic progenitor cells (HPCs) whereas FLT3 ITD/Runx1 DEL/+ did not. FLT3 ITD/NHD13 and FLT3 ITD/Runx1 DEL/+ both induced changes in gene expression and enhancer activation that were not seen with either mutation alone. These cooperative changes were almost entirely non-overlapping. The most notable cooperative change in FLT3 ITD/NHD13 HPCs was activation of a type I Interferon target gene signature. By contrast, this signature was actively repressed in FLT3 ITD/Runx1 DEL/+ HPCs due to mir155 activation. Thus, FLT3 ITD/NHD13 and FLT3 ITD/Runx1 DEL/+ mutations have largely non-overlapping, cooperative effects on hematopoietic cell fate and gene expression.Interferon signaling sustained the FLT3 ITD/NHD13 HPC population by impeding differentiation into more committed myeloid progenitors. When we deleted the type I interferon receptor (Ifnar) on a FLT3 ITD/NHD13 background, the HPC population was virtually eliminated, yet the pre-granulocyte monocyte (pGM) and granulocyte-monocyte progenitor (GMP) populations expanded. HPC proliferation and death did not change in the absence of Ifnar. These findings suggest that type I interferon impedes HPC differentiation into pGMs during the neonatal and juvenile stages of life. Of note, ectopic interferon was insufficient to enhancer HPC numbers in setting of a sole NHD13 mutation. Thus, interferon is necessary to sustain FLT3 ITD/NHD13 HPCs, but not sufficient, and other signaling pathways downstream of FLT3 ITD must cooperate with NHD13 in parallel to drive HPC expansion. To our surprise, Ifnar deletion did not reduce repopulating activity in FLT3 ITD/NHD13 bone marrow - as measured by limiting dilution transplants - it simply shifted repopulating activity to more committed pGMs. Our raise two important conceptual points. First, we show that different initiating mutations can elicit completely different transcriptional responses downstream of FLT3 ITD. Precision medicine strategies must account for these differences as effective therapies for adult FLT3 ITD AML may not yield similar success in the pediatric population. Second, type I interferon shapes pre-leukemic hematopoiesis in the setting of FLT3 ITD/NUP98-t mutations. In doing so, the mutant cells may hijack a program that we recently identified as promoting normal perinatal HPC expansion. DisclosuresNo relevant conflicts of interest to declare.

CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors

Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored.To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription.Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called ‘47S-FISH-Flow’ that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I).In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Plinabulin Rapidly (within 24 Hours) Reverses Myelosuppression Induced By Chemotherapy

