Hematopoiesis In Myelodysplastic Syndromes Research Articles

Successful treatment of concurrent cold agglutinin disease and myelodysplastic syndrome

Herein, we describe a case of severe anemia presenting with myelodysplastic syndrome with cold agglutinin disease that was successfully treated by a moderate dose of steroids followed by cyclosporine. In patients with myelodysplastic syndrome, autoimmunity in erythroid cells is occasionally demonstrated, and autoimmune hemolytic anemia is seen in some patients. However, hemolytic anemia with cold agglutinin in patients with myelodysplastic syndrome is less common, and the effect of corticosteroids for autoimmune hemolytic anemia caused by cold agglutinin is thought to be limited. Although the elevated levels of reticulocytes and LDH are usually caused by ineffective hematopoiesis in myelodysplastic syndrome, clinicians should be aware of latent cold agglutinin disease. In the present case, in addition to the improvement of erythroid dysplasia, the corticosteroid-sparing effect on cold agglutinin disease may have played a role in the mechanism underlying the effectiveness of cyclosporine.

Caspase 8 deletion causes infection/inflammation-induced bone marrow failure and MDS-like disease in mice

Myelodysplastic syndromes (MDS) are a heterogeneous group of pre-leukemic hematopoietic disorders characterized by cytopenia in peripheral blood due to ineffective hematopoiesis and normo- or hypercellularity and morphologic dysplasia in bone marrow (BM). An inflammatory BM microenvironment and programmed cell death of hematopoietic stem/progenitor cells (HSPCs) are thought to be the major causes of ineffective hematopoiesis in MDS. Pyroptosis, apoptosis and necroptosis (collectively, PANoptosis) are observed in BM tissues of MDS patients, suggesting an important role of PANoptosis in MDS pathogenesis. Caspase 8 (Casp8) is a master regulator of PANoptosis, which is downregulated in HSPCs from most MDS patients and abnormally spliced in HSPCs from MDS patients with SRSF2 mutation. To study the role of PANoptosis in hematopoiesis, we generated inducible Casp8 knockout mice (Casp8−/−). Mx1-Cre-Casp8−/− mice died of BM failure within 10 days of polyI:C injections due to depletion of HSPCs. Rosa-ERT2Cre-Casp8−/− mice are healthy without significant changes in BM hematopoiesis within the first 1.5 months after Casp8 deletion. Such mice developed BM failure upon infection or low dose polyI:C/LPS injections due to the hypersensitivity of Casp8−/− HSPCs to infection or inflammation-induced necroptosis which can be prevented by Ripk3 deletion. However, impaired self-renewal capacity of Casp8−/− HSPCs cannot be rescued by Ripk3 deletion due to activation of Ripk1-Tbk1 signaling. Most importantly, mice transplanted with Casp8−/− BM cells developed MDS-like disease within 4 months of transplantation as demonstrated by anemia, thrombocytopenia and myelodysplasia. Our study suggests an essential role for a balance in Casp8, Ripk3-Mlkl and Ripk1-Tbk1 activities in the regulation of survival and self-renewal of HSPCs, the disruption of which induces inflammation and BM failure, resulting in MDS-like disease.

Pathogenesis of myelodysplastic syndromes by excessive mitochondrial fragmentation

Myelodysplastic syndromes (MDS) are a group of heterogenous hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis in which clonal progenitor expansion occurs alongside impaired myelopoiesis. Inflammatory signaling activation due to dysregulated innate immunity is also a hallmark of MDS pathogenesis. We recently established a useful preclinical tool that recapitulates bona fide MDS phenotypes and gene expression profiles based on previously unreported co-mutations discovered during our clinical surveillance of mutations in patients with MDS. Notably, we focused unbiased transcriptome analysis on determining the distinct underlying mediators of MDS etiology, and identified excessive mitochondrial fission-mediated fragmentation in mutant HSCs and progenitors (HSC/Ps). We confirmed excessive mitochondrial fragmentation in HSC/Ps obtained from patients with MDS regardless of the mutational profile. Importantly, in vivo pharmacological inhibition of mitochondrial fission significantly attenuated inflammatory signaling activation, dysplasia formation and ineffective hematopoiesis phenotype, and prolonged survival of MDS mice, suggesting that excessive mitochondrial fragmentation could be a fundamental trigger of MDS pathogenesis. These findings provide new insights into the mechanistic basis of ineffective hematopoiesis, and a clue for targeting bone marrow failure caused by ineffective hematopoiesis in MDS.

