Myelodysplastic Syndromes Progenitor Research Articles

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

In Silico Pan-Cancer Analysis Reveals Prognostic Role of the Erythroferrone (ERFE) Gene in Human Malignancies.

The erythroferrone gene (ERFE), also termed CTRP15, belongs to the C1q tumor necrosis factor-related protein (CTRP) family. Despite multiple reports about the involvement of CTRPs in cancer, the role of ERFE in cancer progression is largely unknown. We previously found that ERFE was upregulated in erythroid progenitors in myelodysplastic syndromes and strongly predicted overall survival. To understand the potential molecular interactions and identify cues for further functional investigation and the prognostic impact of ERFE in other malignancies, we performed a pan-cancer in silico analysis utilizing the Cancer Genome Atlas datasets. Our analysis shows that the ERFE mRNA is significantly overexpressed in 22 tumors and affects the prognosis in 11 cancer types. In certain tumors such as breast cancer and adrenocortical carcinoma, ERFE overexpression has been associated with the presence of oncogenic mutations and a higher tumor mutational burden. The expression of ERFE is co-regulated with the factors and pathways involved in cancer progression and metastasis, including activated pathways of the cell cycle, extracellular matrix/tumor microenvironment, G protein-coupled receptor, NOTCH, WNT, and PI3 kinase-AKT. Moreover, ERFE expression influences intratumoral immune cell infiltration. Conclusively, ERFE is aberrantly expressed in pan-cancer and can potentially function as a prognostic biomarker based on its putative functions during tumorigenesis and tumor development.

Targeting inflammation in lower-risk MDS

The myelodysplastic syndromes (MDS) are a heterogeneous group of malignant hematopoietic stem cell disorders characterized by ineffective growth and differentiation of hematopoietic progenitors leading to peripheral blood cytopenias, dysplasia, and a variable risk of transformation to acute myelogenous leukemia. As most patients present with lower-risk disease, understanding the pathogenesis of ineffective hematopoiesis is important for developing therapies that will increase blood counts in patients with MDS. Various inflammatory cytokines are elevated in MDS and contribute to dysplastic differentiation. Inflammatory pathways mediated by interleukin (IL) 1b, IL-6, IL-1RAP, IL-8, and others lead to growth of aberrant MDS stem and progenitors while inhibiting healthy hematopoiesis. Spliceosome mutations can lead to missplicing of genes such as IRAK4, CASP8, and MAP3K, which lead to activation of proinflammatory nuclear factor κB-driven pathways. Therapeutically, targeting of ligands of the transforming growth factor β (TGF-β) pathway has led to approval of luspatercept in transfusion-dependent patients with MDS. Presently, various clinical trials are evaluating inhibitors of cytokines and their receptors in low-risk MDS. Taken together, an inflammatory microenvironment can support the pathogenesis of clonal hematopoiesis and low-risk MDS, and clinical trials are evaluating anti-inflammatory strategies in these diseases.

TLR9-Selective Therapeutic Targeting of MDS Clones with a CpG Payload-Delivery Strategy

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

Emerging immuno-oncology targets in Myelodysplastic Syndromes (MDS)

Approaches to immunologic therapies in myelodysplastic syndromes (MDS) have generally fallen into 2 categories: therapies that target immune effector cells and enhance or direct an antileukemic effect, and therapies which target immunological markers on MDS progenitors themselves. Examples of the former include immune checkpoint inhibitors, immunomodulatory therapies, and vaccines, among others, while examples of the latter include antibody-drug conjugate therapies and naked antibodies; while bispecific antibodies and modified T-cells (such as CAR-T therapies) bridge both therapeutic modalities. In this review, we will discuss the rationale for the above therapies, clinical results to date, and potential future directions for investigation.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

BackgroundThe identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies.ResultsIn healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy.To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes.Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models.ConclusionWe have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. DisclosuresDick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

