Role In Myelodysplastic Syndrome Research Articles

Establishing a Pathogenic Role for NK Cells As Drivers of Bone Marrow Failure in the Myelodysplastic Syndromes (MDS)

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant bone-marrow (BM) morphology leading to a clonal impairment of hematopoiesis. Worsening BM failure (BMF) and increasing myeloblasts are observed as the predominant consequences of MDS disease progression from very low risk (VLR) to higher risk (HR) to acute myeloid leukemia (AML). Patients are stratified based on the degree of BMF and risk of AML transformation to help guide treatment decisions. Approximately 30% of MDS patients exhibit various autoimmune diseases that can include autoantibodies targeting neutrophils, erythrocytes, and/or platelets. Autoimmune phenomena play an important role in MDS yet are poorly characterized. We hypothesized that early in LR-MDS, dysregulated interactions between stromal cells and developing Natural Killer (NK)/T-cells promote rapid/unregulated development resulting in autoreactivity ( Fig. 1A). We predict dual effector mechanisms: CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) and loss of HLA class I (HLA-I) expression by developing hematopoietic lineage cells making them targets for “missing-self” reactivity ( Fig. 1A). NK cells are important sentinel cells that recognize tumors and dysfunctional cells that lose expression of HLA class I. Such surveillance is guided by interactions between NK inhibitory receptors and HLA class I ligands that ‘train’ NK cells to distinguish diseased cells, exhibiting perturbed HLA expression, from healthy cells with normal expression. The effects of these interactions combined with expression of activating receptors (e.g. CD16, NKG2D, DNAM1, FasL, TRAIL) allow NK cells to engage a target cell through many activating mechanisms, importantly including antibodies eliciting an antibody-dependent cellular cytotoxicity (ADCC) reaction. Autoantibodies are well-established in MDS, yet no one has profiled whether they may trigger ADCC/complement. Further, no one has ever thoroughly characterized BMMCs from LR-MDS patients before. In our study, we observed a clear link between signatures of bone marrow failure at LR-MDS and a significant increase in BM CD57+NK and CD8 T cells expressing a highly activated and dysregulated phenotype. Method: Ex-vivo profiling of 19 MDS patients (N=10 LR/IR-MDS, N=9 HR-MDS) BM mononuclear cells (BMMCs) was performed by mass cytometry (CyTOF)( Fig.1B). Cells were stained with three different antibody panels each consisting of 44 antibodies focused on: specific markers of hematopoiesis and myeloid or lymphoid cell lineages; specific markers of development, effector function, homing, exhaustion, and T-cell memory and proliferation; expression of HLA class I and activating ligands which are critical regulators of NK inhibition. Results: In our study we observed that a major fraction of MDS patients present with clonal-like expansions of BM-derived CD57+CD8+T-cells and natural killer (NK) cells that strongly correlate with autoimmune-cytopenias and BMF-enriched in LR-MDS (Fig.#). We observed that NK-cells and non-classical T-cells were significantly expanded (>2-fold) in LR MDS compared to HR-MDS and healthy-control BM (p<0.01). These non-classical CD57+CD8+T-cells co-expressed HLA-I-reactive and inhibitory NKG2A/KIR receptors as well as activating CD16, NKG2C, NKG2D, NKp46, TRAIL, FasL. In cancers, these cells use innate-like, TCR-independent mechanisms (traditionally ascribed to NK-cells) to target HLA-I-deficient tumor cells. Strikingly, HLA-I was significantly elevated on myeloblasts in LR-MDS compared to HR-MDS (p=0.0007)( Fig.1B), demonstrating NK-evasion strategies, but strongly reduced on dendritic cells, granulocytes, and CD4+T-cells, suggesting susceptible immune targets for destruction by NK-cells (and innate-like CD8+T-cells) (p< 0.0001) ( Fig.1B). BM-derived CD57+NK and CD8+T-cells all co-express very high levels of CD16 making them particularly ADCC reactive. Conclusion: We identified novel innate-like CD8+CD57+ T-cell and NK-cell populations that may play a role in LR-MDS disease pathogenesis. We predict that a strategy to target these cells could help restore hematopoiesis early in the disease state. This may also reduce pressure on myeloblasts to increase HLA-I, permitting improved immune surveillance to control the blast burden and prevention MDS/AML progression. Further experiments are ongoing to validate our observations.

Bone Marrow Microenvironment As a Potential Key Regulator of Hematopoietic Cells Stemness in Myelodysplastic Syndromes (MDS)