▪IntroductionPlinabulin (Plin), a small molecule selective immune-enhancing microtubulin binding agent (SIMBA) has anti-cancer activity, and also prevents chemotherapy (Chemo) induced neutropenia (CIN). Plin has a rapid onset CIN prevention (1 st week) and G-CSF a delayed onset (2 nd week) of the cycle. Single agent Plin has non-inferior protection against CIN compared to Peg. Combining Plin with pegfilgrastim (Peg) has superior protection against CIN throughout the entire Chemo cycle (Blayney ASCO 2021). Plinabulin protected LSK cells in mice receiving myelosuppressive Chemo, which are cells equivalent to CD34+ in humans (Tonra Cancer Chemotherapy and Pharmacology. 2020). We evaluated the mechanism of Plin's rapid onset for CIN prevention in patients (pts).MethodsData from the CIN phase 2/3 Plin trials PROTECTIVE-1 (NCT03102606) with docetaxel 75 mg/m2 in pts with NSCLC, Breast Cancer, Prostate Cancer and PROTECTIVE-2 (NCT0329457) with docetaxel 75 mg/m2, doxorubicin 50 mg/m2, cyclophosphamide 500 mg/m2 (TAC) in early stage Breast Cancer was analyzed for pts receiving either Plin (n=228) or Control (no Plin); n=172). Both studies 105 and 106 had patients randomized to either Plin or Pegfilgrastim, which latter was given on the next day of Chemo, on day (D)2. The safety blood draw, taken on D2 at predose Pegfilgrastim, served as a no-treatment Control on D2 in this blood cell analysis for comparison with Plin, which was given on D1. The Plin dose was 20 mg/m2 or 40 mg, and was given as a single dose per cycle, by 30 min IV infusion, 30 min after Chemo on day (D)1. The 40 mg fixed dose is equivalent to 20 mg/m2 Plin. Absolute neutrophil count (ANC), monocyte (M), eosinophil (E), basophil (B), lymphocyte (L), erythrocyte (RBC), and platelet (P) counts were obtained from peripheral blood draws and analyzed by Central Laboratory (Covance). The primary analysis was to compare cell counts between Plin and Control in blood draws taken 24 hour (hr)post-Chemo (thus D2). Separately, we also established Spearman correlation coefficients (r) of scatter plots between ANC and M,E,B,L,RBC, or P count on D2, in a broader dataset that also included Plin doses of 5,10 and 30 mg/m2 in addition to 0 and 20 mg/m2 (40 mg fixed) doses to allow for dose-response evaluation (total n=451). D2 cell counts were expressed as change from pre-dose D1, in absolute values.ResultsAt predose D1 (baseline), counts of ANC, M,E,B,L,RBC, and P were similar between the Plin and Control groups (p=NS). On D2, at 24 hr post-Chemo, ANC, M,E and B ,had all significantly decreased compared to D1 in the Control group (indicating myelosuppression by Chemo), whereas significantly increased with Plin (suggesting reversal of this myelosuppression), albeit within normal range (Table below). At 24 hr post-Chemo, counts for L and RBC had significantly decreased compared to D1 with both Control and Plin, however this decrease was ~50% less with Plin compared to Control (P<0.001). No difference in P counts were observed between Plin and Control at 24 hr post Chemo (p=0.69). Correlative analyses showed that at 24 hr post-Chemo (D2), ANC was positively correlated with counts of M (r=+0.42; p<0.0001), E (r=+0.16; p=0.0008), B (r=+0.20; p<0.0001), L (r=+0.32; p<0.0001), RBC (r=+0.07; p=0.1187) and P (r=+0.18; p=0.0002). Thus, with larger increase in ANC counts on D2, the larger were the increase in the other blood cell types. The observed results were Plin dose-dependent, and consistent between pts receiving either docetaxel alone or TAC . Shown below are mean (SD) of absolute cell count on D2, expressed as change from predose D1.ConclusionPlin rapidly (within 24 hours) reversed myelosuppression induced by Chemo, by protecting granulocyte-monocyte-progenitor (GMP) stem cells (responsible for producing ANC, M, B and E cells) or progenitor cells further upstream in the hematopoietic lineage tree. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interleukin-1/Toll-like Receptor Inhibition Can Restore the Disrupted Bone Marrow Microenvironment in Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice.Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors.Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p<0.05). In CFU-C assays, NHD13→IL1R1 wt and NHD13→IL1R1 ko demonstrated similar levels of CFU-activity whereas CFU-fibroblast (CFU-F) assays of IL1R1 wt recipients demonstrated lower numbers of large colonies (reported to contain highly proliferative mesenchymal stem cells (MSC)). Flow cytometry analysis of hematopoietic stem and progenitor cells (HSPC) population in IL1R1 wt recipients showed increased myeloid progenitors and decreased long-term HSC (LT-HSC) compared to IL1R1 ko recipients. Cytokine/chemokine profiles revealed that the inflammatory cytokines (IL-6 and TNFα) and macrophage activating cytokines (MCP-1 and M-CSF) were significantly decreased in NHD13→IL1R1 ko compared to NHD13→IL1R1 wt. Next, we treated MDS model mice with inhibitors. The pharmacological targeting of IL1R1 signaling in vivo was associated with decreased NHD13 cell burden and improvement of macrocytic anemia. CFU-C assays demonstrated some decreases in NHD13 CFU capacity post-treatment. Interestingly, non-NHD13 HSPCs assessed by CFU capacity increased in IRAK4 inhibitor treated mice. Consistent with this, flow cytometric analyses of HSPC pools demonstrated decreased NHD13 HSPCs (LT-HSC and granulocyte-monocyte progenitor cells) and increased non-NHD13 HSCs and myeloid progenitors compared to vehicle group. BMME cell populations showed that arteriolar endothelial cells and MSCs were also affected by drug treatment and both IL1R antagonist and MCC950 increased the number of large CFU-F colonies. Analysis of BMEF revealed the decreased IFNγ and IL-18 and upregulated M-CSF in MCC950 treated group. The cytokines of human BM serum revealed higher concentrations of soluble formed IL1R1, IL-18, CXCL1, and osteopontin in MDS compared to young or aged normal donors.Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. DisclosuresNo relevant conflicts of interest to declare.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Discovering Myeloid Cell Heterogeneity in Mandibular Bone - Cell by Cell Analysis.

The myeloid-derived bone marrow progenitor populations from different anatomical locations are known to have diverse osteoclastogenesis potential. Specifically, myeloid progenitors from the tibia and femur have increased osteoclast differentiation potential compared to myeloid progenitors from the alveolar process. In this study, we explored the differences in the myeloid lineage progenitor cell populations in alveolar (mandibular) bone versus long (femur) bone using flow cytometry and high-throughput single cell RNA sequencing (scRNA-seq) to provide a comprehensive transcriptional landscape. Results indicate that mandibular bone marrow-derived cells exhibit consistent deficits in myeloid differentiation, including significantly fewer myeloid-derived suppressor cell (MDSC)-like populations (CD11b+Ly6C+, CD11b+Ly6G+), as well as macrophages (CD11b+F4/80+). Although significantly fewer in number, MDSCs from mandibular bone exhibited increased immunosuppressive activity compared to MDSCs isolated from long bone. Using flow cytometry panels specific for bone marrow progenitors, analysis of hematopoietic stem cells showed no defects in mandibular bone marrow in LSK (Lin–Sca1+cKit+) cell and LK (Lin–Sca1–cKit+) cell populations. While there was no significant difference in granulocyte progenitors, the granulocyte-monocyte progenitors and monocyte progenitor population were significantly decreased in the mandibular bone marrow. T-lymphocyte subsets were not significantly different between mandibular and femoral bone, except for CD4+CD25+Foxp3+ regulatory T lymphocytes, which were significantly increased in the mandible. In addition, B lymphocytes were significantly increased in mandible. Single cell RNA sequencing from mandible and femur BM revealed distinct differences in transcriptomic profiles in myeloid populations establishing previously unappreciated aspects of mandibular bone marrow populations. These analyses reveal site-specific differences in the myeloid progenitor cellular composition and transcriptional programs providing a deeper appreciation of the complex differences in myeloid cell heterogeneity from different anatomical bone marrow sites.

