Megakaryocyte-erythrocyte Progenitors Research Articles

Mapping Lung Hematopoietic Progenitors: Developmental Kinetics and Response to Influenza A Infection.

The bone marrow is a specialised niche responsible for the maintenance of hematopoietic stem and progenitor cells during homeostasis and inflammation. Recent studies however have extended this essential role to the extramedullary and extravascular lung microenvironment. Here, we provide further evidence for a reservoir of hematopoietic stem and progenitor cells within the lung from embryonic day 18.5 until adulthood. These lung progenitors display distinct microenvironment-specific developmental kinetics compared to their bone marrow counterparts, exemplified by a rapid shift from a common myeloid to megakaryocyte-erythrocyte progenitor dominated niche with increasing age. In adult mice, Influenza A viral infection results in a transient reduction in multipotent progenitors within the lungs, with a parallel increase in downstream granulocyte-macrophage progenitors and dendritic cell populations associated with acute viral infections. Our findings suggest lung hematopoietic progenitors play a role in re-establishing immunological homeostasis in the respiratory mucosa, which may have significant clinical implications for maintaining pulmonary health following inflammatory perturbation.

Abstract 5630: Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML

Abstract Acute Myeloid Leukemia (AML) represents a diverse category of blood cancers marked by the clonal proliferation of atypical myeloid precursor cells within the bone marrow. Lysine Specific Demethylase 1 (LSD1), an epigenetic regulator, exhibits elevated expression in AML. Consequently, inhibiting LSD1 has emerged as a promising therapeutic approach for addressing blood and bone marrow cancers. While initial emphasis centered on LSD1's enzymatic inhibition, compelling evidence highlights its pivotal role as a scaffolding protein, orchestrating transcriptional and chromatin remodeling complexes that control cellular differentiation and proliferation. However, our understanding of the precise mechanisms governing these complexes remains limited, necessitating further investigation. The K562 erythroleukemia cell line has served as a prominent model for investigating the molecular factors influencing cell fate determination. In response to specific cues, these cells exhibit a bi-potential capacity, mirroring the behavior of normal hematopoietic megakaryocyte/erythrocyte progenitors (MEP). Notably, when LSD1 is either depleted or inhibited, the erythroid differentiation of K562 cells is hindered, even when exposed to various pro-differentiating triggers, including the BCR-ABL inhibitor Imatinib. In order to gain deeper insights and identify potential new protein partners involved in the K562 differentiation process that interact with LSD1, we employed the erythroleukemic K562 cell model. Using unbiased, proteome-wide proximity labeling we discovered that LSD1 serves as a scaffold for recruitment of the ChAHP complex, comprised of ADNP, CBX3, and CHD4. The ChAHP complex serves fundamental roles in chromatin remodeling, RNA splicing, and transcriptional control. We confirmed these proximity relationships by immunoblotting biotinylated proteins generated in situ by the promiscuous biotin ligase, (BirA*) when anchored to LSD1. We show that depleting ADNP induces spontaneous erythroid differentiation in K562 erythroleukemia cells, which is augmented by BCR-ABL inhibitor Imatinib treatment. ADNP-deficient K562 cells exhibited accelerated erythroid differentiation, as evidenced by a four-fold increase in the surface expression of erythroid markers (CD71, CD235a) and a significant upregulation of beta-globin gene cluster markers. These findings collectively suggest an antagonistic functional relationship between ADNP and LSD1, potentially counterbalancing LSD1's role in lineage allocation. Furthermore, we demonstrate that the loss of the key ChAHP component, ADNP, substantially hampers proliferation and increases apoptosis in K562 cells. These results underscore the potential of ADNP/ChAHP as an appealing target to modulate the choice between erythroid and megakaryocytic fates and offer promise as a target for pro-differentiation therapies in AML Citation Format: Mehraju Din Lone, Mohd Sayeed, Alaa Habieb, Praveen Kumar, Michael E. Engel. Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5630.

Cellular abundance-based prognostic model associated with deregulated gene expression of leukemic stem cells in acute myeloid leukemia.