Background and Rationale. Nuclear Phospholipase C (PLC) beta1 plays a pivotal role in Myelodysplastic Syndromes (MDS) and Azacytidine response [Cocco, L., et al. J Lipid Res. 2015; 56: 1853-60]. Hematopoietic Stem Cells (HSCs) primarily reside within the bone marrow (BM) microenvironment, where mesenchymal stromal cells (MSCs) regulate hematopoiesis through direct interactions with HSCs or cytokine secretion [Blau O, et al. Blood. 2011; 118:5583-92]. The aim of this study was to better elucidate the role of the BM microenvironment in interacting with MDS/AML cells, with particular emphasis on investigating the correlation between PLCbeta1 modulation and the distinctive BM microenvironment response after co-culture, in terms of PLCs gene expression, myeloid differentiation markers and cytokine secretion. Patients and Methods. As it is a very preliminary study, here we analyzed mononuclear cells obtained from 4 higher-risk MDS (RAEB-1, R-IPSS High or Very High, Complex Karyotype), who gave informed consent in accordance with the Declaration of Helsinki. Two patients were treated with Azacytidine (AZA, 75 mg/mq/die for 7 days every 28 days) and one of them reached the fifth cycle with a stable disease. All samples were obtained from the IRCCS-Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy, where clinical evaluations were also conducted. Co-cultures were established with HS-5 MSCs, while Real-Time PCR results were normalized using molecular data obtained from 10 healthy donors (HD). PLCbeta1 overexpression/silencing in vitro model was represented by THP-1 monocytic cells subjected to lentiviral transfection. Both wild-type and PLCbeta1 overexpressing/silencing cells (THP-1 OV/KD) were co-cultured with HS-5 cells for 96 hours. Results. Patients showed distinct basal expression patterns of PLCbeta1: higher in 1/4 MDS and lower in 3/4 MDS, as compared to HD. Irrespective of PLCbeta1 basal expression levels, or response to AZA, in 3/4 patients CD11b increased and CD14 decreased after co-culture. Only one patient, characterized by being a therapy-related MDS, displayed a reduced expression in all myeloid differentiation markers during co-culture. During AZA therapy and after co-culture, one patient showing a stable disease had a significant PLCbeta1 reduction, CD11b increase and CD14 decrease. In THP-1 OV cells co-cultured with HS-5 cells, we observed CD11b reduction, while no significant differences in both PLCs and other myeloid differentiation markers. In contrast, THP-1 KD cells, following co-culture, exhibited a notable increase in CD11b and a decreased CD14 expression. Moreover, THP-1 OV cells alone displayed inhibition of IL-8 secretion, that disappeared after co-culture, in favour of IL-1beta secretion. THP-1 KD cells exhibited suppressed secretion of IL-1alpha, IL-1beta, and IL-8. However, during co-culture, the production of both IL-1alpha and IL-1beta was restored. Conclusions. Our results, that must be confirmed in a larger group of patients, show that the impact of co-culture is not associated with the baseline levels of PLCbeta1, but co-culture recapitulates the PLCbeta1 KD in vitro model and the lack of response to AZA, showing an increase of CD11b and a CD14 reduction. Moreover, the presence of MSCs reversed cytokine secretion, as in co-cultured THP1 KD cells IL-1alpha and IL-1beta were highly secreted again. Considering their multiple roles in both physiological and pathological conditions, ongoing analyses are now further investigating their role in BM microenvironment, also using MDS-MSCs. In addition, we are now exploring the involvement of BM microenvironment in maintaining leukemic cells in a more undifferentiated state and the potential implications of these mechanisms on AZA response/resistance.

LEP promoter methylation in the initiation and progression of clonal cytopenia of undetermined significance and myelodysplastic syndrome

BackgroundIdiopathic non-clonal cytopenia (ICUS) and clonal cytopenia (CCUS) are common in the elderly population. While these entities have similar clinical presentations with peripheral blood cytopenia and less than 10% bone marrow dysplasia, their malignant potential is different and the biological relationship between these disorders and myeloid neoplasms such as myelodysplastic syndrome (MDS) is not fully understood. Aberrant DNA methylation has previously been described to play a vital role in MDS and acute myeloid leukemia (AML) pathogenesis. In addition, obesity confers a poorer prognosis in MDS with inferior overall survival and a higher rate of AML transformation. In this study, we measured DNA methylation of the promoter for the obesity-regulated gene LEP, encoding leptin, in hematopoietic cells from ICUS, CCUS and MDS patients and healthy controls. We investigated whether LEP promoter methylation is an early event in the development of myeloid neoplasms and whether it is associated with clinical outcome.ResultsWe found that blood cells of patients with ICUS, CCUS and MDS all have a significantly hypermethylated LEP promoter compared to healthy controls and that LEP hypermethylation is associated with anemia, increased bone marrow blast percentage, and lower plasma leptin levels. MDS patients with a high LEP promoter methylation have a higher risk of progression, shorter progression-free survival, and inferior overall survival. Furthermore, LEP promoter methylation was an independent risk factor for the progression of MDS in a multivariate Cox regression analysis.ConclusionIn conclusion, hypermethylation of the LEP promoter is an early and frequent event in myeloid neoplasms and is associated with a worse prognosis.

Clinical significance of prognostic nutritional index in myelodysplastic syndrome

ABSTRACTBackgroundPrognostic nutritional index has been found to be related to the clinical outcomes of patients with cancer. However, its role in myelodysplastic syndromes (MDS) patients is unclear. We aimed to assess the value of nutritional status in predicting the prognosis of MDS patients.MethodsTotally 121 MDS patients were analyzed retrospectively. The prognostic nutritional index (PNI) was used to assess nutritional status of the patients. The bio-informatics tool X-tile was used to define the threshold, and accordingly patients were divided into PNIlow and PNIhigh groups, the characteristics were compared between two groups.ResultsThe PNIhigh was associated with better OS (Overall Survival) than PNIlow in MDS patients (Median OS, 28.03 months versus 19.63 months, P = 0.0205). But there were no statistical differences in PFS (Progression-Free-Survival) between the two groups (P = 0.9373). The univariable and multivariable COX proportional hazard analysis adjusted for age, gender, platelet count, HB level and IPSS-R scores, and the results showed that PNI is a useful index in the evaluation of the OS of MDS (HR 0.588, 95%CI 0.374–0.926, P = 0.024).ConclusionPNI would be a simple and immediately available tool for predicting the prognosis of MDS.