Monocyte transcriptomes from patients with axial spondyloarthritis reveal dysregulated monocytopoiesis and a distinct inflammatory imprint

BackgroundIn patients with axial spondyloarthritis (axSpA), monocytes show a pre-activated phenotype. Gut inflammation is a trigger of monocyte activation and may also affect their development in the bone marrow (BM). As gut inflammation is commonly observed in axSpA patients, we performed a detailed analysis of monocyte transcriptomes of axSpA patients in two cohorts and searched for signs of activation and developmental adaptations as putative imprints of gut inflammation.MethodsTranscriptomes of blood CD14+ monocytes of HLA-B27+ axSpA patients and healthy controls (HC) were generated by microarrays from cohort 1 and by RNA-sequencing from cohort 2. Differentially expressed genes from both analyses were subjected to gene set enrichment analysis (GSEA) and to co-expression analysis in reference transcriptomes from BM cells, blood cells and activated monocytes. As serological markers of translocation, 1,3 beta-glycan, intestinal fatty acid binding protein, and lipopolysaccharide binding protein (LBP) were determined by LAL and ELISA.ResultsTranscriptome analysis identified axSpA-specific monocyte signatures showing an imprint of LPS/cytokine-activated monocytes, late granulopoietic BM cells, blood neutrophils, and G-CSF-mobilized blood cells, which suggests LPS/TNF activation and more prominent BM adaptation promoting a neutrophil-like phenotype. GSEA mapped axSpA upregulated genes to inflammatory responses and TNFα signaling and downregulated probe-sets to metabolic pathways. Among translocation markers, LBP levels were significantly increased in axSpA patients vs. HC (p < 0.001).Stratified analysis by disease activity and stage identified an “active disease signature” (BASDAI ≥ 4) with an imprint of LPS/cytokine-activated monocytes and CD16+ monocyte subsets. The “AS signature” (vs. non-radiographic axSpA) showed a reinforced neutrophil-like phenotype due to deprivation of dendritic cell transcripts.ConclusionsThe neutrophil-like phenotype of axSpA monocytes points towards a biased monocytopoiesis from granulocyte-monocyte progenitors. This shift in monocytopoiesis and the LPS/cytokine imprint as well as the elevated LBP levels are indicators of systemic inflammation, which may result from bacterial translocation. The BM adaptation is most prominent in AS patients while disease activity appears to be linked to activation and trafficking of monocytes.

Depleting hypothalamic somatostatinergic neurons recapitulates diabetic phenotypes in mouse brain, bone marrow, adipose and retina.

Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sstcre/gfp mice (SST-DTA). SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2hi) monocytes. SST-DTA mice exhibited a 'diabetic retinopathy-like' phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice. The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation

AbstractHistone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor–like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

Essential role for Gata2 in modulating lineage output from hematopoietic stem cells in zebrafish

The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.

Overexpression of Calreticulin Impairs Self-Renewal Capacity of Hematopoietic Stem Cells

Mutations in JAK2, c-MPL and CALR have all been identified as drivers of MPN pathogenesis. JAK2 and c-MPL are critical for hematopoietic stem cell self-renewal and differentiation, however CALR's physiologic role in hematopoiesis is unknown. To investigate CALR's role in normal and malignant hematopoiesis lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with CALRDEL, CALRWT or empty MSCV-IRES-hCD4 vector retrovirus. As expected, mice transplanted with CALRDEL developed thrombocytosis, mice transplanted with CALRWT and empty vector had normal peripheral blood counts. At 6 months post-transplant mice were sacrificed. The percentage of hCD4+ whole bone marrow cells (marks cells expressing gene of interest) was 76.6% for empty vector, 73.1% for CALRWT, and 8.6% for CALRDEL. hCD4+ hematopoietic stem/progenitor cells (LKS), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP), and megakaryocyte erythroid progenitors (MEP) from empty vector, CALRWT and CALRDEL mice were sorted into methylcellulose supplemented with cytokines (mIL-3, hEPO, and mSCF). MEP from CALRWT mice formed fewer colonies compared to empty vector and CALRDEL (p>0.004). GMP, CMP, and LKS from empty vector, CALRWT and CALRDEL formed equivalent numbers of colonies. To assess self-renewal function colonies were amalgamated and serially replated into methylcellulose at a concentration of 10,000 cells/ml. Upon serial replating CALRWT LKS cells formed fewer colonies compared to CALRDEL and empty vector (p>0.001). To more specifically assess the effect of CALR on HSC self renewal potential whole bone marrow from mice transplanted with CALRDEL, CALRWT or empty MSCV-IRES-GFP (MIG) vector was serially transplanted into lethally irradiated C57B/6 secondary recipient mice and the percentage of GFP-positive cells in the peripheral blood was measured by flow cytometry over time. Mice transplanted with CALRWT bone marrow cells quickly lost GFP positive cells in the peripheral blood over time whereas MIG empty vector and CALRDEL mice retained GFP positive cells for at least 8 months post-transplant.Because mutant CALRDEL interacts with and activates the thrombopoietin receptor (c-MPL) to drive MPN and because the defect in hematopoietic stem cell self-renewal that we observed in mice overexpressing CALRWT is reminiscent of the phenotype of mice lacking c-MPL we hypothesize that CALRWT interacts with c-MPL and negatively affects its function. Using co- immunoprecipitation of Ba/F3 cells doubly transduced with GFP tagged c-MPL and hCD4 tagged empty vector, CALRWT or CALRDEL, we found that CALRWT and CALRDEL interacts with c- MPL.In summary, these data demonstrate that overexpression of CALRWT negatively affects the self-renewal capacity of hematopoietic stem cells, evidenced by impaired ability to reconstitute secondary hosts and reduced serial re-plating ability in methylcellulose. This may be due to CALRWT binding to c-MPL and negatively regulating its function. DisclosuresNo relevant conflicts of interest to declare.