Background: Previous studies have reported that genes highly expressed in leukemic stem cells (LSC) may dictate the survival probability of patients and expression-based cellular deconvolution may be informative in forecasting prognosis. However, whether the prognosis of acute myeloid leukemia (AML) can be predicted using gene expression and deconvoluted cellular abundances is debatable. Methods: Nine different cell-type abundances of a training set composed of the AML samples of 422 patients, were used to build a model for predicting prognosis by least absolute shrinkage and selection operator Cox regression. This model was validated in two different validation sets, TCGA-LAML and Beat AML (n = 179 and 451, respectively). Results: We introduce a new prognosis predicting model for AML called the LSC activity (LSCA) score, which incorporates the abundance of 5cell types, granulocyte-monocyte progenitors, common myeloid progenitors, CD45RA + cells, megakaryocyte-erythrocyte progenitors, and multipotent progenitors. Overall survival probabilities between the high and low LSCA score groups were significantly different in TCGA-LAML and Beat AML cohorts (log-rank p-value = and , respectively). Also, multivariate Cox regression analysis on these two validation sets shows that LSCA score is independent prognostic factor when considering age, sex, and cytogenetic risk (hazard ratio, HR = 2.17; 95% CI 1.40-3.34; p < 0.001 and HR = 1.20; 95% CI 1.02-1.43; p < 0.03, respectively). The performance of the LSCA score was comparable to other prognostic models, LSC17, APS, and CTC scores, as indicated by the area under the curve. Gene set variation analysis with six LSC-related functional gene sets indicated that high and low LSCA scores are associated with upregulated and downregulated genes in LSCs. Conclusion: We have developed a new prognosis prediction scoring system for AML patients, the LSCA score, which uses deconvoluted cell-type abundance only.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Mechanisms of Aging Vary with Developmental Stages in the Human Hematopoietic Trajectory

Background : Hematopoietic stem and progenitor cells (HSPCs) undergo functional changes with age. An unresolved issue is whether aging is induced by a shift in intrinsic state in which all HSPCs undergo coordinated changes in functional potential, or by clonal evolution of a senescent population.Identification of senescent cells in human hematopoiesis poses another challenge. In this study, we have identified lineage-specific, senescence-associated pathways at different developmental stages of HSPCs and have developed an “aging signature HSC” for non-primed human HSCs. Methods: HSPCs (CD34+ cells) were isolated from the bone marrow of healthy human subjects (old = &amp;gt;60 years; young = 21 to &amp;lt;35 years). Single-cell RNA-sequencing (scRNAseq) studies and downstream analysis were performed using different R packages for normalization, dimension reduction, clustering, classification, pseudotime ordering, and developmental trajectory analyses. Gene Set Enrichment Analysis, GSEA (version 4.3.2., Broad Institute, Inc) was applied to identify pathways and processes that were coordinately up- or down-regulated with aging. Results: HSPCs (CD34+ cells) were derived from 13 healthy human subjects (old subjects n = 6; young subjects n = 7). There were 26,076 genes identified. The HSPCs were ordered and classified along their developmental trajectory during differentiation processes from (1) hematopoietic stem cells (HSC) to (2) myeloid-lymphoid progenitors (MLP), (3) megakaryocyte-erythrocyte progenitors (MEP), (4) granulocytic-monocytic progenitors (GMP), (5) granulocyte- (G), and (6) monocyte- (Mono) progenitors, (7) dendritic cell (DC), (8) erythroid progenitors (E), as well as various subsets of lymphocytes (Ly1 to Ly4). The results are depicted in Figure 1A. Remarkable are the increases in HSCs (p &amp;lt; 0.05) and in Ly4 (p &amp;lt; 0.001) in old subjects. The Kolmogorov-Smirnov test showed a significant delay in the developmental “pseudotimes” of HSCs (p &amp;lt; 0.001) and of Ly4 (p &amp;lt; 0.05). We then compared the differences in GSEA between old and young subjects in each of the following clusters: (A) whole HSPCs; (B) HSPCs along myeloid pathway (mye-HSPC); (C) HSC-MLP-MEP; (D) HSC. The major findings relevant to aging are summarized in Fig. 1B. Pathways that are significantly increased in the old subjects and throughout all comparisons include: “Formation of senescence associated heterochromatin foci”, “Aging signature HSC”, “DNA damage telomere stress induced senescence”, and “TNFR1 induced NFKB signaling pathway”. “Hypoxia”, “P53 pathway”, “IL2 signaling”, “TNFA signaling via NFKB” were elevated upon enrichment of the primitive HSC population, while other pathways decreased: “Senescence associated secretory phenotype (SASP)”, “DNA methylation”. “Cell cycle” and “DNA repair” are consistently more abundant in young subjects. We analyzed the 512 cells in the HSC cluster with higher resolution and were able to identify a sub-cluster “MPP2A” that is significantly increased in old subjects (4.5% of all HSPCs and 28.5% of HSCs, versus 1.9% and 12.9% respectively in young subjects; p &amp;lt; 0.005). The results are depicted in the highlighted circles in Fig. 1A. This population is quiescent and is highly enriched in “aging signature HSC” in old subjects (p &amp;lt;0.0001). Prominent examples of these signature genes within this list are shown in Fig. 1C). Further along the trajectory, a unique population of B cells “Ly4” was found predominantly in old subjects. The transcriptome profile Ly4 is similar to immature and age-associated B cells (ABCs). This cluster of lymphoid cells may represent the precursors of ABCs described thus far only in peripheral blood, providing another evidence that the aging mechanisms at various differentiation stages are different. Conclusions: In this study, we have defined specific pathways that distinctly separate aging HSCPs from young ones, and shown that the definition of “aging signature” varies with developmental stages. The accumulation a small cluster in the non-primed HSC compartment, with “aging signature HSC” and cell cycle arrest in old subjects indicates that aging of human hematopoiesis is induced by clonal evolution. The abundance of a unique population among lymphoid precursors with expression profiles similar to ABCs demonstrates that lymphoid and myeloid progenitors age differently.