Gut Microbiome, Metabolome and Immune Changes in Myelodysplastic Syndromes

Introduction. Myelodysplastic syndromes (MDS) are a group of multi-factorial disorders, in which the clonal expansion of somatically mutated hematopoietic stem and progenitor cells plays a crucial role. The influence of the bone marrow microenvironment, including inflammatory and metabolic dysbalances, on disease propagation becomes increasingly recognized. Although changes in gut microbiome have been described in various cancer types, its role in MDS has not been sufficiently elucidated yet. Due to its interconnection with carcinogenesis in solid tumours, systemic inflammation and metabolism, the gut microbiome could possibly play a role in MDS development as well. The goal of our study is to describe the composition of the gut microbiome in MDS patients (pts) and its association with inflammatory-metabolic parameters. Methods. We analysed faecal samples from 16 pts with untreated MDS or chronic myelomonocytic leukemia (median age: 66,5 years [41-81], m/f=13/3) and healthy donors (n=8, median age: 60,5 years [54-70], m/f=3/5). Exclusion criteria were antibiotic treatment within the last 4 weeks, chronic inflammatory bowel disease and other malignant disorders. Gut microbial taxonomic profiles were generated from the frozen faecal samples using 16S rRNA gene amplicon sequencing. For bioinformatic analysis, MapSeq was used. Statistical analysis included diversity analysis (observed microbial richness and Shannon index, differences assessed by Wilcoxon tests); differences in gut microbiome composition were visualized using Principal Coordinate Analysis (PCoA) in which differences were assessed by PERMANOVA. We performed flow cytometry to assess the cellular immune status including B-, T- and NK-cell populations, T/NK-subpopulations and expression of checkpoint molecules. Each parameter was analysed using Mann-Whitney U-test. Metabolite concentrations in plasma and urine were measured using NMR spectroscopy. Multivariate analysis of metabolic parameters was performed as a principal component analysis (PCA) and partial least-square discriminant analysis (PLS-DA), to evaluate the possible discrimination of the two groups. Results. Ten MDS pts were referred to the low-risk group (low and intermediate 1 according to IPSS), 5 pts - high-risk (intermediate 2 and high), in one patient information was not available. All but one patient carried at least one MDS-typical somatic mutation (median: 3 [1-6]). According to cytogenetic analysis 2 pts belong to "very good" prognostic risk, 7 - "good", 4 - "intermediate", 2 - "very bad", in 1 patient the information was not available. The median time from the diagnosis to faecal sampling was 19 months [1-205]. Microbiome composition showed statistically significant differences between MDS and healthy subjects (p<0.05, R2=0.079, PERMANOVA). Gut microbial diversity and richness (the number of genera observed per patient), was lower in MDS, although not reaching statistical significance in this small cohort. Time from MDS diagnosis until faecal sampling did not have an impact on the microbial diversity. Pts with high-risk MDS tended to have the lowest gut microbial diversity and richness compared to pts in the low-risk group, both according to IPSS and IPSS-R scores. Flow cytometry revealed that pts with MDS had reduced numbers of B-lymphocytes (CD19+: 56 vs 184/µL in MDS and healthy, accordingly, p<0.01), increased number of T effector memory cells (0.06 vs 0/µL, p<0.05) and PD-L2 expression on T-lymphocytes (MFI: 150 vs 86, p<0.01), reflecting prolonged inflammatory stimulation. In addition, we observed reduced NK-cell activity, demonstrated by the reduction of the activating receptors on NK cells (NKp30: 92.9 vs 99.05 %CD56dim, p<0.05; DNAM-1: 86.8 vs 97 %CD56bright, p<0.01). PCA applied to plasma and urine metabolites allowed us to separate MDS from healthy counterparts. Further separation was accomplished using a supervised approach with PLS-DA, which highlighted the separation of the groups due to high VIP scores of the parameters, mostly affecting lipid metabolism. Conclusions. We have demonstrated broad, statistically significant changes in microbiome, metabolome and immunome of pts with MDS. Whether these are cause or consequence of the disease development remains to be elucidated.

Human NGFR+/VCAM-1+/Mcam+ Bone Marrow-Derived Stromal Cells (NVML) Provide Enhanced Support for Normal Hematopoiesis and Are Disrupted in Myelodysplastic Syndrome

The bone marrow microenvironment (BMME) is indispensable for support of normal and malignant hematopoiesis. Mesenchymal stem/stromal cells (MSCs) give rise to multiple non-hematopoietic components that play a pivotal role in myelodysplastic syndromes (MDS). However, therapeutic targeting of MSCs has been challenging, in part due to the limited definition of the heterogeneity of MSC populations from human bone marrow. Using bone marrow from normal healthy controls obtained through aspirates rather than surgical specimens (normal bone marrow; NBM), we successfully identified two novel distinctive subsets of human MSCs by combining three MSC cell surface markers: CD271/ Nerve Growth Factor Receptor (NGFR), CD106/ Vascular Cell Adhesion Molecule-1 (VCAM-1) and CD146/ Melanoma Cell Adhesion Molecule (MCAM). We first isolated three BM-derived non-endothelial lineage (CD45/CD235ab/CD34-CD31-) cell populations from NBM: CD271+/CD146- (NGFR+/Lineage- cells: NLCs), CD271+/CD146+/CD106+ (NGFR+/VCAM1+/MCAM+/Lineage- NVML cells), and CD271+/CD146+/CD106- (NGFR+/MCAM+/Lineage-; NML cells). Gene profiling by RNA-seq revealed that NLCs and NVML cells represent distinct populations, whereas NML cells are a transitional population. Transcriptional analysis suggested that NVML cells, compared to NLCs, have stem cell characteristics, including quiescence and multipotentiality, and express higher levels of key genes critical for the support of hematopoietic stem and progenitor cells (HSPCs), including CXCL12 (SDF1A), Kit ligand (KITL) and Angiopoietin-1 (ANGPT1). This signature was compatible with BM-derived Leptin receptor+ MSCs with high capacity of supporting HSPCs based on previously reported single cell RNA-seq analysis in mice (Baryawno et al 2019, Cell). Consistent with their transcriptome, functional analysis showed that NVML cells had higher colony forming units (CFU-F) and tri-lineage differentiation capacity compared to NLCs. Normal NLCs or NVML cells were also co-cultured with NBM or human MDS cell line (MDS-L) in the absence of cytokines/chemokines to evaluate the ability of these novel human MSC populations to provide a supportive niche for HSPCs. NVML cells provided increased support for normal hematopoietic progenitors compared to baseline and NLCs (fold increase over baseline 1.78±0.78(NLCs, p<0.05) vs 2.71±1.71(NVML cells, p<0.001), NLCs vs NVML cells, p<0.05). In contrast, NLCs showed increased ability of supporting MDS-L (fold increase over baseline 2.35±1.35 (NLCs, p<0.01) vs 1.5±0.5 (NVML cells, p=0.26), NLCs vs NVML cells, p=0.053). We next profiled the transcriptome of NLCs and NVML cells from 17 patients with MDS in comparison with age-matched healthy controls (n=8, >50 years old). In spite of mutational heterogeneity in this MDS patient cohort, principal component analysis (PCA) revealed a distinctive shift of MDS-derived NVML cells compared to controls. For upregulated transcripts, pathways and gene ontology analyses of differentially expressed genes in NVML cells from MDS compared to controls identified signals related to DNA/RNA processing, while most of the down-regulated transcripts identified cytokines/chemokines critical for HSPCs. When the transcriptional profiles of NVML cells from MDS patients were compared to age-matched NVML controls, critical genes for HSPC support highly expressed in normal NVML (e.g. SDF1A, ANGPT1) were significantly down-regulated. Moreover, the transcriptional signature of NVML cells from MDS patients was similar to that of normal NLCs. Together these data suggest that, in spite of mutational heterogeneity in the hematopoietic clone, MDS may differentially impact these novel human marrow MSC subsets, decreasing normal HSPC support by disrupting NVML cells, which acquired the transcriptional signature of MDS-supportive normal NLCs. These data highlight a novel paradigm to enrich key human niche cells that provide support for HSPCs. Moreover, our data suggest that these human MSC cell subsets are differentially disrupted in MDS. Mutation specific effects are also likely to impact MDS-dependent disruption of the BMME. Therefore, our novel strategy to enrich critical human BM MSC subsets that differentially support normal and malignant hematopoiesis represents a new paradigm to accelerate therapeutic targeting of the BMME in MDS and other hematologic malignancies.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy

BackgroundAlthough hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated.MethodsWe examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR–DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression.ResultsSALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5′ untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression.ConclusionsBy combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.)

Circulating HMGB1 is increased in myelodysplastic syndrome but not in other bone marrow failure syndromes: proof-of-concept cross-sectional study.

Background:Myelodysplastic syndrome (MDS) is associated with persistent immune activation. High mobility group box-1 (HMGB1) is a ubiquitous, functionally diverse, non-histone intranuclear protein. During acute and chronic inflammatory states, HMGB1 is actively released by inflammatory cells, further amplifying the inflammatory response. A role in MDS and other hypoplastic bone marrow (BM) disorders is incompletely understood.Objectives:The objective of the study is to evaluate whether circulating HMGB1 is elevated in patients with MDS and other BM failure syndromes [namely, aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH)].Design:This is a observational, cross-sectional, single-center, exploratory study.Methods:We evaluated circulating concentrations of HMGB1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in patients with MDS and age-matched hematologically healthy controls as well as patients with AA and PNH.Results:We included 66 patients with MDS and 65 age-matched controls as well as 44 patients with other BM failures (AA = 27, PNH = 17). Circulating levels of HMGB1 were higher in patients with MDS [median, 4.9 ng/ml; interquartile range (IQR): 2.3–8.1] than in AA (median, 2.6 ng/ml; IQR: 1.7–3.7), PNH (median, 1.7 ng/ml; IQR: 0.9–2.5), and age-matched healthy individuals (median, 1.9 ng/ml; IQR: 0.9–2.5) (p = 0.0001). We observed higher concentrations of HMGB1 in the very low/low-risk MDS patients than in the intermediate/high/very high-risk ones (p = 0.046). Finally, in comparison with patients with AA, those with hypocellular MDS (h-MDS) had significantly higher levels of circulating HMGB1 (n = 14; median concentration, 5.6 ng/ml, IQR: 2.8–7.3; p = 0.006). We determined a circulating HMGB1 value of 4.095 ng/ml as a diagnostic cutoff differentiator between h-MDS and AA.Conclusion:These observations indicate that circulating HMGB1 is increased in patients with MDS. HMGB1 (but not IL-1β or TNF-α) differentiated between MDS and other BM failures, suggesting that HMGB1 may be mechanistically involved in MDS and a druggable target to decrease inflammation in MDS.

Plasma IL-33 levels are decreased in patients with high-risk myelodysplastic syndrome and show no correlation with pro-inflammatory IL-6 levels.

Aberrant inflammatory signaling has been shown to be a key pathogenic driver in myelodysplastic syndromes (MDS). Abnormal IL-33 expression has been implicated in inflammatory, immune-related disorders and, some tumors. However, its role in MDS remains widely unknown. This study aimed to evaluate the relationship between plasma levels of IL-33, clinical and prognostic data and, IL-6 levels in 101 patients with MDS. A comparative group of 59 healthy individuals was also evaluated. Plasma levels of cytokines were determined by enzyme-linked immunosorbent assay. Lower levels of IL-33 were found in patients with MDS when compared to the control group (p=0.001), mainly in patients with more advanced stages of the disease and worse prognosis. No significant correlation between the levels of IL-33 and IL-6 was observed (r=0.175; p=0.081). These results reinforce the close association between immunological disorders and the pathogenesis of MDS. A greater understanding of the role of inflammatory cytokines in the disease can potentially provide new diagnosis and prognosis markers and new therapeutic targets.

Circular RNAs in Myelodysplastic Syndromes and Impact of SF3B1 Mutations on Their Expression