MiR-199b-5p Differentially Modulates Hematopoietic Stem Cell Self Renewal in Steady State and Stress Hematopoiesis

The emergence of microRNAs (miRNAs) is providing novel insights into the molecular control networks underlying hematopoiesis and diseases that stem from aberrations in hematopoiesis. We, amongst others, have shown that miR-199b is significantly downregulated in acute myeloid leukemia (AML) and is a promising prognostic factor for FAB-M5 subtype. Herein, to determine miR-199b's role in steady state and stress hematopoiesis, we utilized our recently generated miR-199b knockout (KO) mice (CRISPR approach) to assess frequencies of hematopoietic stem progenitor cells (HSPCs) in WT and miR-199b KO mice through flow cytometry. Under steady state conditions, deletion of miR-199b resulted in significant reduction of Lin- Sca-1+ c-Kit+ (LSK), long-term hematopoietic stem cells (LT-HSCs) (Lin− CD117+ Sca1+ CD150+ CD34low/− CD48−) and increases in multipotent progenitors (MPPs) (Lin− CD117+ Sca1+ CD150− CD34+). However, 5FU induced stress hematopoiesis resulted in significant increases in frequencies of LSK cells, LT-HSCs, and Lin- Sca-1- c-Kit+ (KL) cells compared to wild-type (WT) litter mates. Furthermore, silencing of miR-199b resulted in a significant increase of granulocyte monocyte progenitors (GMP), which was reversed during stress hematopoiesis. The highest expression levels of miR-199b were restricted to the LT-HSC fraction. Mir-199b levels remained moderately high in MPPs and LSK cells. Primarily within the LSK population, miR-199b expression was most pronounced in LT-HSCs and to a lesser extent, MPPs, indicating that miR-199b may play a functional role in LT-HSC activity. Gene profiling studies in HSPCs identified Integrin alpha 8 (Itga8) as a conserved target of miR-199b. We then examined Itga8 transcript levels in HSCs from miR-199b knockout (-/-) mice and found significant elevation of Itga8 transcripts compared to WT mice. One possible mechanism for the differential expansion of miR-199b-/- HSPCs under steady state and stress is an increased or decreased rate of cell cycling. To test this, we examined cell proliferation using Ki67 and Hoechst 33342 markers. MiR-199b-/- LT-HSC at steady state showed significantly decreased proliferation whereas miR-199b-/- LT-HSCs under 5FU induced stress showed significant expansion when compared to wild-type. Next, we tested the adhesion abilities of LT-HSCs from miR-199b KO mice under both steady state and stress hematopoiesis. At steady state, miR-199b-/- LT-HSCs significantly elevated adhesion to vitronectin and fibronectin. Interestingly, like HSPC frequencies, stress hematopoietic-derived miR-199b-/- LT-HSCs showed significantly decreased adhesion to both vitronectin and fibronectin. These findings imply that miR-199b intrinsically regulates HSC behavior, including cell adhesion. To elucidate whether miR-199b's effects on LT-HSCs are mediated by Itga8, we performed an Itga8 knockdown in WT and miR-199b-/- LT-HSCs. Cell proliferation assessed by Ki67 positive cells revealed that downregulation of Itga8 in miR-199b KO LT-HSCs reverts to a miR-199b-induced decrease of LT-HSC proliferation. Intriguingly, we observed that silencing of Itga8 reversed the miR-199b-induced increase of cell adhesion. To corroborate that miR-199b-/- LT-HSCs are fully functional, we performed a competitive repopulation assay. BM derived lineage negative hematopoietic progenitor cells were further purified via immunomagnetic selection for SLAM marker purified (CD150+, CD244+ and CD48-) to enrich the LT-HSC fraction. MiR-199b-/- or miR-199b+/+ LT-HSCs (n=100 LT-HSCs) were transplanted intravenously together with 2.5 X 105 competitor recipient cells. Overall donor BM cellularity was markedly increased in LT-HSCs derived from 5FU treated miR-199 KO mice and overall donor BM cellularity was markedly decreased in LT-HSCs derived from steady state miR-199 KO mice. This suggested that under stress, miR-199b-/- LT-HSCs self-renew extensively after transplantation to repopulate the recipient. The analyses of HSPCs in BM of primary recipients revealed a significant increase in donor LT-HSCs in recipients that were transplanted with LT-HSCs derived from the 5FU induced stress model. Intriguingly, this didn't translate into subsequent increases in MPP, GMP or MEPs, suggesting that miR-199b may govern the self renewal of LT-HSCs and also have progenitor cell specific roles as evident from its expression during early hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute Myeloid Leukemia Classification System That Associates Normal Myeloid-Cell Subset Gene Signatures with Prognosis