Bone Marrow Microenvironment and Hematopoietic Cell Types Are Differentially Affected By Transcriptomic Quality Control Filtering Metrics in Single Cell Data

A crucial step prior to analysis of single cell transcriptomic data includes filtering out low-quality (e.g., dying) cells and artifacts (e.g., empty droplets). Choosing appropriate quality control parameters leads to correct cell assignment, so that the data for downstream analysis is high-quality and is representative of all processed cell types. These metrics include high and low cut-offs for mitochondrial content, total number of genes, number of different genes, and ribosomal content per cell. They have been found to vary based on cell and tissue type, as well biological variables such as age and disease, and technical variables such as sequencing technology. The aim of this study was to investigate the variation of these quality control metrics in a data set that included stem, progenitor, and differentiated cell types of the hematopoietic, mesenchymal, and endothelial lineages. Furthermore, we set out to determine whether applying standard cut-offs may differentially affect different cell types. We calculated individual filtering parameters for each cell type using the R package ddqc (Subramanian et al. Genome Biol. 2022). Thus, any biological variability based on mitochondrial, ribosomal, and gene counts was accounted for. Notably, distribution of measurements for each of the above quality control metrics varied by cluster, and certain cell-type means were adjacent to commonly used cut-off values. For instance, we observed higher content of mitochondrial genes in osteoblasts, sinusoidal endothelial cells, a subset of adipocyte-primed mesenchymal cells (MSPC), as well as hematopoietic progenitor cells, pre-B, and B cells. On the other hand, subsets of erythroblasts, neutrophils, as well as arteriolar endothelial cells had the lowest mitochondrial content. In addition, lower total gene counts and lower gene complexity were found in adipocyte-primed MSPCs, neutrophils, and megakaryocyte-erythrocyte progenitors, while higher counts were found in granulocyte-monocyte progenitor cells, megakaryocyte/erythroid-primed multipotent progenitor cells and subsets of erythroblasts. Applying universal cutoff values to this data set would greatly reduce, and some cases, eliminate cell types that have high mitochondrial content or lower gene counts and complexity, while leaving those cell types on the opposite spectrum unaffected. This would also skew proportions of cells in downstream analyses, thus affecting biological interpretation of the results. Overall, our results demonstrate biological variability of the quality control metric distributions in a single cell transcriptomic data set that includes diverse bone marrow niche and hematopoietic cell types. This reveals novel biological information regarding distinct cell types as well as cautions that applying standard filtering paraments on such a data set would eliminate some cell types and bias downstream analysis for others. Optimization of filtering criteria will be important when broadening single cell analysis to multiplexed approaches incorporating transcriptome, genome, epigenome, and proteome-level data.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4 +/+ and Lama4 -/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4 -/- recipients showed significantly increased platelet counts in blood compared to Lama4 +/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4 -/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1 + cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4 -/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4 -/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4 -/- recipients (33.6%) than that in Lama4 +/+ recipients (41.5%), raising a possibility that Lama4 -/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4 -/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.

