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Patients with lower-risk myelodysplastic syndromes (LR-MDS) have a generally favorable prognosis; however, a small proportion of cases progress rapidly. This study aimed to define molecular biomarkers predictive of LR-MDS progression and to uncover cellular pathways contributing to malignant transformation. The mutational landscape was analyzed in 214 LR-MDS patients, and at least one mutation was detected in 137 patients (64%). Mutated RUNX1 was identified as the main molecular predictor of rapid progression by statistics and machine learning. To study the effect of mutated RUNX1 on pathway regulation, the expression profiles of CD34 + cells from LR-MDS patients with RUNX1 mutations were compared to those from patients without RUNX1 mutations. The data suggest that RUNX1-unmutated LR-MDS cells are protected by DNA damage response (DDR) mechanisms and cellular senescence as an antitumor cellular barrier, while RUNX1 mutations may be one of the triggers of malignant transformation. Dysregulated DDR and cellular senescence were also observed at the functional level by detecting γH2AX expression and β-galactosidase activity. Notably, the expression profiles of RUNX1-mutated LR-MDS resembled those of higher-risk MDS at diagnosis. This study demonstrates that incorporating molecular data improves LR-MDS risk stratification and that mutated RUNX1 is associated with a suppressed defense against LR-MDS progression.

Background/Aim: Prediction of response to azacitidine (AZA) treatment is an important challenge in hematooncology. In addition to protein coding genes (PCGs), AZA efficiency is influenced by various noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs), circular RNAs (circRNAs), and transposable elements (TEs). Materials and Methods: RNA sequencing was performed in patients with myelodysplastic syndromes or acute myeloid leukemia before AZA treatment to assess contribution of ncRNAs to AZA mechanisms and propose novel disease prediction biomarkers. Results: Our analyses showed that lncRNAs had the strongest predictive potential. The combined set of the best predictors included 14 lncRNAs, and only four PCGs, one circRNA, and no TEs. Epigenetic regulation and recombinational repair were suggested as crucial for AZA response, and network modeling defined three deregulated lncRNAs (CTC-482H14.5, RP11-419K12.2, and RP11-736I24.4) associated with these processes. Conclusion: The expression of various ncRNAs can influence the effect of AZA and new ncRNA-based predictive biomarkers can be defined.

Simple Summary Epigenetic drugs, such as azacitidine (AZA), hold promise in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), however, the mechanisms predicting the patients’ response to AZA is not completely understood. Quiescence of hematopoietic CD34+ progenitors has been proposed as a predictive factor for AZA therapy failure in MDS/AML patients, but the interplay between CD34+ cell cycle status and their metabolic signature in a predisposition to AZA (non)responsiveness remains unclear. Our data on patients with MDS or AML with myelodysplasia-related changes (AML-MRC) suggest that AZA-responders have actively cycling CD34+ cells poised for erythro-myeloid differentiation, with high metabolic activity controlling histone acetylation. Conversely, the patients who progressed early on AZA therapy revealed quiescence signature of their CD34+ cells, with signs of reduced metabolically-controlled acetylation of histones needed for transcription-permissive chromatin configuration. Our study delineates plasma citrate levels and CD34+ cells’ transcriptional signatures associated with cycling status and metabolic characteristics as factors predicting the response to AZA monotherapy in MDS/AML-MRC patients. Abstract To better understand the molecular basis of resistance to azacitidine (AZA) therapy in myelodysplastic syndromes (MDS) and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), we performed RNA sequencing on pre-treatment CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from 25 MDS/AML-MRC patients of the discovery cohort (10 AZA responders (RD), six stable disease, nine progressive disease (PD) during AZA therapy) and from eight controls. Eleven MDS/AML-MRC samples were also available for analysis of selected metabolites, along with 17 additional samples from an independent validation cohort. Except for two patients, the others did not carry isocitrate dehydrogenase (IDH)1/2 mutations. Transcriptional landscapes of the patients’ HSPCs were comparable to those published previously, including decreased signatures of active cell cycling and DNA damage response in PD compared to RD and controls. In addition, PD-derived HSPCs revealed repressed markers of the tricarboxylic acid cycle, with IDH2 among the top 50 downregulated genes in PD compared to RD. Decreased citrate plasma levels, downregulated expression of the (ATP)-citrate lyase and other transcriptional/metabolic networks indicate metabolism-driven histone modifications in PD HSPCs. Observed histone deacetylation is consistent with transcription-nonpermissive chromatin configuration and quiescence of PD HSPCs. This study highlights the complexity of the molecular network underlying response/resistance to hypomethylating agents.