Background: Acute Myeloid Leukemia (AML) represents a group of heterogeneous clonal disorders of hematopoietic progenitor cells that are driven by multiple genetic events that impair cell differentiation and apoptosis and invoke uncontrolled cell proliferation. Consequently, immature myeloid blasts rapidly accumulate in the bone marrow and block the generation of mature end stage cells, which eventually causes potentially fatal infections and hemorrhages. AML is characterized by extensive genetic and phenotypic heterogeneity, which currently is diagnostically classified based on blast morphology defined by the French-American-British (FAB) Cooperative Group, cytogenetic abnormalities, and improved knowledge of genomic and epigenomic alterations. However, this has not yet resulted in treatment progress. AML patients are still routinely treated with a standard first-line combinatorial chemotherapy regimen with an overall survival rate remaining below 20%. Therefore, there is an unmet need for novel methods to stratify patients into therapy-relevant subtypes.Concept and Aims: We hypothesize that phenotypic heterogeneity of AML is a consequence of dysregulated differentiation and that classification based on transcriptional reminiscence of cell of origin (COO) during AML transformation is of pathogenetic impact and therefore provide diagnostic and prognostic value. It is the aim of this project to generate and validate a COO classification system based on normal myeloid-cell-subset-associated gene signatures (MAGS) to stratify AML patients into therapy-relevant and prognostic subtypes.MAGS Generation: Based on gene expression profiles of publicly available sorted normal myeloid subpopulations, we generated a COO classifier including the CD34+/CD38- hematopoietic stem cells (HSC), CD34+/CD38+/CD45RA- megakaryocyte-erythroid progenitors (MEP), and CD34+/CD38+/CD45RA+ granulocytic-monocytic progenitors (GMP) using regularized multinomial regression with 3 discrete outcomes and the elastic net penalty. The regularization parameters were chosen by cross validation.Results: MAGS prediction accuracy was validated in an independent dataset (n = 38; accuracy = 0.79) of sorted normal myeloid subpopulations. The prognostic value of MAGS assignment was studied by survival analyses using the Kaplan Meier method, log-rank test statistics, and simple and multiple Cox proportional hazards regression analysis in 2 clinical cohorts (TCGA: n = 115; GSE6891: n = 431). MAGS assignment had significant prognostic associations with overall survival (Figure 1; Table 1A) revealing that the linage committed MAGS subtypes GMP and MEP showed superior prognosis compared to the undifferentiated AMLs captured by the MAGS HSC subtype. Furthermore, multiple Cox regression analysis with MAGS assignment (HSC, GMP, MEP), FAB score (M0-M6), cytogenetics (good, intermediate, poor), and age (dichotomized into <55 and ≥55) as independent explanatory variables showed that MAGS assignment provides independent prognostic information (Table 1B), indicating that the MAGS classification captures distinct pathogenetic and prognostic knowledge not already explained by the FAB- or cytogenetic classifications. Molecular characterization of MAGS subtypes by differential gene expression analysis, gene set enrichment analysis, and identification of subtype specific mutation patterns of well-documented AML driver mutations indicated a reduced proliferation rate as well as overrepresentation of EVI1 mutations associated with the poor responding HSC subtype.Conclusion: In accordance with our previous reports in malignant B-cell disorders, we have documented a novel classification system that associates normal myeloid progenitor subsets with AML subtypes of distinct pathogenetic and prognostic importance not already explained by the FAB- or cytogenetic classifications. Further pathogenetic studies are needed to develop a diagnostic platform using MAGS-assigned subtypes to improve disease management. [Display omitted] DisclosuresSeverinsen:ARIAD: Research Funding; Celgene: Research Funding; Novartis: Research Funding. El-Galaly:Roche: Other: Travel funding; Takeda: Other: Travel funding.

Severe Congenital Neutropenia-Associated Mutations Reveal Stage-Specific Gfi1-Dependent Checkpoints in Myeloid Development