Intracerebral Hemorrhage Leads to Anemia By Disrupting Erythropoiesis and Erythromyeloblastic Islands

Intracerebral hemorrhage (ICH), caused by the rupture of a cerebral vessel and subsequent entry of blood into the brain parenchyma, is a major form of acquired brain injury and contributes a remarkable proportion of stroke-related morbidity and mortality. Due to the few proven effective emergency and preventive treatments, patients who survived suffer varying degrees of poor prognosis that affect their quality of life. There is an urgent need to explore the cellular and molecular basis of the differences in the severity and prognosis of ICH. Anemia is a common manifestation of ICH. However, the mechanisms underlying ICH-induced anemia as we as the correlation between anemia and ICH progression/prognosis remain unclear. In this study, we found that approximately 35.1% (39 of 111) of ICH patients exhibited anemia and that decreased hemoglobin levels were positively associated with higher scores on the National Institutes of Health Stroke Scale, which quantifies the degree of neurological deficit, at admission. Furthermore, patients with decreased hemoglobin levels at admission showed higher modified Rankin scale scores at 3 months, which reflect the global disability of daily living at this time, suggesting that anemia was associated with a more severe progression and poor patient prognosis. To investigate the effects of ICH on bone marrow (BM) erythropoiesis, we conducted experiments in 12-month-old mice with collagenase-induced striatal ICH. Our results showed that ICH caused apparent anemia on day 3, associated with larger hematomas in the striatum and more severe neurologic deficits. We first evaluated the phenotype of hematopoietic progenitors and found that mice suffering ICH exhibited increased granulocyte monocyte progenitors but a significant decrease in common myeloid progenitors and megakaryocyte erythrocyte progenitors compared to control mice, suggesting a reduced source of erythroid progenitors. Further analysis revealed that the colony-forming ability of erythroid progenitors and subsequent terminal differentiation was significantly suppressed after ICH. To assess whether ICH-induced suppression of erythropoiesis is associated with erythromyeloblastic islands (EMBIs), the niche for erythropoiesis/myelopoiesis, we performed ICH using 12-month-old EpoR-tdTomato-Cre mice and found that the number of BM EMBIs and EpoR + macrophages decreased significantly. Moreover, the number and proportion of developing erythroid cells surrounding EMBIs decreased while LY6G + cells representing granulocytes increased prominently, indicating an altered microenvironment, with EMBI favoring granulopoiesis over erythropoiesis after ICH. Studies have shown that inflammation plays a crucial role in secondary brain injury after ICH. We hypothesize that inflammatory cytokines may contribute to the suppression of erythropoiesis observed in ICH mouse models. To test this, we measured inflammatory factors in both peripheral blood and BM fluid using Quantibody Mouse Inflammation Array. In particular, IL-6 concentrations in peripheral blood and BM fluid in ICH mice were significantly higher than controls. Treatment with MR16-1, an IL-6 antagonist, alleviated ICH-induced anemia and improved BM erythropoiesis in ICH mice, as evidenced by increased peripheral red blood cells and hemoglobin levels, elevated numbers of erythroid progenitors and terminal erythroblast. MR16-1 treatment also improved BM EMBI formation and increased EpoR + macrophages. In addition, MR16-1 treatment effectively improved neurologic deficits and reduced brain hematoma after ICH. Administration of recombinant IL-6 to wild-type mice resulted in decreased levels of red blood cells and hemoglobin along with suppression of erythropoiesis in the BM. Collectively, these results suggest that ICH modulates erythropoiesis and erythromyeloblastic islands function to ultimately lead to anemia and reveal a possible therapeutic opportunity to reverse ICH-induced anemia and improve ICH prognosis by targeting IL-6.

Hyperactivity of platelets and increased megakaryopoiesis in COVID-19 patients with acute respiratory distress syndrome

The Severe Acute Respiratory Syndrome (SARS)-CoV-2-induced Coronavirus Disease 2019 (COVID-19) pandemic has caused an overwhelming influence on public health because of its high morbidity and mortality. Critical-illness cases often manifest as acute respiratory distress syndrome (ARDS). Previous evidence has suggested platelets and thrombotic events as key mediators of SARS-CoV-2-associated ARDS. However, how the balance of platelet regeneration from the hematopoietic system is changed in ARDS remains elusive. Here, we reported a more severe inflammation condition and hyperactivity of platelets in COVID-19 ARDS patients compared with those infected but without ARDS. Analysis of peripheral blood revealed an increased proportion of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), megakaryocyte-erythrocyte progenitors (MEPs), and megakaryocyte progenitors (MkPs) in ARDS patients, suggesting a megakaryocytic-differentiation tendency. Finally, we found altered gene expression pattern in HSCs in COVID-19 ARDS patients. Surprisingly, genes representing platelet-primed HSCs were downregulated, indicating these cells are being stimulated to differentiate. Taken together, our findings shed light on the response of the hematopoietic system to replenish platelets that were excessively consumed in COVID-19 ARDS, providing a mechanism for disease progression and further therapeutic development.Graphical