Myelodysplastic Syndrome Associated Hemophagocytic Lymphohistiocytosis: A Retrospective Study of 15 Cases in a Single Center

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by excessive cytokine secretion, resulting in hyperinflammation and multiorgan dysfunction. It is classified into primary HLH, which is mainly caused by genetic defects, and secondary HLH, triggered by various underlying conditions. Malignancy-associated HLH (M-HLH) is a secondary form commonly observed in patients with hematological malignancies, such as lymphomas, T/NK-cell disorders, acute leukemias, lymphoproliferative diseases, and myelodysplastic syndrome (MDS). M-HLH can occur in up to 1% of patients with hematologic malignancies, with lymphoma being the most frequently associated malignancy. HLH initiated by MDS is a rare entity with only a few cases reported, leading to limited knowledge regarding its pathogenesis, diagnosis and optimal treatment strategies. Methods: In this retrospective study, we analyzed a consecutive series of MDS-HLH patients admitted to our center between March 1, 2019, and March 1, 2023. Bio-clinical data related with MDS and HLH were collected at diagnosis of MDS-HLH. Additional observational indicators encompassed the following: basic clinical characteristics of the patients (age at onset, gender, duration of MDS prior to HLH), correlation between the onset of HLH and MDS, treatment and response, and genetic defects associated with HLH and MDS. MDS risk classification was according to the Revised International Prognosis Scoring System (IPSS-R) and the WHO classification-based Prognosis Scoring System, while patients were categorized into two groups as higher-risk and lower-risk MDS-HLH group. The follow-up period started at the time of MDS-HLH diagnosis and ended at the date of death or last examination. Overall survival (OS) was calculated from the time of HLH diagnosis to the date of death from any cause. Results: A total of 15 patients diagnosed with MDS-HLH were included in the study, with a median age of 66 years (range, 33-83 years). The majority of patients (10/15, 67%) were over 60 years old. All patients exhibited typical features of aggressive HLH: fever, cytopenia and elevated serum ferritin in all patients, hepatomegaly/splenomegaly in 87% (13/15), hypofibrinogenemia/hypertriglyceridemia in 73% (11/15), hemophagocytosis in 73% (11/15), low NK-cell activity in 21% (3/14), and elevated levels of sCD25 in 93% of patients (14/15). Additionally, four patients (27%) had concurrent Epstein-Barr virus (EBV) infection. Further analysis comparing the lower-risk and higher-risk MDS-HLH groups revealed interesting findings. In the lower-risk group, all patients developed HLH concurrently with the diagnosis of MDS. However, in the higher-risk group, the majority of patients (6/8, 62.5%) experienced HLH onset during MDS progression or relapse (p=0.014). Among the 15 patients, 8 patients (53.3%) achieved remission of HLH. During the study period, 8 deaths occurred, all directly attributed to HLH or its complications. The majority of deaths (75%, 6/8) occurred within the first two months after HLH diagnosis. The median overall survival was 12.3 weeks (95%CI, 1.1-23.5). Patients who achieved complete or partial response had significantly prolonged overall survival compared to those who did not respond to treatment (p=0.002). In the multivariate analysis of prognosis, the remission status of HLH was the only factor which was significantly associated with survival (p=0.018, Exp(B)=0.003). Conclusion: This is the first comprehensive study focusing specifically on MDS-HLH, providing valuable insights into the clinical characteristics, treatment outcomes, and prognosis of MDS-HLH. The findings emphasize the need of considering revision of diagnostic criteria specially for MDS-HLH. The pathogenesis of HLH in MDS-HLH differs based on the risk classification of MDS, with higher-risk cases driven by malignancy and lower-risk cases associated with immune dysfunction. The prognosis of MDS-HLH is generally poor. Achieving remission of HLH is crucial for improving the prognosis of MDS-HLH patients. Conventional chemotherapy regimens, including etoposide, have shown limited efficacy and safety in this context. Chemotherapy-free regimens should be preferred considering the features of ineffective hematopoiesis in MDS. The role of EBV infection in MDS-HLH development should be recognized.