Growth factor independent-1 (GFI1) is a zinc-finger transcription factor that is critically required for both murine and human granulopoiesis. Mutations in GFI1 zinc fingers (e.g. N382S in zinc finger 5) have previously been found in humans with severe congenital neutropenia (SCN). We noted that clinical resequencing of neutropenic patients identified a number of novel GFI1 sequence changes of unknown significance. To clarify their impact, we used viral vectors to force expression of 13 GFI1 polymorphisms/mutations in primary-murine progenitors followed by colony-forming-unit assays for differentiation. While these assays infer new potential clinical significance to some genetic alterations, we focused on the sixth zinc finger of GFI1. While Gfi1 zinc-finger 6 was previously found to be dispensable for DNA binding and repression of a high affinity GFI1 DNA-binding-site reporter, the biological impact of expressing GFI1 zinc finger 6 mutants suggests a functional role for this domain in granulopoiesis. Therefore, we generated mice with patient-derived SCN-associated mutations (zinc finger 5; N382S, zinc finger 6; K403R, and R412X) in the murine Gfi1 locus. The resulting defects in murine neutrophil production are proportional to the severity of SCN in patients with the same GFI1 mutation (ranging from mild to profound neutropenia); with neonates displaying more profound defects. Neonatal and adult Gfi1N382S/- and Gfi1R412X/- mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. While Gfi1-/- HSC are profoundly abnormal, SCN-associated mutant HSC are phenotypically normal; dissociating Gfi1 control of hematopoietic stem cell biology from granulopoiesis. Similar to Gfi1-/-, the Gfi1N382S/- and Gfi1R412X/- mice accumulate cells within the “granulocyte monocyte progenitor” (GMP) gate as well as those that express Sca1 (known as emergency GMP; eGMP); however, unlike Gfi1-/- mice, the serum levels of known cytokine inducers of eGMP are normal in SCN-associated mutants. Moreover, isolated progenitors from all three mutants form significantly more monocytic colonies at the expense of granulocytic colonies in methylcellulose assays. Together, these data suggest that the mutations induce a cell-intrinsic defect.We recently utilized single-cell RNA-Seq (scRNA-Seq) to identify a gene regulatory network (GRN) centered on the Gfi1 and Irf8 transcription factors that underlies the granulocytic versus monocytic lineage decision of myeloid progenitors at steady state. To pinpoint the molecular deregulation underlying Gfi1-mutant progenitors, we performed scRNA-Seq. Notably, Gfi1R412X/- induced deregulation of gene expression which increases in amplitude and complexity as myeloid differentiation proceeds; coincident with induction of Gfi1 expression. ChIP-Seq analysis reveals that most of the deregulated genes are normally bound by Gfi1. Using murine fluorescent-protein knock-in reporter alleles for Gfi1 and Irf8, we demonstrate the stage-specific regulation of the granulocyte-monocyte lineage decision, and how SCN mutations deregulate the Gfi1-Irf8 network. Finally, we developed an alternative flow-cytometric strategy that highlights individual Gfi1-dependent checkpoints in myeloid differentiation (identified by comparative analysis of Gfi1-/- versus knock-in mutants). We find that the accumulated Gfi1N382S/- GMP are unspecified (similar to Gfi1-/-); however, Gfi1R412X/- GMP exhibit only a modest reduction in the granulocytic specified population and instead display a profound block at granulocytic commitment. The latter effects can be partially explained by the protein-destabilizing effect of mutations in zinc finger 6; suggesting that there are dose-dependent roles for Gfi1 in granulopoietic specification and commitment. In summary, Gfi1 knock-in mice provide a mouse model of human SCN, and both flow-cytometric and scRNA-Seq dissection of hematopoiesis reveal myelopoietic-stage-specific deregulation to both block granulopoiesis and deregulate monocytic subsets. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Population-Based Genetic Fine Mapping and Single-Cell Epigenomics Provide Insights into the Regulation of Human Hematopoiesis

A wide spectrum of variation exists in blood cell traits, such as hemoglobin levels and blood cell counts, yet how this variation is genetically regulated across the hematopoietic lineage remains largely unknown. Single-cell transcriptomics has recently found that hematopoietic lineage commitment occurs in earlier populations of progenitor cells than previously thought, but the functional consequences of these refined models have not yet been explored. Here, we integrate population-based genetics with single-cell chromatin landscapes to gain novel insights into the lineage-specific regulation of human hematopoiesis.First, we conducted a genome-wide association study (GWAS) using data from the UK Biobank, consisting of 113,000 individuals of predominantly European descent. We measured the effects of genetic variation on 16 blood cell traits, including red blood cell counts, platelet counts, and white blood subtype cell counts. Genetic correlations revealed significant correlations between traits from the same lineage (Fig. 1A), such as red blood cell count and hemoglobin (R2 = 0.91, Bonferroni p < 0.001), as well as others associated with distinct lineages, such as platelet count and lymphocyte count (R2 = 0.29, Bonferroni p < 0.001), suggesting that regulation of hematopoiesis occurs across various stages of lineage commitment.A total of 2,178 regions (each 3 megabases in size) contained sentinel variants with genome-wide significant associations across the 16 blood cell measurements. We performed Bayesian fine mapping on all common variants (MAF > 0.1%) within each region to resolve specific variants driving phenotypic differences. 36,919 variants had >1% posterior probability of being causal for a trait association, and 1,110 regions (51%) contained a top variant with >50% causal probability, indicating that fine mapping successfully pinpointed a large number of causal variants.A major advantage of our approach was the ability to identify multiple causal hits per association locus. For example, in addition to a primary hit in an intronic region, our analysis confidently identified a secondary variant (rs4739140) near IL7, a cytokine critical for B lymphopoiesis (Fig. 1B). This variant fails to reach genome-wide significance independently (p = 9.2e-07), but is likely causal due to its low linkage disequilibrium and distinct effect size relative to nearby associations. Interestingly, when we overlay rs4739140 with chromatin dynamics in 20 primary human hematopoietic cell types using assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq), we find that this variant falls within a nucleosome depleted region (NDR) in early progenitors such as hematopoietic stem cells, multipotent progenitors, and lymphoid-primed multipotential progenitors, but not lineage restricted cells such as B cells. These findings suggest that rs4739140 may affect an enhancer for IL7 that impacts early lymphoid commitment.In addition to 20 bulk ATAC-seq populations, we quantified enrichment of fine mapped GWAS variants in the chromatin landscapes of 2,034 single cells isolated from 8 immunophenotypically-defined hematopoietic progenitor populations. Enrichment analyses identified that most genetic variation was enriched for NDRs within relevant lineage restricted precursors, such as mean corpuscular volume within megakaryocyte-erythroid progenitors and eosinophil count within granulocyte-monocyte progenitors (Fig. 1C, D). However, one subgroup of common myeloid progenitor (CMP) cells was enriched for genetic variation associated with eosinophil count but not other granulocyte traits such as basophil count or neutrophil count, suggesting that eosinophil lineage commitment may actually occur in a distinct subpopulation contained in the CMP population.By integrating GWAS fine mapping with bulk and single-cell chromatin data, we identified novel putative causal variants affecting 16 blood traits and refined our understanding of the exact stages at which hematopoietic lineages are specified. To our knowledge, this is the first study to characterize the phenotypic consequences of genetic variation across the hematopoietic lineage at single-cell resolution. A better understanding of human hematopoiesis can contribute to knowledge of how these processes go awry in diseases of blood cell production, including cytopenias and malignant disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MLL3 Deletion Enhances HSC Self-Renewal