Genome-wide transcription factor–binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs

AbstractHematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type–specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell–specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type–specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

Ribonuclease Inhibitor 1 (RNH1) Regulates Myeloid Lineage Choice through Cyclin-Dependent Kinase 1

Background: Myelopoiesis is a complex process, it begins with myeloid lineage choice determination in haemopoietic stem cells (HSCs). Still we do not have complete molecular understanding of how myeloid lineage choices were determined. Although transcription factors expression majorly determine the lineage choices at HSCs level, recent study suggest that ratios of total PU.1:GATA1 protein levels are not the central mechanism that initiates HSPC lineage decisions . Further, cell fate commitment is linked to the cell cycle regulation, however whether the cell cycle itself regulates lineage choices in HSCs is not known. Previously, we have published that Ribonuclease inhibitor (RNH1) deletion in mice leads to defect inembryonic erythropoiesis. Here, we report that RNH1 regulates myeloid lineage choice through cyclin-dependent kinase 1 (CDK1) mediated cell cycle regulation. Methods: We have generated hematopoietic-specific conditional knockout mice by crossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Phenotypic analysis, Bone marrow transplantation (BMT) experiments, cell cycle and CFU assays were performed. To understand molecular mechanism RNA seq, mass spectrometry, confocal microscopy and biochemical studies were performed. Histopathology performed in AML bone marrow biopsies. RNH1 was knocked out in several AML cell lines and analyzed myeloid differentiation and performed CFU assays. Results: Rnh1-deficiency in both Vav1-iCre and Mx1-Cre models resulted in elevated levels of myeloid cells but decreased lymphoid cells and haemoglobin (HGB) levels in peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type (WT) and Rnh1-/- mice. However, the number of erythroid cells and lymphoid cells was significantly decreased, whereas myeloid cells were significantly increased. We analysed the progenitor populations in the BM. In line with increased myelopoiesis phenotype, granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-/- mouse BM, suggesting that RNH1 could affect HSC function. Supporting this, Rnh1-/- HSCs failed to engraft lethally irradiated mice in competitive and non- competitive BMT experiments. Intriguingly, despite increased myelopoiesis in Rnh1-/- mice, conventional myeloid genes that support myelopoiesis were not increased in Rnh1-/- HSCs by RNA-Seq studies. Rather we found increased expression of genes related to cell cycle, kinetochore and DNA damage in Rnh1-/- HSCs. Corroborating this, Rnh1-/- HSCs showed significant increase in G1 cycle phase compared to WT. At the molecular level, we found that RNH1 directly binds to CDK1 and also inhibits CDK1 kinase function. Supporting this, molecular docking studies suggest that RNH1 potentially binds with CDK1. In agreement with this, loss of RNH1 in HSCs increased CDK1 protein levels as well as increased CDK1 phosphorylation at Thr161. Further, CDK1 inhibitor treatment decreased myelopoiesis and rescued erythropoiesis and lymphopoiesis in Rnh1-/- mice. We checked whether RNH1 mediated myelopoiesis is relevant in any disease models and can be targeted. Interestingly, we found increased RNH1 expression in AML patients bone marrow biopsies independent of their genetic mutation. AML blasts have myeloid differentiation arrest and therapies that resolve differentiation arrest represent a powerful treatment strategy. Interestingly, RNH1 deletion in AML cell lines leads to increased expression of the myeloid markers, increased cell death and formed less colonies compared to wild type cells. Summary: Collectively, our studies suggest that loss of RNH1 in HSCs leads to dysregulation in cell cycle through increased CDK1 activity. Dysregulation in cell cycle decreases HSCs stemness and repopulating potential, and influences cell fate decisions by increasing myelopoiesis. Since loss of RNH1 increased myelopoiesis without increase of myeloid transcription factors expression and dependent on CDK1, we call this pathway 'alternative myelopoiesis'. Targeting alternative myelopoiesis could be a potential therapy for AML.