PI3-Kinase Deletion Dysregulates Autophagy in HSCs and Promotes Myelodysplasia

▪Myelodysplastic Syndrome (MDS) is a heterogeneous clonal malignancy arising in hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis, cytopenias, and the potential to progress to acute myeloid leukemia (AML). However, the perturbations in HSCs that lead to MDS initiation are poorly understood. It has been reported that HSCs are particularly dependent on autophagy for the maintenance of differentiation and self-renewal. We observed that, compared to healthy donor bone marrow hematopoietic stem and progenitor cells (HSPCs), MDS patient stem and progenitor cells (Lin-CD33-CD34+CD38-) have abnormal levels of autophagic degradation, as demonstrated by abnormal intracellular LC3II and P62 staining (Figure 1A). Autophagy is known to be regulated by the (PI3K)/AKT pathway, which transduces hematopoietic growth factor and cytokine signals in HSCs. PI3K/AKT is frequently activated in AML, but its role in MDS is less clear. Surprisingly, we found that CD34+ cells from a subset of MDS patients have upregulated expression of PTEN, the major negative regulator of the PI3K/AKT pathway, suggesting that PI3K/AKT may be downregulated in MDS stem cells. Therefore, we hypothesized that the Class IA PI3K isoforms (P110α, β, and δ) are required to maintain HSC differentiation and self-renewal.To understand the consequences of PI3K downregulation in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. Surprisingly, we found that PI3K deletion causes transplantable pancytopenia and decreased survival, despite the abnormal expansion of donor TKO HSCs (Figure 1 B,C). Consistent with this inefficient hematopoiesis, TKO bone marrow cells exhibited dysplastic features in multiple blood lineages and multiple chromosomal abnormalities (Figure 1 E,F), suggesting that PI3K inactivation in HSCs can promote MDS initiation. To determine whether impaired HSC differentiation in TKO mice could be due to dysregulated autophagy, we assessed autophagy in TKO HSCs by flow cytometry and immunofluorescence with the autophagosomal marker, LC3II. Our results showed that, compared to the WT controls, TKO HSCs have inefficient autophagic flux and decreased degradation of the cargo protein P62. We also discovered that TKO HSCs have significantly enlarged autophagic vesicles (Figure 1 G), and impaired fusion of autophagosomes with lysosomes, consistent with a marked defect in autophagic degradation. Treatment of TKO mice with two pharmacologic inducers of autophagy, rapamycin or metformin, improved HSC differentiation with an increase in Flk2+ MPPs (Figure 1 H), reduced dysplasia, and decreased the size of the TKO mutant clone in chimeric mice. Thus, our results uncover an important role for PI3K in regulating autophagy in HSCs to maintain the proper balance between self-renewal and differentiation. Our new mouse model of MDS will be a useful tool to study the mechanisms of MDs initiation. In addition, our findings open exciting avenues for future investigations of autophagy-inducing agents in MDS. [Display omitted] DisclosuresVerma: Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; BMS: Research Funding; GSK: Research Funding. Gritsman: iOnctura: Research Funding.

Unique Metabolic Vulnerabilities of Myelodysplastic Syndrome Stem Cells

Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study.Methods: Bone marrow specimens from MDS patients at various disease stages, including serial samples during progression, were obtained. Single cell techniques including mass cytometry, antibody based single cell RNA sequencing (CITE-Seq) and transcriptional profiling with RNA sequencing were used to elucidate novel mechanisms of progression. Selective targeting of primitive MDS cells was tested using several agents.Results: Our previous work characterizing MDS stem cells (MDSC) showed significant similarities between MDSCs and AML LSCs (Stevens et al, Nat. Communications, 2018). However, little is known about lower risk disease. In order to understand transcriptional changes and their relationship to metabolism across pathogenesis, the transcriptome of blasts from patients with LR, intermediate (INT), or HR IPSS scores was investigated. The first major transcriptional difference identified was enrichment of glycolysis pathway at LR and INT stage. In contrast, HR MDS demonstrated enrichment of oxidative phosphorylation. Furthermore, comparison of intermediate to HR MDS showed increased RNA polymerase and Ribosome pathways at the HR stage. These changes demonstrate the progressive alteration of metabolic properties during MDS pathogenesis with cells first relying on mechanisms associated with normal stem cells (i.e. glycolysis) and later transitioning to a state associated with AML stem cells (i.e. reliance on oxidative phosphorylation).Using serial specimens of patients of who progressed from LR to HR MDS we performed CITE-Seq and mass cytometry. CITE-seq in serial specimens showed up-regulation of protein translation and oxidative phosphorylation in a subset of MDS stem and progenitor cells (CD34+ at transcript and antibody level) present at LR stage and conserved at HR stage (Fig 1A-C). MDSCs also acquired surface antigens including CD99 and CD52 upon progression from LR to HR. Analysis of the mass cytometry data showed significant overlap with CITE-Seq data including increased CD123+ and MCL1 expression in MDS stem cells upon progression.In order to understand therapeutic vulnerabilities as they relate to progression, we investigated ex vivo drug response in LR and HR specimens. MDS samples were challenged with two regimens, Ven/Aza, a regimen known to inhibit OXPHOS; and omacetaxine and azacitidine (Oma/Aza), which inhibits translation. CITE-seq showed that MDSC were selectively sensitive to these agents (Fig 1D). Importantly, addition of either drug regimen caused ablation of MDSC at LR and HR stages and these changes were most profound in cells with LSC properties.Based on preclinical findings, we are investigating MDS patients treated with Ven/Aza or Oma/Aza via CITE-seq and metabolomics for correlation of clinical response with properties of MDSC. Preliminary studies show that patients that respond to Oma/Aza present with a population of MDSC with transcriptional signatures of protein translation and LSCs (Fig. 1E). Studies are underway to investigate overlapping properties of ven/aza resistance in AML to resistance in MDS specifically investigating fatty acid metabolism in MDSC.Conclusions: Analysis of MDS patient bone marrow reveals acquisition of aberrant metabolic properties at both low and high risk stages of disease. These distinct aspects of MDSC biology create unique and targetable features. [Display omitted] DisclosuresPollyea: Genentech: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Consultancy; Glycomimetics: Other.

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.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

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Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.

Despite evidence of chronic inflammation in myelodysplastic syndrome (MDS) and cell-intrinsic dysregulation of Toll-like receptor (TLR) signaling in MDS hematopoietic stem and progenitor cells (HSPCs), the mechanisms responsible for the competitive advantage of MDS HSPCs in an inflammatory milieu over normal HSPCs remain poorly defined. Here, we found that chronic inflammation was a determinant for the competitive advantage of MDS HSPCs and for disease progression. The cell-intrinsic response of MDS HSPCs, which involves signaling through the noncanonical NF-κB pathway, protected these cells from chronic inflammation as compared to normal HSPCs. In response to inflammation, MDS HSPCs switched from canonical to noncanonical NF-κB signaling, a process that was dependent on TLR-TRAF6-mediated activation of A20. The competitive advantage of TLR-TRAF6-primed HSPCs could be restored by deletion of A20 or inhibition of the noncanonical NF-κB pathway. These findings uncover the mechanistic basis for the clonal dominance of MDS HSPCs and indicate that interfering with noncanonical NF-κB signaling could prevent MDS progression.

Inhibition of LSD1 in MDS progenitors restores differentiation of CD141Hi conventional dendritic cells.

The use of immunotherapy to treat patients with myelodysplastic syndromes (MDS) shows promise but is limited by our incomplete understanding of the immunologic milieu. In solid tumors, CD141Hi conventional dendritic cells (CD141Hi cDCs) are necessary for anti-tumor immunosurveillance and the response to immunotherapy. Here, we found that CD141Hi cDCs are reduced in MDS bone marrow and based on the premise established in solid tumors, we hypothesized that reduced numbers of CD141Hi cDCs are associated with inferior overall survival in MDS patients. We found that MDS patients with reduced numbers of CD141Hi cDCs, but not other DC populations, showed reduced overall survival. To examine the basis for reduction in CD141Hi cDCs, we found fewer numbers of progenitors committed to DC differentiation in the MDS bone marrow and these progenitors expressed lower levels of interferon regulatory factor-8 (IRF8), a master regulator of CD141Hi cDC differentiation. To rescue impaired CD141Hi cDC differentiation, we used pharmacologic inhibition of lysine-specific demethylase 1A (LSD1) to promote CD141Hi cDC differentiation by MDS progenitors. These data reveal a previously unrecognized element of the MDS immunologic milieu. Epigenetic regulation of CD141Hi cDC differentiation offers an intriguing opportunity for intervention and a potential adjunct to immunotherapy for patients with MDS.

Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes

Myelodysplastic Syndromes (MDS) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, Nlrp3 inflammasome (IFM) driven pyroptotic cell death and the induction of interferon-stimulated genes (ISG). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p&amp;lt;0.03).We next confirmed that TLR9 interacts with ox-mtDNA by immunofluorescence (IF) microscopy in MDS samples and murine somatic gene mutation (SGM) models (Tet2-/- and Srsf2P95H). We further investigated the relationship between TLR9 and ox-mtDNA during IFM activation. Treatment of SKM1 and U937 cells with the TLR4 ligand LPS and priming agents ATP/nigericin initiates a robust increase in ox-mtDNA that co-localized intracellularly with TLR9 by IF microscopy. Furthermore, upon incubation with ox-mtDNA, ox-mtDNA bound TLR9 is internalized. Additionally, interferon regulatory factor 7 (IRF7), an ISG transcription factor induced by TLR9, translocates to the nucleus following treatment, confirming TLR9 activation in response to ox-mtDNA. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent. 50ng/mL ox-mtDNA was incubated with 3 leukemic cell lines: SKM1, U937, and THP1 that display varying levels of endogenous TLR9 expression, as well as corresponding TLR9-KO cells generated by CRISPR-Cas9 editing. We found that TLR9 was necessary for IFM initiated caspase-1 cleavage in response to ox-mtDNA, thereby demonstrating the specificity of ox-mtDNA for the TLR9 sensor. Notably, receptor density determined response tempo. SKM1 cells, which have the highest TLR9 receptor density, responded within 1 hour, U937 cells which have an intermediate receptor density respond in 2 hours, while THP1 cells, which do not express endogenous TLR9 never responded to ox-mtDNA treatment. TLR9 knockout abrogated IFM activation in response to ox-mtDNA as demonstrated by caspase-1 glo ® assay (n=5, p&amp;lt;0.03). By western blot, TLR9 knockout cells no longer demonstrate phosphorylated NFk-B, cleaved caspase-1, and cleaved IL-1β in response to ox-mtDNA treatment. We conclude that MDS HSPC display functionally competent TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Blocking TLR9 activation may prove to be a novel therapeutic strategy for MDS and suppression of inflammatory BM failure. Disclosures Hou: Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson &amp; Johnson, Kirin, Merck Sharp &amp; Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Inhibition of LSD1 in Myelodysplastic Syndrome Progenitors Restores Differentiation of CD141Hi Conventional Dendritic Cells

Background: Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hi conventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141Hi cDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141Hi cDCs due to impaired myeloid differentiation. Methods: Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results: We found fewer CD141Hi cDCs (p&lt;0.0001), CD1c+ cDCs (p&lt;0.005) and plasmacytoid DCs (CD123+ pDCs; p&lt;0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141Hi cDCs had superior survival (p &lt; 0.05). No differences in survival were seen in patients stratified by the CD1c+ cDCs (p = 0.96) and CD123+ pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141Hi cDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p&lt;0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141Hi cDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p&lt;0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141Hi cDCs compared to those with higher levels of IRF8 (n=10; p&lt;0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141Hi cDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8 expression in myeloid leukemia cells, would induce CD141Hi cDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+ cells, and in HD and MDS CD34+ progenitors (p&lt;0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p&lt;0.05) at a region -70 kb to the IRF8 transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8 locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+ cells increased the number of mature CD141Hi cDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+ cells increased the number of CD141Hi cDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+ cells from Irf8 knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+ cells resulted in increased numbers of CD141Hi cDCs in vitro (p&lt;0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+ cells did not result in differentiation of CD141Hi cDCs. These data suggest that LSD1 inhibition drives CD141Hi cDCs differentiation through IRF8. Conclusion: These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141Hi cDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients. Disclosures Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.

SNP-Array Genome Wide Association Study Meta-Analysis Identifies Innate Immune Susceptibility Loci Associated with Non-Del(5q) Myelodysplastic Syndromes Predisposition