Abstract 5150: Improvement of hematopoiesis in primary low risk MDS with the TGFBR1 investigational inhibitor TP-6379

Abstract Background: The inflammaging modulator, S100A9, mediates immunosuppression and plays a key role in the pathogenesis of low-risk myelodysplastic syndrome (MDS). Specifically, S100A9’s feedforward activation in the bone marrow (BM) induces pyroptotic cell death of hematopoietic stem and progenitor cells (HSPC) and the activation and accumulation of myeloid-derived suppressor cells (MDSC). This process creates a suppressive microenvironment including the secretion of transforming growth factor β (TGFβ) that signals through the TGFβ receptor 1 (TGFBR1) to induce inhibitory processes that contribute to this phenotype. We hypothesize that targeting with an investigational TGFBR1 inhibitor, TP-6379, reduces immune suppression and restores hematopoiesis in MDS. Methods: Primary low risk MDS BM mononuclear cells (BMMNC) were obtained from the Moffitt Total Cancer Care Protocol and cultured in vitro with TP-6379. Healthy BMMNC were purchased, treated with or without recombinant S100A9 and TP-6379. Results: Treatment of primary MDS BMMNC (n=15) and S100A9-treated healthy BMMNC (n=5) with TP-6379 for 48 hours was observed to significantly improve proliferation of hematopoietic progenitor cells as measured by colony forming capacity. Total colonies, and BFU-E specific colonies, showed significant increase. This expansion of progenitors with treatment was validated by flow cytometric analysis of Lineage−HLA-DR−CD34+ HSPC and showed a reduction in genomic instability, measured by γH2AX. In addition, TP-6379 was observed to reduce the numbers of suppressive mediators; MDSC, T regs and senescent lymphocytes and increase the number of cytotoxic cells, including NK cells. Our analysis revealed that BMMNC with spliceosomal mutations, including SF3B1, have elevated sensitivity to TP-6379 treatment. Therefore, we tested wild type and SF3B1 K700E CRISPR knock-in K562 cells in the presence of TP-6379 for 48 hours. K562 is a well-established cell line model for study of MDS. The SF3B1 mutant expressing cells have significantly reduced BFU-E colony forming capacity, compared to wild type cells, concordant with higher pyroptosis activation and consistent with the BMMNC results. Hence, we measured phospho-SMAD activity, as a direct measure of TGFBR1 inhibition. SF3B1 mutant expressing cells were shown to be highly sensitive to TP-6379, with a significant loss of SMAD activation. Expression analysis of primary MDS, S100A9-treated healthy BMMNC cells and K562 SF3B1 mutant cells revealed elevated expression of SERPINE1 which was rescued by TP-6379 treatment. Conclusions: TP-6379 restores healthy hematopoiesis in low-risk MDS, which may translate into a strong therapeutic option in a disease with few clinical options. Importantly, our work also suggests a specific role for spliceosome dysfunction in bone marrow failure and a novel role for SERPINE1 in MDS and S100A9-induced suppression. Citation Format: Erika A. Eksioglu, Gabriela M. Wright, Jason M. Foulks, Matthew Lalonde, Steven L. Warner, Kenneth L. Wright. Improvement of hematopoiesis in primary low risk MDS with the TGFBR1 investigational inhibitor TP-6379. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5150.