Erythroid and Megakaryocytic Differentiation Program in JAK2-Mutated Acute Myeloid Leukemia with or without Antecedent Myeloproliferative Neoplasm

JAK2 V617F is the most common driver mutation for Ph-negative MPN and related blast transformation (MPN-BP). In contrast, JAK2 V617F is rare (<5%) in acute myeloid leukemia (AML) lacking an antecedent MPN (nonMPN AML). JAK2 V617F skews the differentiation of hematopoietic stem/progenitor cells (HSPC) towards erythroid and megakaryocytic lineages and in conjunction with TP53 deficiency/mutations results in acute erythroid leukemia in mice (Rampal et al., PNAS 2014). These phenotypes, although also reported in patients with MPN-BP (Mesa et al., Blood 2005), remain incompletely understood. In this study, we set out to study these phenotypes at the protein, epigenomic and transcriptomic levels in JAK2-mutated AML comparing MPN-BP to nonMPN AML. The study cohort included 58 MPN-BP (16 PV, 16 ET, 14 PMF and 12 MPN-U) and 27 nonMPN AML patients (11 de novo AML, 12 AML-MRC, 4 t-AML) with JAK2 V617F mutation (Table 1). In addition, 100 patients with JAK2 WT AML were also included as control group. The median age at diagnosis in MPN-BP and nonMPN AML subgroups as well as in JAK2 WT AML was similar (70 years vs 71 years vs 70 years, respectively) and showed a male preponderance. The median interval between MPN diagnosis and blast transformation was 4.3 years (range, 0-20.5 years). Overall survival was inferior in MPN-BP compared to nonMPN AML (median survival: 7.4 vs 19.3 months). MPN-BP showed a higher rate of MF-2 or 3 myelofibrosis (78% vs 33%, p<0.05), osteosclerosis (50% vs 15%, p<0.05) and marrow necrosis (7% vs 0%) on bone marrow biopsies than nonMPN AML. On immunohistochemistry, 28% (10/32) MPN-BP cases (vs 0% nonMPN AML, p<0.05) were classified as acute leukemia with erythroid/megakaryocytic differentiation (M6b and/or M7) based on expression of E-cadherin, CD71, Glycophorin A, and CD61 on leukemic blasts. ERG, FLI1, and LMO2, transcription factors that are often expressed in HSPCs, were also expressed in leukemic blasts of MPN-BP and nonMPN AML. Aberrant TP53 expression by immunohistochemistry was seen in 31% of MPN-BP (5/9 and 5/21 cases with and without erythroid/megakaryocytic differentiation, respectively) and 17% (3/18) of nonMPN AML. The most common mutations on next-generation sequencing included TP53, RUNX1, ASXL1, TET2, DNMT3A, SRSF2, IDH1, IDH2 and EZH2 in each group. However, of these, TP53 mutations were more frequent in MPN-BP (34% vs 11% in nonMPN AML, p<0.05) and TET2 mutations more frequent in nonMPN AML (48% vs 24% in MPN-BP, p<0.05). Interestingly, NPM1 and FLT3, which are among the most mutated genes in AML were not detected in either MPN-BP or nonMPN AML. Based on the over-representation of erythroid/megakaryocytic phenotype in MPN-BP seen on immunohistochemistry, we hypothesized that MPN-BP blasts might exhibit an upregulated epigenetic and transcriptional program involved in erythroid/megakaryocytic differentiation. RNA-seq and ATAC-seq analysis were performed on flow cytometry sorted CD34+ leukemic blast populations from 11 MPN-BP and 6 nonMPN AML, as well as similar sorted populations from normal controls and JAK2 WT AML. Principal component analysis showed overlapping MPN-BP and nonMPN AML samples, both of which were separated from JAK2 WT AML. Differential expression analysis confirmed nearly identical gene expression between blasts from MPN-BP and nonMPN AML. In addition, consistent with findings by immunohistochemistry, the expression of genes specifically involved in erythroid/megakaryocytic differentiation (TFRC, GYPA, HBB, SPTA1, EPOR, ITGA2B, ITGB3, PF4, ALAS2, GP1BA, ENG and MPL) were upregulated in MPN-BP. Hierarchical clustering using Ward's method on both RNA-seq and ATAC-seq normalized counts revealed that the JAK2 WT AML cluster was distinct from the MPN-BP and nonMPN AML cluster (Figure 1). Lineage deconvolution of ATAC-seq using hematopoietic differentiation stages (Glass et al., Blood 2017, Dilip et al., Blood 2021) to evaluate lineage characteristics of the blasts showed hematopoietic stem cell (HSC) predominance in JAK2 WT AML while MPN-BP and nonMPN AML displayed enrichment of multipotent progenitor (MPP), megakaryocyte-erythrocyte progenitor (MEP) and erythroid signatures. Collectively, our data demonstrates enrichment of erythroid and megakaryocytic programs in both JAK2-mutated MPN-BP and JAK2-mutated nonMPN AML, suggesting that chromatin accessibility and transcription may be driven by mutated JAK2 regardless of antecedent MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vitamin C deficiency reveals developmental differences between neonatal and adult hematopoiesis.

