Key Points• Key biological features of MDSs are explained by NLRP3 inflammasome activation, which drives pyroptotic cell death and b-catenin activation.• Alarmin signals and founder gene mutations license this redox-sensitive inflammasome platform.Despite genetic heterogeneity, myelodysplastic syndromes (MDSs) share features of cytological dysplasia and ineffective hematopoiesis. We report that a hallmark of MDSs is activation of the NLRP3 inflammasome, which drives clonal expansion and pyroptotic cell death. Independent of genotype, MDS hematopoietic stem and progenitor cells (HSPCs) overexpress inflammasome proteins and manifest activated NLRP3 complexes that direct activation of caspase-1, generation of interleukin-1b (IL-1b) and IL-18, and pyroptotic cell death. Mechanistically, pyroptosis is triggered by the alarmin S100A9 that is found in excess in MDS HSPCs and bone marrow plasma. Further, like somatic gene mutations, S100A9-induced signaling activates NADPH oxidase (NOX), increasing levels of reactive oxygen species (ROS) that initiate cation influx, cell swelling, and b-catenin activation. Notably, knockdown of NLRP3 or caspase-1, neutralization of S100A9, and pharmacologic inhibition of NLRP3 or NOX suppress pyroptosis, ROS generation, and nuclear b-catenin in MDSs and are sufficient to restore effective hematopoiesis. Thus, alarmins and founder gene mutations in MDSs license a common redox-sensitive inflammasome circuit, which suggests new avenues for therapeutic intervention. (Blood. 2016;128(25):2960-2975

Myelodysplastic syndromes (MDS) are characterized by bone marrow cytological dysplasia and ineffective hematopoiesis in the setting of recurrent somatic gene mutations and chromosomal abnormalities. The underlying pathogenic mechanisms that drive a common clinical phenotype from a diverse array of genetic abnormalities have only recently begun to emerge. Accumulating evidence has highlighted the integral role of the innate immune system in upregulating inflammatory cytokines via NF-κB activation in the pathogenesis of MDS. Recent investigations implicate activation of the NLRP3 inflammasome in hematopoietic stem/progenitor cells as a critical convergence signal in MDS with consequent clonal expansion and pyroptotic cell death though caspase-1 maturation. Specifically, the alarmin S100A9 and/or founder gene mutations trigger pyroptosis through the generation of reactive oxygen species leading to assembly and activation of the redox-sensitive NLRP3 inflammasome and β–catenin, assuring propagation of the MDS clone. More importantly, targeted inhibition of varied steps in this pathway restore effective hematopoiesis. Together, delineation of the role of pyroptosis in the clinical phenotype of MDS patients has identified novel therapeutic strategies that offer significant promise in the treatment of MDS.

Anemia is characteristic of myelodysplastic syndromes (MDS). The mechanisms of anemia in MDS are unclear. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, encoded on chromosome 5q and commonly deleted in MDS (del(5q) MDS), causes an age-related anemia and ineffective erythropoiesis mimicking human MDS. We demonstrate that the ageing bone marrow microenvironment is important for the development of ineffective erythropoiesis in these mice. Damage-associated molecular pattern molecules (DAMPs), whose levels increase in ageing bone marrow, induced TNFα and IL-6 upregulation in myeloid-derived suppressor cells (MDSCs) in mDia1/miR-146a double knockout mice. Mechanistically, we reveal that pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and apoptosis. Treatment of the mDia1/miR-146a double knockout mice with all-trans retinoic acid, which promoted the differentiation of MDSCs and ameliorated the inflammatory bone marrow microenvironment, significantly rescued anemia and ineffective erythropoiesis. Our study underscores the dual roles of the ageing microenvironment and genetic abnormalities in the pathogenesis of ineffective erythropoiesis in del(5q) MDS.

Key Points• mDia1 deficiency led to a cellautonomous overexpression of CD14 on granulocytes and a hypersensitive innate immune response.• mDia1 heterozygous and knockout mice developed age-dependent MDS that was accelerated by chronic stimulation of the innate immunity.The myelodysplastic syndromes (MDSs) include a spectrum of stem cell malignancies characterized by an increased risk of developing acute myeloid leukemia. Heterozygous loss of chromosome 5q (del[5q]) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin proteins, mDia1, which is involved in linear actin polymerization. Mice with mDia1 deficiency develop hematologic features with age mimicking human myeloid neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 heterozygous and knockout mice develop MDS phenotypes with age. In these mice, CD14 was aberrantly overexpressed on granulocytes in a cell-autonomous manner, leading to a hypersensitive innate immune response to lipopolysaccharide (LPS) stimuli through CD14/Toll-like receptor 4 signaling. Chronic stimulation with LPS accelerated the development of MDS in mDia1 heterozygous and knockout mice that can be rescued by lenalidomide. Similar findings of CD14 overexpression were observed on the bone marrow granulocytes of del(5q) MDS patients. Mechanistically, mDia1 deficiency led to a downregulation of membrane-associated genes and a specific upregulation of CD14 messenger RNA in granulocytes, but not in other lineages. These results underscore the significance of mDia1 heterozygosity in deregulated innate immune responses in del(5q) MDS. (Blood. 2014;124(5):780-790)

Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = −0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).

Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes. A key myeloid-related inflammatory protein, S100A9, serves as a Toll-like receptor ligand regulating tumor necrosis factor-α and interleukin-1β production. The role of myelodysplastic syndrome-related inflammatory proteins in endogenous erythropoietin regulation and response to erythroid-stimulating agents or lenalidomide has not been investigated. The HepG2 hepatoma cell line was used to investigate in vitro erythropoietin elaboration. Serum samples collected from 311 patients with myelodysplastic syndrome were investigated (125 prior to treatment with erythroid-stimulating agents and 186 prior to lenalidomide therapy). Serum concentrations of S100A9, S100A8, tumor necrosis factor-α, interleukin-1β and erythropoietin were analyzed by enzyme-linked immunosorbent assay. Using erythropoietin-producing HepG2 cells, we show that S100A9, tumor necrosis factor-α and interleukin-1β suppress transcription and cellular elaboration of erythropoietin. Pre-incubation with lenalidomide significantly diminished suppression of erythropoietin production by S100A9 or tumor necrosis factor-α. Moreover, in peripheral blood mononuclear cells from patients with myelodysplastic syndromes, lenalidomide significantly reduced steady-state S100A9 generation (P=0.01) and lipopolysaccharide-induced tumor necrosis factor-α elaboration (P=0.002). Enzyme-linked immunosorbent assays of serum from 316 patients with non-del(5q) myelodysplastic syndromes demonstrated a significant inverse correlation between tumor necrosis factor-α and erythropoietin concentrations (P=0.006), and between S100A9 and erythropoietin (P=0.01). Moreover, baseline serum tumor necrosis factor-α concentration was significantly higher in responders to erythroid-stimulating agents (P=0.03), whereas lenalidomide responders had significantly lower tumor necrosis factor-α and higher S100A9 serum concentrations (P=0.03). These findings suggest that S100A9 and its nuclear factor-κB transcriptional target, tumor necrosis factor-α, directly suppress erythropoietin elaboration in myelodysplastic syndromes. These cytokines may serve as rational biomarkers of response to lenalidomide and erythroid-stimulating agent treatments. Therapeutic strategies that either neutralize or suppress S100A9 may improve erythropoiesis in patients with myelodysplastic syndromes.

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.