Mlkl Mediates Age-Related Attrition of Hematopoietic Stem Cells and Ineffective Hematopoiesis in Myelodysplastic Syndrome

Aging causes anemia, perturbed immunity, and increased propensity to age-related hematological diseases such as myelodysplastic syndrome (MDS). Age-dependent functional decline in hematopoietic stem cells (HSCs), which is accompanied by morphological and functional changes in their organelles as well as sterile inflammation in the bone marrow (BM), is one of the main reasons why the hematopoietic system ages. However, molecular mechanisms by which HSCs alter their function during aging remain unclear. Necroptosis is a form of programmed necrosis that underlies sterile inflammation and can take place upon exposure to inflammatory stimuli in apoptosis-resistant cells including HSCs. MLKL, the effector molecule of necroptosis that can form channel-like structure on lipid bilayers upon activation, not only induces cell death but also injures membrane-bound organelles such as mitochondria, though its significance in HSC aging remains elusive. Our quantitative RT-PCR revealed abundant expression of MLKL in murine BM immature hematopoietic cells including HSCs (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+), which was validated by reanalysis of the published proteome datasets (Zaro et al., eLife 2020). Using FRET-based necroptosis reporter mice and Western blotting analysis, we found that while MLKL was inactivated at the steady state, it was readily activated in HSCs upon poly I:C injection. To study the effect of MLKL activation on HSCs, we assessed cell death of HSCs isolated from poly I:C-treated MLKL-deficient (Mlkl-/-) mice. Unexpectedly, Annexin V/DAPI staining revealed no changes in the frequencies of dying and dead cells between wild-type (WT) and Mlkl-/- HSCs. Nonetheless, PVA-based HSC culture revealed that the growth suppression observed in poly I:C-treated WT HSCs largely depended on MLKL (p=0.0003). Furthermore, HSC transplantation assay revealed that reduced engraftment and skewed myeloid differentiation were observed in poly I:C-treated WT HSCs but largely alleviated by MLKL deficiency (p=0.011). These results indicate that inflammation-induced MLKL activation does not cause HSC death but impairs their growth and lymphopoietic potential. To assess the relevance of MLKL activation in HSC aging, we treated WT and Mlkl-/- mice with serial rounds of 5-FU treatment, which were shown to induce age-related alterations in HSCs (Beerman et al., Cell Stem Cell 2013). Of note, we observed marked attenuation of age-related features such as myeloid-skewed hematopoiesis and expansion of phenotypic HSCs in 5-FU-treated Mlkl-/- mice (p=0.0051). HSC transplantation experiments further revealed that MLKL deficiency alleviated 5-FU-induced decrease in engraftment and lymphopoietic potential of HSCs (p=0.013). Indeed, analysis of 18-month-old WT and Mlkl-/- mice demonstrated that MLKL deficiency largely rescued myeloid-skewed hematopoiesis and loss of BM CLPs (Lin-/cKitlo/Sca1lo/Flk2+/IL-7Rα+) (p=0.0047), and serial HSC transplantation revealed better self-renewal and lymphopoietic potential of aged Mlkl-/- HSCs (p=0.0055). Importantly, multiplex ELISA assay using BM supernatants revealed no changes in concentrations of inflammatory cytokines, indicating that the observed effects in Mlkl-/- HSCs were likely caused by cell intrinsic changes. In line with this, electron microscopy analysis revealed that age-related changes in mitochondria, such as elongated and swollen structure with disorganized cristae, were significantly attenuated in aged Mlkl-/- HSCs (p<0.0001). JC-1 dye staining further revealed that age-related accumulation of damaged mitochondria was significantly attenuated in Mlkl-/- HSCs (p=0.049). Finally, to evaluate the effect of MLKL on development of age-related hematological disease, we established MDS mouse models by transplantation of RUNX1S291fs-transduced WT and Mlkl-/- HSCs. We found that WT MDS mice mainly died of severe anemia due to ineffective hematopoiesis, whereas Mlkl-/- MDS mice showed milder anemia and better overall survival (p=0.044), indicating that MLKL promotes ineffective hematopoiesis in MDS. Taken together, our results identify MLKL as a key mediator of age-related functional decline in HSCs and ineffective hematopoiesis in MDS. They also suggest that pharmacological inhibition of MLKL activity is a promising strategy to better control aging of the hematopoietic system and age-related hematological disease.

A Phase I Open Label Study of Fostamatinib, a SYK Inhibitor, in Patients with Lower-Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

INTRODUCTION: Ineffective hematopoiesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) have been associated to upregulated innate immune and inflammatory signals. Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is highly expressed in hematopoietic cells and mediates signaling through Toll-like receptors and pattern recognition receptors leading to activation of the ERK, AKT, NF-kB and p38 signaling cascades. Given prior data suggesting that inhibition of these signals might be associated with improved hematopoiesis in MDS we hypothesized SYK inhibition might have therapeutic efficacy in lower-risk MDS and CMML. METHODS: To study this we designed an open label phase I clinical trial of fostamatinib, a SYK inhibitor, for patients with lower-risk MDS or CMML (defined as those with a score <3.5 by the revised International Prognostic Scoring System [IPSS-R]) and no response to prior erythroid stimulating agents or hypomethylating agents (HMAs). Patients with MDS with del(5q) must have received prior therapy with lenalidomide and those with MDS with ring sideroblasts must have received luspatercept. Patients had to be transfusion dependent, defined as prior RBC or platelet transfusions within the past 28 days, or have hemoglobin <10g/dL or platelets <75x109/L. Patients with profound neutropenia (ANC <0.5x109/L) where excluded. The study includes an intra-patient dose escalation design by which patients start therapy at a dose of fostamatinib of 100mg po bid on days 1-28 of a 28-day cycle. If no response to therapy, by IWG 2006 response criteria, is observed after 8 weeks of therapy dose will be escalated to 150mg po bid on days 1-28 of a 28-day cycle and then, in the absence of response after an additional 8 weeks of therapy, to 200mg po bid on days 1-28 of a 28 day cycle. Patients achieving no response after the second dose escalation will be discontinued from study (Figure 1a). The primary endpoint of the study is to assess the safety and tolerability of fostamatinib. Secondary objectives included overall response rates, transfusion independence and survival outcomes. The study includes futility and safety stopping rules. This study is registered at clinicaltrials.gov (NCT05030675). RESULTS: To date, a total of 5 patients have been treated on study as of July 2022 including 3 patients with MDS and ring sideroblasts. Patient characteristics are detailed in Figure 1b. Median age is 71 years (range 59-82). Median number of prior therapies was 5 (range 4-6) including one patient with a prior allogeneic stem-cell transplant. Four (80%) patients had received prior therapy with HMA. Two patients were transfusion dependent of RBC at the time of enrollment with one patient who was eligible based on the presence of thrombocytopenia. At the current data cut off, three patients developed treatment emergent adverse events the main including grade 1-2 diarrhea or constipation. One patient developed COVID-19 infection during the course of therapy and had mild symptomatology. Fostamatinib was not discontinued in this patient. No episodes of expected on-target uncontrolled hypertension or transaminitis have been observed so far. To date, only 4 patients have completed at least 2 cycles of therapy (8 weeks) and are eligible for response, and only 2 patients have dose escalated beyond 100mg po bid: 1 currently at the 150mg po bid dose level, and 1 at the maximum dose of 200mg po bid. With a median follow up of only 2.7 months (95% CI 0-5.6), no responses have been observed. All 5 patients remain on study: 3 (60%) at the 100mg po bid dose, 1 (20%) at 150mg po bid and 1 (20%) at 200mg po bid. CONCLUSIONS: Preliminary data suggests that treatment with fostamatinib at doses of up to 200mg po bid can be safely administered in patients with lower-risk MDS who have failed available therapies. Enrollment is ongoing to determine the potential efficacy of doses of up to 200mg bid. Additional correlative studies to determine predictors of response are underway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

Mitochondrial Fragmentation Triggers Ineffective Hematopoiesis in Myelodysplastic Syndromes.