Background: Myelodysplastic Syndromes (MDS) are genetically and hematologically diverse stem cell malignancies pathogenetically linked to constitutive innate immune activation. Other than rare germline mutations and age, the only known predispositions to adult MDS include prior cytotoxic therapy, clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. Few studies investigating the genetic susceptibility to MDS have been performed owing to the limitations of SNP-array sample size. Here, we report the results from the first unbiased genome wide analysis of germline polymorphisms associated with non-del(5q) MDS using a multinational curated data set.Methods: Association analyses were performed on 2 sample sets (set 1: 555 cases, 2,964 controls; set 2: 352 cases, 2,640 controls) and combined by meta-analysis. Standard SNP- and sample-level QC was applied. Haplotype reference consortium (HRC) imputation was done by the Michigan imputation server (Rsq>0.4) providing 23,278,269 markers for analysis. Gene expression sequencing was performed on an independent sample set from the National Taiwan University Hospital (213 MDS cases, 20 healthy donors; HumanHT-12 v4 Expression BeadChip; Chuang et al. Leukemia 2015). Functional analyses were performed as described.Results: Eight MDS associated loci were identified with lead variants, rs6683416, rs34539210, rs341274, rs1634783, rs7099032, rs2947170, rs4404050, and rs1206818, at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2), respctively. Odds ratio (OR) and p-values of each are listed in Table 1. Using gene expression profiling in an independent MDS sample set, we found expression of these five candidate genes was significantly increased in MDS vs controls (p<0.01). Using the same independent sample set, we found higher PLA2G4A and lower EYA2 expression were associated with reduced overall survival (p=0.039 and p=0.037, respectively). Importantly, EYA2 was an independently favorable risk factor irrespective of age, gender and IPSS-R (RR, 0.665; p=0.048). High PLA2G4A expression was associated with NRAS (p<0.001), RUNX1 (p=0.012), ASXL1 (p=0.007), and EZH2 (p=0.038) somatic mutations whereas higher FAM19A4 expression associated with KRAS mutations (p=0.045). Importantly, PLA2G4A, FAM19A4, GRID1, and EYA2 are involved in the regulation of innate immune signaling. We previously reported that across all somatic gene mutation classes, MDS cases biologically converge upon the redox-sensitive, Nlrp3 inflammasome to drive pyroptosis, a caspase-1 mediated innate immune cell death (Basiorka et al. Blood 2016). PLA2G4A encodes phospholipase A2 (PLA2) that inhibits inflammasome activation. Further, PLA2 is required for MAPK phosphorylation of S100A9, a key danger associated molecular pattern (DAMP) and TLR4 ligand implicated in the induction of pyroptosis in MDS progenitors. FAM19A4 encodes a cytokine whose expression is upregulated in response to endotoxin exposure suggesting a role in immune modulation. EYA members activate innate immune response in part through recognition of cytosolic double-stranded DNA, which can serve as a DAMP triggering inflammasome assembly. To investigate the possible role of EYA2 in inflammasome activation in MDS, we overexpressed EYA2 in the monocytic cell line, THP1. EYA2 overexpression induced caspase-1 and maturation of IL-1β, indicating inflammasome activation. These findings were accompanied by NFκB phosphorylation consistent with innate immune signal priming. We next treated MDS primary bone marrow specimens (n=3) with the EYA2 inhibitor, MLS000544460, demonstrating a significant, dose-dependent improvement in colony recovery consistent with restoration of effective hematopoiesis.Conclusion: We describe here the first MDS susceptibility loci ever identified the majority of which have a direct relationship to innate immune activation, a driver of MDS pathogenesis. Expression of genes housing these loci is increased in MDS with demonstrable prognostic and biological relevance. Further, functional studies implicate EYA2 as a novel, biologically rational target for MDS treatment. The direct functions of each polymorphism as well as the potential relationship between these predisposition loci to age-related clonal hematopoiesis merits further investigation. [Display omitted] DisclosuresCluzeau:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Menarini: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau. Sallman:Celgene: Research Funding, Speakers Bureau. Sokol:Spectrum Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Mallinckrodt Pharmaceuticals: Consultancy. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. List:Celgene: Research Funding.

The NLRP3 Inflammasome as a Driver of the MDS Phenotype

Myelodysplastic syndromes (MDS) share features of cytological dysplasia and ineffective hematopoiesis while demonstrating profound molecular and genetic heterogeneity. Our investigations show that many of the hallmark features of MDS arise from activation of the NLRP3 inflammasome that directs clonal expansion and pyroptotic cell death, a caspase-1–dependent pro-inflammatory, lytic form of cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPC) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct the activation of caspase-1, IL-1β and IL-18 generation, and pyroptosis. NLRP3 is a redox-sensitive, cytosolic sensors of danger signals, which upon stimulation, undergoes a conformational change fostering homotypic pyrin domain interaction with the adaptor protein ASC [apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)] and ASC polymerization to create large, cytoplasmic filaments. The ASC speck formed by ASC filament clusters creates abundant caspase-1 activation sites, thereby acting as an inflammasome signal amplification complex. Mechanistically, pyroptosis is triggered by the alarmin S100A9 found in excess in MDS HSPC, myeloid-derived suppressor cells (MDSC) and bone marrow plasma, which like founder gene mutations, induces reactive oxygen species (ROS) to initiate cation influx, cell swelling and β-catenin activation. Accordingly, knockdown of caspase-1, neutralization of S100A9 in BM plasma, and pharmacologic inhibition of the NLRP3 inflammasome or NADPH oxidase suppresses pyroptosis, ROS generation and nuclear β-catenin in MDS HSPC and restores effective hematopoiesis. NLRP3 inflammasome activation appears specific to MDS compared to other bone marrow malignancies, with ASC specks detected and quantified in the peripheral blood providing an index of ineffective hematopoiesis and pyroptotic cell death in MDS patients These data indicate that DAMP signals and oncogenic mutations in MDS HSPC license a common redox-sensitive inflammasome platform, and that targeted disruption of the inflammasome signal can restore effective hematopoiesis.

66 - Allogeneic Transplantation of Hematopoietic Progenitors in Myelodysplastic Syndromes

66 - Allogeneic Transplantation of Hematopoietic Progenitors in Myelodysplastic Syndromes