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with a high risk of transformation to acute myeloid leukemia (AML). One of the processes implicated in MDS pathogenesis is RNA splicing. Its alterations are caused by somatic mutations in splicing factor genes. Mutations in SF3B1 (Splicing Factor 3b Subunit 1) gene are the most frequently found mutations in MDS. Circular RNAs (circRNAs) are covalently closed RNAs that are produced by back-splicing process. CircRNAs can regulate multiple biological processes through various molecular mechanisms, such as microRNA sponging. Their deregulation is frequently found in cancer. It is likely that they also contribute to the development of MDS, however, their role in MDS has not been researched yet. Therefore, our aim was to explore circRNA levels in MDS and analyze their association with patient prognosis. We further hypothesized that mutations in splicing factor genes can affect production of circRNAs and thus, we examined circRNA levels with respect to the mutational status. We explored transcriptome of 78 MDS patients, 7 AML patients, and 13 healthy donors using Illumina RNA-seq of total RNA isolated from CD34+ bone marrow cells. To associate circRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Of 8,620 circRNAs identified by RNA-seq, 204 circRNAs were deregulated in MDS (e.g., MENTRNL, EBF1, and PPM1L-derived circRNAs) and 246 circRNAs were altered between lower- and higher-risk patients (e.g., CHST15, TMTC2, and PDE3B-derived circRNAs). Most of the progression-related circRNAs (n = 234) showed elevated levels in higher-risk patients, suggesting that the back-splicing process might be stimulated during the disease progression. In MDS patients with SF3B1 mutations, other 40 circRNAs were deregulated (e.g., ZNF91, ZEB1, and ZNF124-derived circRNAs). This circRNA profile was substantially different from the profiles associated with the rest of recurrently mutated splicing factor genes (SRSF2, U2AF1, and ZRSR2). To study alterations in forward- and back-splicing in SF3B1-mutated patients, we examined transcriptional differences on the levels of whole genes, transcript variants, and circRNAs and searched for specificities in transcription within individual gene loci. A set of circRNAs whose levels differed specifically without affecting expression of corresponding forward-spliced mRNAs included several oncology/hematopoiesis-relevant genes (e.g., ATM, CBL, ERCC5, ETV6, FLT3, and MAPK6). Gene loci with changed expression of both, alternative mRNA transcripts and circRNAs, but stable transcription on whole gene level included for example CDK14, KDM1A, and ZEB1. Because ZEB1 (Zinc Finger E-Box Binding Homeobox 1) serves as an essential hematopoietic transcription factor, we focused on ZEB1-derived circRNAs (hsa_circ_0000228 and hsa_circ_0003793) in more detail. We demonstrated that upregulation of these circRNAs is SF3B1-specific and not related to any other clinical or molecular characteristics. Finally, using RNA-seq data from CRISPR/Cas9 edited K562 cells (Liberante FG, et al., Sci Rep. 2019; 9:2678), we confirmed that SF3B1 K700E mutation leads to strong upregulation of ZEB1 circRNAs. To conclude, this is an early report showing for the first time that the levels of specific circRNAs are altered in MDS. We demonstrated that particular circRNAs may have potential to become markers that would contribute to more accurate prognosis of MDS patients. Further, we identified circRNAs with deregulated levels specifically in MDS patients with SF3B1 mutation, suggesting that this mutation affects circRNA production.Supported by GA CR (N20-19162S) and MH CZ-DRO (UHKT, 00023736). 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.

Transcriptomic Signitures of Azacitidine (AZA) and Decitabine (DAC) Resistance in MDS and CMML

INTRODUCTION: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are heterogeneous myeloid neoplastic disorders characterized by ineffective hematopoiesis leading to cytopenias and increased risk of transformation to acute myeloid leukemia (AML) . The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve the natural history of MDS and CMML patients (Fenaux et al 2009). However, over half of the cases experience primary failure defined by a lack of response to HMA treatment which is associated with poor prognosis and a median survival of 4-6 months (Garcia-Manero et al. 2016, Jabbour et al. 2010). The highly heterogeneous pathophysiology of myeloid neoplasms and poorly understood mechanisms underlying therapeutic action of HMAs pose substantial challenges in understanding the biology of HMA failure in MDS and CMML.METHODS: We established a cohort of baseline bone marrow (BM) cells that were collected from 17 CMML and 34 MDS patients with excessive blasts (MDS-EB) prior to their HMA based therapies (Figure 1). RNA-Seq based transcriptomic analysis was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs) of patients, known to be the cellular origin of these diseases, to identify biological signatures of primary HMA resistance.RESULTS: Similar to the recent reports regarding the lack of common gene expression signatures in HMA resistant AML cell lines (Leung et al. 2019) , RNA-Seq in patient cohort detected fewer than 0.5% of the total number of differentially expressed genes in non-responders in common for both AZA and DAC. Of note, all the AZA and a large portion of DAC associated genes with down-regulations in non-responders encode immunoglobulins, which is consistent with several recent findings indicating impaired differentiation B cells in association with unfavorable outcomes in MDS and CMML (Ribeiro et al. 2006 and Kahn et al. 2015). We performed flow cytometry analysis in BM cells available for 13 patients prior to start of HMA treatments (5 non-responders and 8 responders), and detected that HMA non-responders had a strong tendency (P=0.07) of decreased baseline frequencies of B cells in their BM than HMA responders.GSEA analysis based on leading edge genes identified over 200 and 300 biological signaling pathways to be associated with AZA and DAC failure respectively, with 78 pathways commonly correlated with treatment failure of both drugs (28 up-regulated and 60 down-regulated, Table 1). Clustering of these commonly altered pathways based on biological functions revealed that most of them are known to have a role in MDS and CMML pathogenesis and/ or drug resistance. For instance, neurotransmitter, olfactory pathways, and associated G-protein coupled receptor signals was recently reported to play a role in regulating the maintenance and differentiation of BM HSPCs (Shao et al. 2021 and Shim et al. 2013), whereas cell junction signaling that involves integrins and increased MAP2K-MAPK signaling were also reported to be associated with AZA resistance in MDS and CMML (Unnikrishnan et al. 2017).Among commonly down-regulated biological pathways in non-responders of AZA and DAC, there were clusters of immunoglobulin-associated signals, protein translation regulatory pathways that involve ribosomal proteins, cell cycle signaling, respiratory chain, and Golgi transport signals. Decreased expression of ribosomal proteins and related impairment of ribosomal functions were known mechanism in MDS and CMML development (Ebert et al. 2008 and Schneider et al. 2016). Furthermore, decreased respiratory chain and Golgi transport signals were also identified by transcriptomic investigation in the DAC resistant TF1-RES cell lines.In addition to the commonly altered signals for both AZA and DAC resistance, innate immune signaling pathways including interferon and toll-like receptor signals were significantly up-regulated in DAC non-responders but down-regulated in AZA non-responders.CONCLUSIONS: In this study, the transcriptomic data between responders and non-responders to AZA and DAC were investigated separately, thereby drug-specific as well as the common biomarkers associated with treatment failures of both drugs could be identified. The relatively low proportion of genes and pathways shared by AZA and DAC non-responders suggest the difference underlying biological mechanisms of AZA and DAC failure. [Display omitted] DisclosuresSasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wei: Daiichi Sanko: Research Funding.