Mixed lineage leukemia 3 (MLL3 ) is one of the most commonly mutated genes in human cancer, and it is believed to function as a tumor suppressor. It is one of many genes affected as a result of mono-allelic 7q deletions that occur commonly in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). MLL3 encodes a histone methyltransferase that catalyzes the monomethylation of Histone 3 lysine 4 (H3K4), a known feature of enhancer elements. MLL3 also nucleates a COMPASS-like complex that removes repressive H3K27me3 marks, recruits the histone acetyltransferase p300/CBP to enhancers and promotes transcript elongation at promoters. For these reasons, MLL3 binding at cis-regulatory elements is thought to potentiate enhancer activity and transcription. Previous studies have shown that Mll3 deficiency causes a mild myeloproliferative disorder in mice (PLoS ONE 11:e0162515), and shRNA knockdown of Mll3 has been shown to cooperate with loss of Nf1 and p53 to promote AML (Cancer Cell 25:652). In the latter study, Mll3 silencing was associated with a hematopoietic stem cell (HSC)-like gene expression signature within the AML cells. These studies implicate MLL3 as a hematopoietic tumor suppressor, though its precise function in HSC self-renewal and lineage commitment has not been described.To better understand how Mll3 regulates HSC self-renewal, we developed a novel loss-of-function mouse allele in which a 5-base pair deletion and frameshift mutation was created near the N-terminus of the protein. Homozygous Mll3-/- mice are viable through most of gestation, but they die just prior to birth. Heterozygous Mll3+/- mice are born at normal Mendelian ratios, their development is normal after birth and they are fertile. To test whether Mll3 regulates hematopoiesis, we determined the frequencies and absolute numbers of HSCs, lineage restricted hematopoietic progenitors (HPCs), and granulocyte-monocyte progenitors (GMPs) in control, Mll3+/- and Mll3-/- fetal livers (E18.5) and control and Mll3+/- adult bone marrow. Heterozygous and homozygous Mll3 deletion caused a modest increase in HSC numbers and a modest decrease in HPC frequency in fetal livers. Mll3 heterozygosity resulted in a significant expansion of the HSC population in adult bone marrow. To test whether these increases in HSC numbers reflected enhanced self-renewal, we performed serial transplantation assays with fetal and adult HSCs. While primary transplants showed only a modest increase in repopulating activity in Mll3+/- and Mll3-/- HSCs, this effect was greatly enhanced in secondary transplants. Thus, Mll3 deficiency enhances HSC self-renewal capacity. Despite this phenotype, RNAseq showed no differences in the transcriptomes of wild type and Mll3+/- HSCs or HPCs. This suggests that Mll3 loss may reduce the probability of lineage commitment during successive HSC divisions without altering the commitment pathways themselves. Ongoing experiments will test this hypothesis. Enhanced self-renewal, particularly under conditions of hematopoietic stress, may convey a selective advantage to Mll3 -heterozygous HSCs and enhance progression to MDS. DisclosuresNo relevant conflicts of interest to declare.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The C-terminal hypervariable region (HVR) of Ras proteins specifies post-translational modifications required for their subcellular localization and biologic function. The HVR of all four Ras isoforms terminates with a CAAX motif that is prenylated by farnesyltransferase, producing a weak membrane binding affinity that is further stabilized by a second signal motif. For K-Ras4b, this signal is provided by a polybasic lysine domain that allows direct localization of the protein at the plasma membrane (PM). By contrast, H-Ras, N-Ras, and K-Ras4a PM localization requires palmitoylation at cysteine(s) adjacent to the CAAX motif and is maintained through a dynamic cycle regulated by depalmitoylation and repalmitoylation. Importantly, these differences raise the provocative possibility that inhibiting palmitoylation could selectively target NRAS mutant cancer cells without toxicity to normal cells that retain intact K-Ras4b function.Indeed, Nras oncogenes with a cysteine-to-serine second site amino acid substitution at the palmitoylation site (C181S) exhibited defective transforming activity in hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo (Cuiffo et al., Blood 2010; Xu et al., Blood 2012). However, these studies relied on retroviral promoters to express oncogenic N-RasG12D that resulted in supra-physiological protein levels in HSPCs. To overcome this limitation, we retargeted embryonic stem cells that were used to generate Lox-STOP-Lox (LSL) NrasG12D/+ knock in mice ( Haigis et al., Nat Genet 2008 ) to create a NrasG12D,C181S allele (hereafter called NrasC181S). We then performed intercrosses to generate Mx1-Cre, NrasC181S/C181S mice as well as cohorts of congenic Mx1-Cre, NrasWT/WT and Mx1-Cre, NrasG12D/G12D control animals. As expected (Li et al ., Blood 2011; Wang et al ., Cell Cycle 2011), Mx1-Cre, NrasG12D/G12D mice that were injected with poly(I:C) at weaning had a mean white blood cell (WBC) count of 58.3 x 103/µL at six months of age (Fig.1 A) and died from hematologic disease after 225 days (range 119-267 days, n= 14, Fig. 1B). By contrast, Mx1-Cre, NrasC181S/C181S mice had normal WBC counts at the same time point (mean: 11.34x103 WBC/µL) and remained well until 19-20 months of age (n=12). The difference in survival between these groups was highly significant (p=3x10-6 by Kaplan-Meier analysis). Detailed analysis of the hematopoietic compartment at six months of age (n=5 per genotype) revealed profound splenomegaly (mean weight: 1.272g) in NrasG12D/G12D mice, but normal spleen weights in the NrasWT/WT and NrasC181S/C181S animals (Fig. 1C). The expansion of granulocyte-monocyte progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and more primitive CD48+ Lin- c-Kit+ Sca1+ (LKS) cells previously observed in NrasG12D/G12D mice was also rescued by the C181S second site mutation. Furthermore, in vitro culture of HSPC-enriched bone marrow cells over a range of granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations revealed normal cytokine sensitivity of NrasC181S/C181S cells, while NrasG12D/G12D cells were characterized by cytokine-independent growth and GM-CSF hypersensitivity as previously described. Importantly, Ras-GTP levels were similarly elevated in bone marrow cells from both NrasG12D/G12D and NrasC181S/C181S mice compared to wild-type cells. This indicates that the C181S mutation abrogates the cytokine hypersensitivity and increased cell proliferation that results from oncogenic N-RasG12D expression without reverting the canonical biochemical consequence of the oncogenic G12D substitution. This observation, in turn, suggests that abnormal subcellular localization and trafficking of oncogenic N-Ras potently suppresses myeloid transformation. Taken together, these in vivo studies directly validate the palmitoylation/depalmitoylation cycle as a therapeutic target in hematologic malignancies and potentially in other cancers characterized by oncogenic NRAS mutations. [Display omitted] DisclosuresFirestone:Calico Life Sciences: Employment.