Ineffective hematopoiesis is a fundamental process leading to the pathogenesis of myelodysplastic syndromes (MDS). However, the pathobiological mediators of ineffective hematopoiesis in MDS remain unclear. Here, we demonstrated that overwhelming mitochondrial fragmentation in mutant hematopoietic stem cells and progenitors (HSC/P) triggers ineffective hematopoiesis in MDS. Mouse modeling of CBL exon deletion with RUNX1 mutants, previously unreported comutations in patients with MDS, recapitulated not only clinically relevant MDS phenotypes but also a distinct MDS-related gene signature. Mechanistically, dynamin-related protein 1 (DRP1)-dependent excessive mitochondrial fragmentation in HSC/Ps led to excessive reactive oxygen species production, induced inflammatory signaling activation, and promoted subsequent dysplasia formation and impairment of granulopoiesis. Mitochondrial fragmentation was generally observed in patients with MDS. Pharmacologic inhibition of DRP1 attenuated mitochondrial fragmentation and rescued ineffective hematopoiesis phenotypes in mice with MDS. These findings provide mechanistic insights into ineffective hematopoiesis and indicate that dysregulated mitochondrial dynamics could be a therapeutic target for bone marrow failure in MDS. SIGNIFICANCE: We demonstrated that excessive mitochondrial fragmentation is a fundamental pathobiological phenomenon that could trigger dysplasia formation and ineffective hematopoiesis in MDS. Our findings provide mechanistic insights into ineffective hematopoiesis and suggest dysregulated mitochondrial dynamics as a therapeutic target for treating MDS.This article is highlighted in the In This Issue feature, p. 1.

Targeting ineffective hematopoiesis in myelodysplastic syndromes.

Data sharing not applicable - no new data generated, or the article describes entirely theoretical research.

Utility of plasma cell-free DNA for de novo detection and quantification of clonal hematopoiesis.

Although cell-free DNA (cfDNA) tests have emerged as a potential non-invasive alternative to bone marrow biopsies for monitoring clonal hematopoiesis in hematologic diseases, whether commercial cfDNA assays can be implemented for the detection and quantification of de novo clonal hematopoiesis in place of blood cells is uncertain. In this study, peripheral plasma cfDNA samples available from patients with aplastic anemia (n=25) or myelodysplastic syndromes (n=27) and a healthy cohort (n=107) were screened for somatic variants in genes related to hematologic malignancies using a Clinical Laboratory Improvement Amendments-certified panel. Results were further compared to DNA sequencing of matched blood cells. In reported results, 85% of healthy subjects, 36% of patients with aplastic anemia and 74% of patients with myelodysplastic syndromes were found to have somatic cfDNA variants, most frequently in DNMT3A, TET2, ASXL1 and SF3B1. However, concordance between cfDNA and blood cell findings was poor for the detection of clonal hematopoiesis when the allele frequency of the variants was <10%, which was mostly observed in the healthy and aplastic anemia cohorts but not in patients with myelodysplastic syndromes. After filtering data for potential artifacts due to low variant allele frequency and sequencing depth, the frequency of clonal hematopoiesis in cfDNA from healthy individuals and patients with aplastic anemia decreased to 52% and 20%, respectively. cfDNA and matched blood cells were not interchangeable for tracking changes in allele burdens as their agreement by Bland-Altman analysis was poor. A commercial cfDNA assay had good performance for de novo detection of clonal hematopoiesis in myelodysplastic syndromes, but showed no advantage over blood cells in diseases with low allele burdens or in healthy individuals.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Oxidized mitochondrial DNA released after inflammasome activation is a disease biomarker for myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell malignancies that can phenotypically resemble other hematologic disorders. Thus, tools that may add to current diagnostic practices could aid in disease discrimination. Constitutive innate immune activation is a pathogenetic driver of ineffective hematopoiesis in MDS through Nod-like receptor protein 3 (NLRP3)–inflammasome-induced pyroptotic cell death. Oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis, acts as a danger signal, and triggers inflammasome oligomerization via DNA sensors. By using immortalized bone marrow cells from murine models of common MDS somatic gene mutations and MDS primary samples, we demonstrate that ox-mtDNA is released upon pyroptosis. ox-mtDNA was significantly increased in MDS peripheral blood (PB) plasma compared with the plasma of healthy donors, and it was significantly higher in lower-risk MDS vs higher-risk MDS, consistent with the greater pyroptotic cell fraction in lower-risk patients. Furthermore, ox-mtDNA was significantly higher in MDS PB plasma compared with all other hematologic malignancies studied, with the exception of chronic lymphocytic leukemia (CLL). Receiver operating characteristic/area under the curve (ROC/AUC) analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for patients with MDS compared with healthy donors (AUC, 0.964), other hematologic malignancies excluding CLL (AUC, 0.893), and reactive conditions (AUC, 0.940). ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and apoptosis-associated speck-like protein containing caspase recruitment domain (CARD) specks, which provide an index of medullary pyroptosis. Collectively, these data indicate that quantifiable ox-mtDNA released into the extracellular space upon inflammasome activation serves as a biomarker for MDS and the magnitude of pyroptotic cell death.

The Metabolomic Status of the Differentiating Myeloid Lineage in MDS with Low and High Bone Marrow Blast Counts

Despite its major role in cellular biology, metabolism has only recently acquired a principal role in the research of the most profound cellular cycle disturbance, cancerous transformation. Myelodysplastic syndromes (MDS), a massively heterogeneous group of Hematopoietic Stem/ Progenitor Cell (HSC/HPC) disorders lie at the interface of normal differentiation and malignant transformation and have thus drew great attention due to their polymorphic presentation and elusive pathophysiology. Failure to establish a direct etiopathogenic relationship with specific genetic aberrations, along with the novel finding of a highly deregulated HIF1 activity by several unrelated research groups worldwide, including ours, urged us to investigate the metabolomic status of human bone marrow derived differentiating myeloid lineage in comparison with one another as well as with control samples. BM aspiration samples collected from 14 previously untreated MDS patients (10 patients with &amp;lt;5% (1 SLD, 8MLD, 1del5q, group 1- G1) and 4 with &amp;gt;5% BM blasts (2 EB1, 2 EB2group 2 - G2)) and 5 age matched controls. Myeloid lineage cells were isolated through ficoll bilayer protocol. All samples contained homogenous myeloid lineage subpopulations, assessedthrough optical microscopy. Two different metabolite extraction protocols were applied. The one with the best metabolites yield (50% MeOH, 30% ACN, 20% H2O) was chosen. LC-MS/MS analysis was performed using UPLC 1290 system (Agilent Technologies) coupled to a TripleTOF 5600+ mass spectrometer (SCIEX) equipped with SWATH acquisition, SelexION technology and an electrospray ionization source (ESI). A threshold of a minimum of three samples expressing a given metabolite was set against data sparsity. Data tables were scaled by data centering and setting unit variance. Log2 Foldcalculation and PLS analysis were performed for the two datasets (positive and negative ion-modes). R2 and Q2 for positive ion-mode and negative-ion mode analyses were determined. Both datasets were merged in a unique data table by taking into account maximum absolute log2 foldvalues, when a metabolite was found in both datasets. Warburg effect was evidently present in both the G1 and G2 vs control comparisons, yet the role of this stem like aerobic glycolysis seems markedly different in the two groups. While in the G2 group it serves to rescue glucose from complete burn in the mitochondrion and thus shuts it towards nucleotide synthesis (Pentose Phosphate Pathway found upregulated) with the added benefit of increased reduced Glutathione synthesis and improved redox state, in the G1 group proves detrimental. This greatly variable effect of the same phenomenon in the cellular fate lies upon the quality and functionality of the cellular mitochondrial content. G2 precursors presented functional mitochondrial (decreased NAD/NADH and FAD/FADH2) contrary to the G1 ones (Table). Failing TCA cycle, with increased NAD/NADH and FAD/FADH2 ratios and markedly increased ADP/ATP levels leads to FAs accumulation due to failure of effective adequate β oxidation. The uncontrolled increase in the NAD/NADH ratio stimulates upper glycolysis into a turbo mode further increasing the ADP/ATP, depleting cellular energy contents, engaging it to a never-ending deadly metabolism. The enormous abundance of upper glycolytic intermediates is relieved through phospholipid and ceramide synthesis, all found massively upregulated in both the MDS vs control yet also in the G1 vs G2 comparisons. FAs, mostly phospholipid and ceramide accumulation, interrupt the mitochondrial membrane lipidome further incapacitating metabolic integrity and inducing their autophagic degradation which further stimulates the Warburg effect. This type of metabolic reprogramming is eventually targeted to epigenetic modifier production, increased S-adenosyl-methionine, the major methyl group donor, 2-HydroxyGlutarate, a potent epigenetic modifier and notorious oncometabolite, Acetyl-Lysine, the major acetyl- group donor, even glutathione. We therefore present a model of an uncontrolled Warburg effect which in the G1 group confers premature death of the hematopoietic precursors, the ineffective hematopoiesis of MDS. Yet, under the pressure of the vastly upregulated epigenetic modifiers cellular fate changes, the G1 precursors adapt and transform to the G2 ones yet eventually to Acute Myeloid Leukemia blasts. Table Disclosures Vassilopoulos: Genesis pharma SA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (&gt;26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents &amp; Royalties: royalties for the invention of NUP98-HOXD13.