TIRAP Drives Marrow Failure through an Ifnγ-Hmgb1 Axis That Disrupts the Endothelial Niche

The myelodysplastic syndrome (MDS) are a group of hematological malignancies with the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). Dysregulation of immune and inflammatory responses has been implicated in MDS and other BMF disorders. There is significant evidence for IFNγ playing a key role in MDS and BMF syndromes. However, there are conflicting theories regarding the mechanism by which IFNγ promotes BMF. There is also very little information on the triggers that underlie upregulation of IFNγ in these BMF syndromes. Interstitial deletion of chromosome 5q is the most common cytogenetic abnormality observed in MDS, accounting for approximately 10% of all cases. Our lab has previously shown that miR-145, which is located on the minimally deleted region of chromosome 5q, targets Toll/Interleukin-1 receptor domain containing adaptor protein (TIRAP) - an innate immune adaptor protein. However, the role of TIRAP in marrow failure has not been well elucidated. In this study, we identify a novel role for TIRAP in dysregulating normal hematopoiesis through activation of Ifnγ.Using bone marrow transplants in wild-type mice, we showed that constitutive expression of TIRAP in wild-type hematopoietic stem and progenitor cells (HSPC) caused peripheral blood cytopenia, suppressed the bone marrow endothelial niche and significantly reduced overall survival of the mice (median survival 9 weeks post-transplant) compared to controls (p < 0.0001). RNA-seq analysis of TIRAP expressing HSPC identified several proinflammatory cytokines to be significantly overrepresented. Geneset enrichment analysis (GSEA) identified the Ifnγ response as the single most significantly enriched pathway of the Hallmark genesets. To test the functional role of Ifnγ in TIRAP-mediated BMF, wild-type recipient mice were transplanted with TIRAP- HSPC from Ifnγ-/- donor mice. Mice that received TIRAP-transduced Ifnγ-/- HSPC were rescued from BMF, as evidenced by normalized blood cell counts and improved median survival (median survival 48.6 weeks) (Ifnγ-/- TIRAP vs. wild-type TIRAP: p = 0.0004). Interestingly, in our model of TIRAP induced BMF, myeloid rather than the conventional T and NK cells were the cells most responsible for the increased production of Ifnγ. Further, when we transplanted TIRAP expressing wild-type HSPC into NSG recipient mice, which are deficient in functional B, T and NK cells, the NSG mice developed BMF with pancytopenia in a similar time-frame as wild-type mice. This suggested that T and NK cells are not central for the development of TIRAP induced BMF.Delving deeper into the mechanism by which the TIRAP-Ifnγ axis causes BMF, we saw that while Ifnγ played a direct role in suppressing erythropoiesis and megakaryopoiesis, it played an indirect, Ifnγ receptor independent role on myelopoiesis. TIRAP-induced activation of Ifnγ released the alarmin, Hmgb1, which suppressed the marrow endothelial niche, which in turn promoted myeloid suppression. Overexpression of TIRAP in Ifnγ -/- background blocked Hmgb1 release. Further, blocking Hmgb1 in presence of TIRAP expression was sufficient to reverse the marrow endothelial defect and restore myelopoiesis in vivo.Our findings highlight a novel, non-canonical effect of aberrant TIRAP expression via the Ifnγ-Hmgb1 axis on the endothelial cell component of the marrow microenvironment and hematopoiesis. Further understanding of this pathway would open up avenues for developing new therapies for BMF. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders.Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs.To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies.Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Display omitted] DisclosuresFrank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

Hypomethylating Chemotherapeutic Agents as Therapy for Myelodysplastic Syndromes and Prevention of Acute Myeloid Leukemia.

Myelodysplastic Syndromes (MDSs) affect the elderly and can progress to Acute Myeloid Leukemia (AML). Epigenetic alterations including DNA methylation and chromatin modification may contribute to the initiation and progression of these malignancies. DNA hypomethylating agents such as decitabine and azacitidine are used as therapeutic treatments and have shown to promote expression of genes involved in tumor suppression, apoptosis, and immune response. Another anti-cancer drug, the proteasome inhibitor bortezomib, is used as a chemotherapeutic treatment for multiple myeloma (MM). Phase III clinical trials of decitabine and azacitidine used alone and in combination with other chemotherapeutics demonstrated their capacity to treat hematological malignancies and prolong the survival of MDS and AML patients. Although phase III clinical trials examining bortezomib’s role in MDS and AML patients are limited, its underlying mechanisms in MM highlight its potential as a chemotherapeutic for such malignancies. Further research is needed to better understand how the epigenetic mechanisms mediated by these chemotherapeutic agents and their targeted gene networks are associated with the development and progression of MDS into AML. This review discusses the mechanisms by which decitabine, azacitidine, and bortezomib alter epigenetic programs and their results from phase III clinical trials.

The Clinical Significance of Chromosome 17 Abnormalities in Myelodysplastic Syndrome Treated with 5-Azacytidine. Results from the Hellenic 5-Azacytidine Registry