IL-6 contributes to metastatic switch via the differentiation of monocytic-dendritic progenitors into prometastatic immune cells

BackgroundMetastasis is the major cause of death in patients with cancer. Myeloid skewing of hematopoietic cells is a prominent promoter of metastasis. However, the reservoir of these cells in the...

Impact of aging on monocyte subset production by bone marrow progenitors

Abstract Monocytes can patrol the circulation or be recruited into tissues to become macrophages or monocyte-derived dendritic cells (moDCs). We previously showed that monocytes arise via two independent pathways in mouse bone marrow: Granulocyte-Monocyte Progenitors (GMPs) produce neutrophils and monocytes, and Monocyte-DC Progenitors (MDPs) yield monocytes and conventional and plasmacytoid DCs (cDCs and pDCs). Notably, both pathways produce monocytes that can differentiate into macrophages, but moDCs arise exclusively via the MDP pathway. We also showed that the microbial stimuli LPS and CpG promote monocyte production by GMPs and MDPs, respectively. We are now investigating how aging impacts the balance of monocyte production by GMPs and MDPs. scRNAseq analysis revealed elevated Cd74 and H2 gene expression by steady-state classical monocytes from old mice compared to young mice, with a larger proportion of monocytes expressing these genes. Consistent with this, we observed proportionally more monocytes expressing CD74 and MHCII in the bone marrow and circulation of old mice by flow cytometry, and a higher yield of CD11c+ MHCII+ moDCs in GM-CSF cultures of MDPs and monocytes from old bone marrow. These findings are indicative of elevated moDC production during aging, perhaps reflecting the more inflammatory microenvironment. In ongoing studies, we are investigating whether imbalanced monocyte production by the GMP and MDP pathways underlies increased moDC production during aging.

Angiotensin II modulates the murine hematopoietic stem celland progenitors cocultured with stromal S17 cells.

Although the existence of the renin-angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1+ /Mac-1+ cells without changes in Terr119+ cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67+ protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans.