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Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS., (© 2019 by The American Society of Hematology.)

Background: NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes., Methods: This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis., Findings: Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log 10 -transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97)., Interpretation: Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes., Funding: 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)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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-1β (IL-1β) 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 β-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 β-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., (© 2016 by The American Society of Hematology.)

Inflammatory and immune dysregulation are crucial in the initiation and development of myelodysplastic syndromes (MDS). It is noted that clonal T-cell large granular lymphocyte (T-LGL) proliferation associated with MDS is not uncommon. However, clinicopathological features, and prognostic and predictive value of presence of T-LGL proliferation in MDS patients is not very clear. This study compared 35 MDS patients with T-LGL proliferation with 36 MDS patients without T-LGL proliferation and summarized clinicopathologic features, including peripheral blood LGL cell counts, immunophenotype, T cell receptor gene rearrangement, bone marrow hematopoietic status, and adjuvant immunosuppressive therapy. The peripheral blood CD3+/CD57+ cell counts were significantly different (p<0.01) between the two groups. Notably, on examination of the bone marrow, MDS patients with T-LGL proliferation showed more frequent hypocellularity and/or lineage hypoplasia, particularly erythroid hypoplasia. On survival analysis, no overall difference was noted between MDS patients with T-LGL proliferation and those without T-LGL proliferation, and between the patients who received therapy for LGL and those who did not receive adjuvant therapy for LGL in the same risk group. In conclusion, T-LGL proliferation present in MDS patients can be associated with bone marrow hypocellularity and lineage hypoplasia. Although immunosuppressive therapy to eliminate T-LGL cells is potentially beneficial to the MDS patients with associated T-LGL proliferation, there is no overall survival benefit to the patients who received such treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome.

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.

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-β) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-β signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3'UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3'UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-β signaling in a TGF-β-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-β signaling in MDS.

We examined whether lenalidomide exposure up-regulates miRNAs and mRNAs, previously shown to play a role in the disease phenotype of del(5q) myelodysplastic syndrome, in pre-treatment CD34(+) marrow cells. We hypothesized that increased expression would predict for clinical response. Changes in miR-143, miR-145, miR-146a, miR-146b, miR-378, miR-584, SPARC and RPS14 were examined in del(5q) (n=10) and non-del(5q) (n=18) myelodysplastic syndrome patient samples. Significantly increased expression of miR-143 (1.8-fold and 1.5-fold in del(5q) and non-del(5q), respectively), and miR-145 (1.9-fold and 1.6-fold in del(5q) and non-del(5q), respectively) was observed. In the del(5q) myelodysplastic syndrome cohort, transfusion independence correlated with a 1.3-fold or more increase in miR-145 expression and response over 12 months correlated with a 1.5-fold or more increase. Knockdown of miR-143 and miR-145 in cord blood CD34(+) cells resulted in increased erythroid progenitor activity. Lenalidomide selectively abrogated progenitor activity in cells depleted of miR-143 and miR-145 supporting a key role for miR-143/145 in the sensitivity to lenalidomide of del(5q) myelodysplastic syndrome patients.

Large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of antigen-activated cytotoxic T cells (CTL). Patients frequently exhibit seroreactivity against a human T-cell leukemia virus (HTLV) epitope, BA21. Aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome are bone marrow failure diseases that can also be associated with similar aberrant CTL activation (LGL-BMF). We identified a BA21 peptide that was specifically reactive with LGL leukemia sera and found significantly elevated antibody reactivity against the same peptide in LGL-BMF sera. This finding of shared seroreactivity in LGL-BMF conditions and LGL leukemia suggests that these diseases might share a common pathogenesis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS., (© 2011 Blackwell Publishing Ltd.)

Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases, Cdc25C and PP2Acalpha, which are coregulators of the G(2)-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyotypes. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G(2) arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (shRNA) suppression of Cdc25C and PP2Acalpha gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G(2) arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acalpha phosphatases in the selective drug sensitivity of del(5q) MDS.