IntroductionAberrations of chromosome 17 have been correlated to poor prognosis and resistance to treatment in several hematologic malignancies. Their role in myelodysplastic syndrome (MDS) has not yet been fully elucidated. Inversion of chromosome 17 (inv17) has been characterized as an intermediate risk cytogenetic abnormality in the revised International prognostic scoring system (IPSS-R), but chromosome 17 monosomy and other chromosome 17 abnormalities have not yet been fully characterized. Moreover, mutations of TP53 located on chromosome 17p, have been correlated to poor prognosis and resistance to treatment.AimTo determine the incidence and clinical significance of acquired abnormalities of chromosome 17 in adult patients with MDS treated with 5-azacytidine, in the context of the Hellenic 5-azacytidine registry.Patients and methodsPatients diagnosed with myelodysplastic syndrome per the 2008 WHO classification were included in the study. Conventional cytogenetic testing was performed at diagnosis. All patients were treated with 5-azacytidine during the course of their disease according to common clinical practice. Demographic, clinical and laboratory data were recorded retrospectively (Hellenic 5-azacytidine registry). Statistical analysis was performed using IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA).ResultsWe analyzed the results of the cytogenetic analysis of 465 patients with MDS at diagnosis. Patients with chronic myelomonocytic leukemia, MDS/myeloproliferative neoplasm (MDS/MPN), and low-blast acute myeloid leukemia were excluded from the analysis. The main characteristics of the patients are shown in Figure 1A. We found 23/465 (4.9%) patients with chromosome 17 aberrations (6 with chromosome 17 inversion, 13 with a complex karyotype including chromosome 17 monosomy, 3 with a complex karyotype with add(17) and 1 with t(17;19)). All cases of inv(17) were found as an isolated cytogenetic abnormality, while all cases of chromosome 17 monosomy were found in the context of a complex karyotype. Abnormalities of chromosome 17 were the 7th more common cytogenetic abnormalities after complex karyotype and abnormalities of chromosomes 7, 8, 3, 5 and 12.Patients with chromosome 17 abnormalities from our registry tended to be higher risk per both the IPSS and the IPSS-R. The median follow-up of the patients was 23.1 (0.7-203.8) months. Patients with chromosome 17 aberrations had a median overall survival (OS) of 20.7 months versus 35.3 months for patients without chromosome 17 aberrations (log Rank 2-sided p<0.0001, Figure 1B). Patients with any type of chromosome 17 abnormalities carried a worse prognosis than patients without abnormalities of chromosome 17 (OS was as follows: inv(17), 28.9 months; chromosome 17 monosomy, 20.7 months; add(17), 14.8 months; t(17;19), 5.7 months). Patients with a complex karyotype carried a poor prognosis irrespective of chromosome 17 aberrations. The presence of chromosome 17 abnormalities did not affect response to 5-azacytidine, and the rate of transformation to AML was comparable to that of the rest of the studied population. Moreover, t(17;19) has been correlated with poor prognosis in patients with acute lymphoblastic leukemia. Here we report for the first time the same chromosome abnormality in a patient with RAEB1 with poor prognosis.DiscussionThe present study evaluates the cytogenetic analysis of the largest, to our knowledge, cohort of patients with MDS treated with 5-azacytidine. Chromosome 17 abnormalities in patients with MDS comprise a group of cytogenetic disorders with poor prognosis since most of them are found in the context of a complex karyotype with the exception of inv(17) that occurs typically as a single cytogenetic abnormality. 5-azacytidine seems to be equally effective in patients with a complex karyotype with and without chromosome 17 abnormalities. Nevertheless, overall survival and survival after 5-azacytidine treatment remain short, although patients with inv(17) have a better prognosis from patients carrying other chromosome 17 abnormalities, with survival rates exceeding the historical control of 12 months for patients with high-risk MDS treated with best supportive care. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of Toll-like Receptor 2 Results in Accelerated Leukemogenesis in the NUP98-HOXD13 Mouse Model of MDS

The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis and a high risk of leukemic transformation. Stem cell transplantation is the only curative treatment available to a subset of patients and is associated with significant morbidity and mortality; thus, new therapies are needed. Multiple studies have shown a correlation between MDS and enhanced toll-like receptor 2 (TLR2) signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains unclear. Increased TLR2 signaling has been associated with low-risk disease, higher rates of apoptosis associated with improved prognosis, and longer survival (Zeng et al ., Exp Cell Res 2016; Wei et al., Leukemia 2013), suggesting TLR2 may, in fact, have a protective role in MDS. Thus, we are exploring how TLR2 signaling regulates cell fate in MDS to shape efforts to target this pathway in patients.To elucidate the contribution of TLR2 signaling to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS (expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter). These “ NHD13” mice recapitulate many features of human MDS and die of leukemia or cytopenias (Lin et al ., Blood 2005). Similar to CD34+ cells in humans, we found a nearly two-fold increase of TLR2 on NHD13 HSCs as compared to controls. NHD13 mice were crossed to Tlr2-/- mice to generate NHD13;Tlr2-/-, NHD13;Tlr2+/+, Tlr2-/-, and wild-type (WT) groups. Interestingly, loss of TLR2 in NHD13 mice did not confer an improvement in peripheral cytopenias, but was associated with accelerated leukemogenesis and significantly worse survival (p=0.016, with a median survival of 335 days in NHD13;Tlr2-/- mice vs. 370 days for NHD13;Tlr2+/+ mice; in both cohorts, leukemia was the primary cause of death- 12/20 for NHD13;Tlr2-/- and 10/13 for NHD13;Tlr2+/+). An even more significant acceleration of leukemogenesis and death was observed in NHD13 mice crossed to mice lacking myeloid differentiation primary response gene 88 (MyD88), an adaptor required for most TLR signaling, including TLR2 (p<0.0001, with a median survival of 218 days in NHD13;MyD88-/- mice vs. 370 days in NHD13 mice).To investigate the potential mechanism by which TLR2 signaling loss accelerates leukemogenesis in NHD13 mice, hematopoietic stem and progenitor cells (c-Kit+, Sca-1+, Lin-; HSPCs) of premalignant NHD13;Tlr2+/+ and NHD13;Tlr2-/- mice were evaluated for cell cycling, cell death, and DNA damage. While loss of TLR2 in the NHD13 mice did not confer a significant difference in HSPC cycling, it did reduce cell death (HSPCs of NHD13+;Tlr2+/+ mice display 18.4% Annexin V+ cells, while those of NHD13+;Tlr2-/- mice display only 6.2%, p=0.0008). An assessment of caspase-1 and caspase-3/7 activities showed a significant reduction in activated caspase-1 in the HSPCs of NHD13;Tlr2-/- mice compared to NHD13; Tlr2+/+ mice (average luminescence units: 7310 vs. 10164), suggesting that TLR2 specifically promotes inflammasome-dependent cell death in the NHD13 mice. RNA sequencing of sorted HSPCs also demonstrated an enrichment of apoptosis-related gene pathways in the NHD13 compared to the NHD13;Tlr2-/- cells. Moreover, the accumulated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of γH2AX staining as compared to NHD13; Tlr2+/+ mice and WT controls, indicating TLR2 loss promotes the survival of damaged HSPCs.As apoptosis of HSPCs is characteristic of low-risk MDS and is lost as the disease progresses to leukemia, these data suggest that some TLR2 signaling may be protective against leukemic transformation via promotion of apoptosis of damaged premalignant HSCs. Further supporting this idea, we found that in 38% of cases evaluated, TLR2 surface expression was lost from the malignant cells upon transformation to acute leukemia in the NHD13 mice. Thus, down-regulation of TLR2 may be a later event in the progression of MDS to acute leukemia, and complete inhibition of this pathway may, in fact, facilitate the survival of pre-leukemic HSPCs. Ongoing experiments are aimed at elucidating the mechanism by which TLR2 signaling promotes premalignant cell death in MDS and understanding how TLR2 is down-regulated upon transformation to leukemia. DisclosuresNo relevant conflicts of interest to declare.