Hematopoiesis, a process that results in the differentiation of all blood lineages, is essential throughout life. The production of 1x1012 blood cells per day, including 200x109 erythrocytes, is highly dependent on nutrient consumption. Notably though, the relative requirements for micronutrients during the perinatal period, a critical developmental window for immune cell and erythrocyte differentiation, have not been extensively studied. More specifically, the impact of the vitamin C/ascorbate micronutrient on perinatal as compared to adult hematopoiesis has been difficult to assess in animal models. Even though humans cannot synthesize ascorbate, due to a pseudogenization of the L-gulono-γ-lactone oxidase (GULO) gene, its generation from glucose is an ancestral mammalian trait. Taking advantage of a Gulo-/- mouse model, we show that ascorbic acid deficiency profoundly impacts perinatal hematopoiesis, resulting in a hypocellular bone marrow (BM) with a significant reduction in hematopoietic stem cells, multipotent progenitors, and hematopoietic progenitors. Furthermore, myeloid progenitors exhibited differential sensitivity to vitamin C levels; common myeloid progenitors and megakaryocyte-erythrocyte progenitors were markedly reduced in Gulo-/- pups following vitamin C depletion in the dams, whereas granulocyte-myeloid progenitors were spared, and their frequency was even augmented. Notably, hematopoietic cell subsets were rescued by vitamin C repletion. Consistent with these data, peripheral myeloid cells were maintained in ascorbate-deficient Gulo-/- pups while other lineage-committed hematopoietic cells were decreased. A reduction in B cell numbers was associated with a significantly reduced humoral immune response in ascorbate-depleted Gulo-/- pups but not adult mice. Erythropoiesis was particularly sensitive to vitamin C deprivation during both the perinatal and adult periods, with ascorbate-deficient Gulo-/- pups as well as adult mice exhibiting compensatory splenic differentiation. Furthermore, in the pathological context of hemolytic anemia, vitamin C-deficient adult Gulo-/- mice were not able to sufficiently increase their erythropoietic activity, resulting in a sustained anemia. Thus, vitamin C plays a pivotal role in the maintenance and differentiation of hematopoietic progenitors during the neonatal period and is required throughout life to sustain erythroid differentiation under stress conditions.

A glycan-based approach to cell characterization and isolation: Hematopoiesis as a paradigm.

Cell surfaces display a wide array of molecules that confer identity. While flow cytometry and cluster of differentiation (CD) markers have revolutionized cell characterization and purification, functionally heterogeneous cellular subtypes remain unresolvable by the CD marker system alone. Using hematopoietic lineages as a paradigm, we leverage the extraordinary molecular diversity of heparan sulfate (HS) glycans to establish cellular "glycotypes" by utilizing a panel of anti-HS single-chain variable fragment antibodies (scFvs). Prospective sorting with anti-HS scFvs identifies functionally distinct glycotypes within heterogeneous pools of mouse and human hematopoietic progenitor cells and enables further stratification of immunophenotypically pure megakaryocyte-erythrocyte progenitors. This stratification correlates with expression of a heptad of HS-related genes that is reflective of the HS epitope recognized by specific anti-HS scFvs. While we show that HS glycotyping provides an orthogonal set of tools for resolution of hematopoietic lineages, we anticipate broad utility of this approach in defining and isolating novel, viable cell types across diverse tissues and species.

Microbiota-Derived Propionate Modulates Megakaryopoiesis and Platelet Function

Rheumatoid arthritis (RA) is associated with an increased risk for cardiovascular events driven by abnormal platelet clotting effects. Platelets are produced by megakaryocytes, deriving from megakaryocyte erythrocyte progenitors (MEP) in the bone marrow. Increased megakaryocyte expansion across common autoimmune diseases was shown for RA, systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS). In this context, we evaluated the role of the microbial-derived short chain fatty acid (SCFA) propionate on hematopoietic progenitors in the collagen induced inflammatory arthritis model (CIA) as we recently showed attenuating effects of preventive propionate treatment on CIA severity. In vivo, propionate treatment starting 21 days post immunization (dpi) reduced the frequency of MEPs in the bone marrow of CIA and naïve mice. Megakaryocytes numbers were reduced but increased the expression of the maturation marker CD61. Consistent with this, functional analysis of platelets showed an upregulated reactivity state following propionate-treatment. This was confirmed by elevated histone 3 acetylation and propionylation as well as by RNAseq analysis in Meg-01 cells. Taken together, we identified a novel nutritional axis that skews platelet formation and function.