A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe &amp; Dohme: Research Funding.

U2AF1 Driver Mutations in Hematopoietic Disorders Alter but Do Not Abrogate RNA Binding and Enlighten Structural Dependencies of the U2AF-RNA Complex

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

Single-Cell RNA Sequencing Reveals Distinct Hematopoietic Stem Cell Hierarchies in MDS

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by dysplastic and ineffective hematopoiesis that can result from aberrant expansion and activation of myeloid-derived suppressor cells (MDSCs) within the bone marrow (BM) niche. MDSCs produce S100A9, which mediates premature death of hematopoietic stem and progenitor cells (HSPCs). The PD-1/PD-L1 immune checkpoint impairs immune responses by inducing T-cell exhaustion and apoptosis, but its role in MDS is uncharacterized. Here we report an increased expression of PD-1 on HSPCs and PD-L1 on MDSCs in MDS versus healthy donors, and that this checkpoint is also activated in S100A9 transgenic (S100A9Tg) mice, and by treatment of BM mononuclear cells (BM-MNC) with S100A9. Further, MDS BM-MNC treated with recombinant PD-L1 underwent cell death, suggesting that the PD-1/PD-L1 interaction contributes to HSPC death in MDS. In accordance with this notion, PD-1/PD-L1 blockade restores effective hematopoiesis and improves colony-forming capacity in BM-MNC from MDS patients. Similar findings were observed in aged S100A9Tg mice. Finally, we demonstrate that c-Myc is required for S100A9-induced upregulation of PD-1/PD-L1, and that treatment of MDS HSPCs with anti-PD-1 antibody suppresses the expression of Myc target genes and increases the expression of hematopoietic pathway genes. We conclude anti-PD-1/anti-PD-L1 blocking strategies offer therapeutic promise in MDS in restoring effective hematopoiesis.

Impairment of the Stromal SDF-1-Mediated Hematopoietic Support By GDF-11 in MDS Is Rescued By Luspatercept

Introduction:Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology.Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown.Aim:We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs).Methods:MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo.Results:GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment.Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p< 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p< 0.05, n=6).These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1).Conclusion:These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS. DisclosuresPlatzbecker:Celgene: Research Funding.