Clinical Response to Traditional Chinese Herbs Containing Realgar (As2S2) is Related to DNA Methylation Patterns in Bone Marrow DNA from Patients with Myelodysplastic Syndrome with Multilineage Dysplasia.

PurposeDNA methylation is known to play an important role in myelodysplastic syndrome (MDS). We previously showed that Chinese herbs (CHs) containing realgar (As2S2) were effective at treating MDS with multilineage dysplasia (MDS–MLD). We tested whether the response to CH treatment was related to changes in DNA methylation in MDS–MLD.Patients and MethodsFirst, the Illumina methylation 850K array BeadChip assay was used to assess the pretreatment methylation status in bone marrow cells from eight MDS–MLD patients and 3 healthy donors. The eight MDS–MLD patients were then treated with CHs for six months, the arsenic concentration was measured following treatment. The patients were subsequently divided into “effective” and “ineffective” treatment response groups and the DNA methylation patterns of the two groups were compared. Finally, the BeadChip data were validated by pyrosequencing.ResultsFive of the eight MDS–MLD patients showed hematological improvement (effective-treatment group), while three showed disease progression (ineffective-treatment group) (positive response rate: 62.5%). The arsenic concentrations in the patients ranged from 26.60 to 64.16 μg/L (median 48.4 μg/L) and were not significantly different between the two groups (p = 0.27). Compared with the healthy controls, three genes were hypomethylated and 110 were hypermethylated in the ineffective-treatment group. However, in the group showing hematological improvement, 102 genes were markedly hypomethylated and 87 hypermethylated. The effective-treatment group had a higher proportion of hypomethylated sites than the ineffective-treatment group (53.9% vs 2.6%, respectively; chi-square test) (p < 0.0001). Two hypermethylated and two hypomethylated genes were selected for validation by pyrosequencing (all p < 0.05).ConclusionMDS–MLD patients may present different DNA methylation subtypes. CHs containing realgar may be effective for treating MDS–MLD patients with the hypomethylation subtype.

Profile of Checkpoint Molecules Expression on Bone Marrow Cell Populations in Patients with High-Risk Myelodysplastic Syndrome

Background Immune checkpoint (IC) inhibitors (ICI) is a promising group of agents with potential effect in myelodysplastic syndrome (MDS). However, there is not enough data on the pattern of IC expression in MDS bone marrow, though this can guide future therapies with ICI. Methods We prospectively included 57 patients with high risk MDS during 2019-2020 years after signing informed consent for participation in research. We used 7 eight-colored panels in each patient to define IC expression on T- and NK-cells (CD3, CD8, CD4, CD56, CD16), myeloid precursors (CD117, CD34), T-regulators (CD25, CD4), myeloid-derived suppressor cells (CD15, CD11b, CD14, HLA-DR, CD33). In each population we assessed expression of CD279, CD152, CD223, TIM3, CD273, CD274, CD275, CD80. Results In lymphoid populations we observed an extremely high percentage of PD-1 positive lymphocytes (10.5 ± 8.0% of all nucleated cells), including PD-1 positive CD4 lymphocytes (6.3 ± 3.7%) and CD8 lymphocytes (4.7 ± 3.5%). Interestingly, on average 63 ± 34% of all lymphocytes were PD-1 positive (Figure 1A), which indicates an extreme degree of T-cell exhaustion and significant exploitation of the PD-1 system by a malignant tumor. Among the PD-1 ligands PD-1L predominated, however it was present only on a small percentage of myeloid precursors (2.2 ± 0.39% of nucleated cells), but most of the PD-1L ligand expression was observed on granulocytes in the process of their differentiation from myeloid precursors (5.8 ± 4.1% of nucleated cells), and in some patients the proportion of such granulocytes reached more than 20%. Moreover, on the cells of the monocytic series, PD-1L is practically not expressed, but CD80 is expressed, a ligand for CTLA4 (1.6 ± 0.7% of nucleated cells). Interestingly, despite the presence of expression of CD80 on monocytic and dendritic cells, the receptor for this ligand on T-lymphocytes was practically absent. The proportion of such cells was only 0.02 ± 0.01%, i.e. the expression of CD80 facilitates the activation of lymphocytes through interaction with CD28 rather than the suppression of the immune response through CD152. Thus, it can be suggested that CTLA4 does not have a clinically significant role in MDS. At the same time, a certain number of TIM3 positive lymphocytes were observed, the proportion of which was 0.42 ± 0.16%, and the percentage of lymphocytes carrying this receptor was 3.6 ± 1.2% (Figure 1A). However, significantly greater TIM3 expression was detected on NK cells. The proportion of such cells was 1.3 ± 0.6% of all nucleated cells, while 30% of all NK cells carried the TIM3 receptor (Figure 1A). Only 0.1% of cells from early myeloid progenitors that expressed TIM3 receptor were detected during flow cytometry, while the proportion of TIM3 positive myeloid cells increased during maturation just in the same way as the PD-1L. Expression of Gal9 was negligible, as it is a protein secreted into the intercellular space. Only a small percentage is associated with cell membranes. The analysis of the correlation matrix showed synchronous changes in the number of most bone marrow T-cell populations. A negative correlation of the level of myeloid tumor precursors actively expressing IC ligands and activated T cells, expressing CD223, NK cells, NKT was observed. The total number of myeloid tumor precursors positively correlated with the number of PD-1 positive lymphocytes (Figure 1B). Conclusion It was found that IC system plays a role in high-risk MDS. Moreover, the expression of ligands is mainly realized during maturation of blast cells into granulocytes, which are the main population expressing these inhibitory molecules. The leading role in the suppression of the T-cell response in MDS is played by the PD-1-PD-1L system, the CTLA4 system plays a minor role in our study cohort. The second most important system is GAL9-TIM3, effecting predominantly NK cells, while T cell are less affected. Thus, the data obtained justify the trials of dual blockade with anti-PD-1 and anti-TIM3 agents in high-risk MDS. Figure 1 Disclosures No relevant conflicts of interest to declare.

SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS

AbstractSETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation–seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.