Sepsis promotes splenic production of a protective platelet pool with high CD40 ligand expression.

Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen was a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyte-erythrocyte progenitors (MEPs) from the bone marrow to the spleen, where IL-3 induced their differentiation into megakaryocytes (MKs). In the spleen, immune-skewed MKs produced a CD40 ligandhi platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

Hematopoietic Stem Cells (HSC) and Granulocyte Macrophage Progenitors (GMP) are the Oxidative Stress Targets in Low/Intermediate-1 Risk Myelodysplastic Syndromes.

Myelodysplastic Syndromes (MDS) are a heterogenous group of clonal hematopoietic stem cell malignancies. Previous studies showed that Reactive Oxygen Species (ROS) play a role in the pathogenesis and clinical evolution of MDS, contributing to Hematopoietic Stem and Progenitor Cells (HSPC) genetic instability. Less is known about ROS levels in the various sub-populations of MDS HSPC and how they correlate with clinical data in MDS patients. Our study aims to analyze ROS levels in MDS Hematopoietic Stem Cells (HSC), common myeloid progenitors (CMP), Granulocyte Macrophages Progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP); afterwards, we looked at the relationship between ROS levels and clinical data. thirty-eight MDS and 27 Normal Bone Marrow (NBM) samples were collected; ROS levels were analyzed via multicolor flow cytometry. In both NBM and MDS, HSC showed much higher ROS levels than progenitors (3 to 4 folds, p < 0.0001); HSC ROS were significantly more elevated in MDS-no excess blasts versus MDS with excess blasts and versus NBM. GMP from MDS-no excess blasts showed higher ROS compared to NBM GMP. The 3 MDS with Ringed Sideroblasts (RS) showed more elevated ROS in HSC and GMP compared to the not RS low/intermediate-1 MDS; the 2 monosomy 7 patients displayed higher ROS levels in each subpopulation compared to the normal karyotype MDS; the only del(5q) patient did not show relevant differences in ROS levels compared to the median of the normal karyotype MDS ROS. The 9 high transfusion burden patients exhibited higher ROS in HSC and GMP compared to NBM HSC and GMP. These data were not confirmed in low transfusion burden (n:2) and non-transfused patients (n:26). In low/intermediate-1 MDS, a direct correlation between ferritin values and ROS levels in progenitors, but not in HSC, was detected. Interestingly, low/intermediate-1 risk patients that are no longer responding to recombinant human erythropoietin (rh-EPO) showed higher ROS levels in GMP and HSC. Our data showed that ROS can play a role in the pathogenesis and maintenance of low and intermediate-1 risk MDS clone; ROS status can be influenced by several clinical factors as ferritin levels and rh-EPO treatment. In this scenario, high ROS levels can contribute to genetic instability and influence progression to AML. Further biological studies are needed to elucidate ROS role in MDS pathogenesis and analyze the possible benefit of antioxidant drugs added to the standard MDS treatments.

CD62L expression level determines the cell fate of myeloid progenitors.

SummaryHematopoietic cells differentiate through several progenitors in a hierarchical manner, and recent single-cell analyses have revealed substantial heterogeneity within each progenitor. Although common myeloid progenitors (CMPs) are defined as a multipotent cell population that can differentiate into granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs), and GMPs generate neutrophils and monocytes, these myeloid progenitors must contain some lineage-committed progenitors. Through gene expression analysis at single-cell levels, we identified CD62L as a marker to reveal the heterogeneity. We confirmed that CD62L-negative CMPs represent “bona fide” CMPs, whereas CD62L-high CMPs are mostly restricted to GMP potentials both in mice and humans. In addition, we identified CD62L-negative GMPs as the most immature subsets in GMPs and Ly6C+CD62L-intermediate and Ly6C+CD62L-high GMPs are skewed to neutrophil and monocyte differentiation in mice, respectively. Our findings contribute to more profound understanding about the mechanism of myeloid differentiation.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-β.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Whole-Genome CRISPR Screening Identifies N-Glycosylation As an Essential Pathway and a Potential Novel Therapeutic Target in CALR-Mutant MPN

Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis.We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values <0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings.Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis.We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells.To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells.We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p<.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03).We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p<0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells.Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN.In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN.AEM and JSJ contributed equally. [Display omitted] DisclosuresMullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.