Thalidomide and lenalidomide belong to the proprietary group of immunomodulatory drugs (IMiDs) that display broad biologic and pharmacologic properties. Encouraging results of clinical studies that evaluated the efficacy of thalidomide in patients with myelodysplastic syndromes (MDSs) led to the investigation of its structural analogue, lenalidomide, in patients with lower-risk MDS. The cumulative results of studies that tested lenalidomide in patients with interstitial deletion of chromosome 5q, ie, del(5q), showed a high frequency of both erythroid and cytogenetic responses (approximately 75% of patients), which led to US Food and Drug Administration approval of this agent for this cytogenetically defined MDS subset. A multicenter phase III study (MDS-002) that investigated the frequency of transfusion response in lower-risk non-del(5q) MDS patients showed that lenalidomide had significant erythropoietic activity, albeit less robust in lower-risk MDS without del(5q). These studies established lenalidomide as an active erythropoietic-remitting agent with novel cytogenetic-remitting activity in lower-risk MDS patients who would not otherwise benefit from therapy with erythropoietic growth factors. The National Comprehensive Cancer Network Clinical Practice Guidelines recently added lenalidomide to the therapeutic algorithm for MDS as front-line therapy for lower-risk MDS patients with del(5q) and transfusion-dependent anemia.

The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38alpha by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38alpha isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

Lenalidomide has been approved by the US Food and Drug Administration for the treatment of patients with myelodysplastic syndromes (MDS) with an interstitial deletion of the long arm of chromosome 5 and, more recently, in combination with dexamethasone for multiple myeloma in patients who received at least one prior therapy. This discussion examines several clinically relevant, practical considerations regarding dosing, monitoring, follow-up evaluation, adverse events, and available support for lenalidomide recipients and their prescribing physicians in the MDS setting.

The management strategy for patients with myelodysplastic syndromes (MDS) has evolved from sole reliance on supportive measures to active treatment guided by disease risks. Recent progress in understanding the molecular pathogenesis of MDS has accelerated the discovery of new therapeutic targets, and consequently launched the development of several novel therapeutics that are currently in varied stages of clinical testing. One such agent is lenalidomide, which has shown remarkable effectiveness in the cytogenetically defined subset of MDS with the chromosome 5q31 deletion. The advent of new and effective targeted therapeutics may beneficially affect outcomes of an ever-increasing number of patients with MDS. This discussion summarizes the preliminary results of selected novel therapeutics.

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. [ABSTRACT FROM AUTHOR]

Lenalidomide is the first karyotype-selective therapeutic approved for the treatment of myelodysplastic syndromes (MDS) owing to high rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)]. Although haploinsufficiency for the RPS14 gene and others encoded within the common deleted region (CDR) have been implicated in the pathogenesis of the del(5q) phenotype, the molecular basis of the karyotype specificity of lenalidomide remains unexplained. We focused our analysis on possible haplodeficient enzymatic targets encoded within the CDR that play key roles in cell-cycle regulation. We show that the dual specificity phosphatases. Cdc25C and PP2Acα, which are coregulators of the G2-M checkpoint, are inhibited by lenalidomide. Gene expression was lower in MDS and acute myeloid leukemia (AML) specimens with del(5q) compared with those with alternate karyo- types. Lenalidomide inhibited phosphatase activity either directly (Cdc25C) or indirectly (PP2A) with corresponding retention of inhibitory phospho-tyrosine residues. Treatment of del(5q) AML cells with lenalidomide induced G2 arrest and apoptosis, whereas there was no effect in nondel(5q) AML cells. Small interfering RNA (5hRNA) suppression of Cdc25C and PP2Acα gene expression recapitulated del(5q) susceptibility to lenalidomide with induction of G2 arrest and apoptosis in both U937 and primary nondel(5q) MDS cells. These data establish a role for allelic haplodeficiency of the lenalidomide inhibitable Cdc25C and PP2Acα phosphatases in the selective drug sensitivity of del(Sq) MDS. [ABSTRACT FROM AUTHOR]