Simple Summary Although lncRNAs have been increasingly recognized as regulators of hematopoiesis, only several studies addressed their role in myelodysplastic syndrome (MDS). By genome-wide profiling, we identified lncRNAs deregulated in various groups of MDS patients. We computationally constructed lncRNA-protein coding gene networks to associate deregulated lncRNAs with cellular processes involved in MDS. We showed that expression of H19, WT1-AS, TCL6, and LEF1-AS1 lncRNAs associate with higher-risk MDS and proposed processes related with these transcripts. Abstract Background: myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder with an incompletely known pathogenesis. Long noncoding RNAs (lncRNAs) play multiple roles in hematopoiesis and represent a new class of biomarkers and therapeutic targets, but information on their roles in MDS is limited. Aims: here, we aimed to characterize lncRNAs deregulated in MDS that may function in disease pathogenesis. In particular, we focused on the identification of lncRNAs that could serve as novel potential biomarkers of adverse outcomes in MDS. Methods: we performed microarray expression profiling of lncRNAs and protein-coding genes (PCGs) in the CD34+ bone marrow cells of MDS patients. Expression profiles were analyzed in relation to different aspects of the disease (i.e., diagnosis, disease subtypes, cytogenetic and mutational aberrations, and risk of progression). LncRNA-PCG networks were constructed to link deregulated lncRNAs with regulatory mechanisms associated with MDS. Results: we found several lncRNAs strongly associated with disease pathogenesis (e.g., H19, WT1-AS, TCL6, LEF1-AS1, EPB41L4A-AS1, PVT1, GAS5, and ZFAS1). Of these, downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with adverse outcomes in MDS patients. Multivariate analysis revealed that the predominant variables predictive of survival are blast count, H19 level, and TP53 mutation. Coexpression network data suggested that prognosis-related lncRNAs are predominantly related to cell adhesion and differentiation processes (H19 and WT1-AS) and mechanisms such as chromatin modification, cytokine response, and cell proliferation and death (LEF1-AS1 and TCL6). In addition, we observed that transcriptional regulation in the H19/IGF2 region is disrupted in higher-risk MDS, and discordant expression in this locus is associated with worse outcomes. Conclusions: we identified specific lncRNAs contributing to MDS pathogenesis and proposed cellular processes associated with these transcripts. Of the lncRNAs associated with patient prognosis, the level of H19 transcript might serve as a robust marker comparable to the clinical variables currently used for patient stratification.

Circular RNAs (circRNAs) constitute a recently recognized group of noncoding transcripts that function as posttranscriptional regulators of gene expression at a new level. Recent developments in experimental methods together with rapidly evolving bioinformatics approaches have accelerated the exploration of circRNAs. The differentiation of hematopoietic stem cells into a broad spectrum of specialized blood lineages is a tightly regulated process that depends on a multitude of factors, including circRNAs. However, despite the growing number of circRNAs described to date, the roles of the majority of them in hematopoiesis remain unknown. Given their stability and disease-specific expression, circRNAs have been acknowledged as novel promising biomarkers and therapeutic targets. In this paper, the biogenesis, characteristics, and roles of circRNAs are reviewed with an emphasis on their currently recognized or presumed involvement in hematopoiesis, especially in acute myeloid leukemia and myelodysplastic syndrome.

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). Disclosures No relevant conflicts of interest to declare.

Changes in DNA methylation are characteristic for patients with acute myeloid leukemia (AML) and many studies have reported the prognostic significance of these epigenetic aberrations. We aimed for a complex evaluation of DNA methylation changes in AML patients at diagnosis. Therefore, we designed a sequencing panel targeting 239 regions annotated to 186 genes previously described in literature as having a prognostic impact or being commonly associated with AML pathogenesis (e.g. WT1, HOX genes). We used diagnostic whole-blood DNA samples of adult AML patients who were treated with curative intent starting with 3+7 induction regimen. In the testing cohort, we sequenced 128 AML patients and 11 samples from healthy donors. We analyzed another 50 AML patients from partner institution University Hospital Brno as an independent validation cohort. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Data were processed in Linux opensource software and further analyzed in R. For each sample, we measured the methylation level of nearly 50 000 CpGs. In the testing cohort, we used the Cox regression to evaluate the effect of each CpG's methylation level on overall survival (OS). As a result, we found 1961 CpGs significantly effecting the OS (p To validate these results, we counted MethScore and MethScore-MVA for 50 patients from the validation cohort and performed the same Cox multivariate analysis. MethScore remained highly significant for both OS and EFS (p=0.002 and p=0.004, respectively). MethScore-MVA was significant for OS (p=0.02) but not for EFS (p=0.09). Here, we present the MethScore as a novel complex characterization of predictive DNA methylation changes in patients with AML. MethScore might be used as a new surrogate marker that could enrich the currently used cytogenetic and genetic markers to refine the prognostication of newly diagnosed AML patients. This study was supported by the Ministry of Health of the Czech Republic, project for conceptual development of research organizations (00023736, IHBT). Disclosures Šálek: Amgen: Consultancy, Honoraria, Research Funding. Mayer:Celgene: Research Funding.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with large heterogeneity at the clinical and molecular levels. As diagnostic procedures shift from bone marrow biopsies towards less invasive techniques, circulating small noncoding RNAs (sncRNAs) have become of particular interest as potential novel noninvasive biomarkers of the disease. We aimed to characterize the expression profiles of circulating sncRNAs of MDS patients and to search for specific RNAs applicable as potential biomarkers. We performed small RNA-seq in paired samples of total plasma and plasma-derived extracellular vesicles (EVs) obtained from 42 patients and 17 healthy controls and analyzed the data with respect to the stage of the disease, patient survival, response to azacitidine, mutational status, and RNA editing. Significantly higher amounts of RNA material and a striking imbalance in RNA content between plasma and EVs (more than 400 significantly deregulated sncRNAs) were found in MDS patients compared to healthy controls. Moreover, the RNA content of EV cargo was more homogeneous than that of total plasma, and different RNAs were deregulated in these two types of material. Differential expression analyses identified that many hematopoiesis-related miRNAs (e.g., miR-34a, miR-125a, and miR-150) were significantly increased in MDS and that miRNAs clustered on 14q32 were specifically increased in early MDS. Only low numbers of circulating sncRNAs were significantly associated with somatic mutations in the SF3B1 or DNMT3A genes. Survival analysis defined a signature of four sncRNAs (miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p measured in EVs) as the most significantly associated with overall survival (HR = 5.866, p &lt, 0.001). In total plasma, we identified five circulating miRNAs (miR-423-5p, miR-126-3p, miR-151a-3p, miR-125a-5p, and miR-199a-3p) whose combined expression levels could predict the response to azacitidine treatment. In conclusion, our data demonstrate that circulating sncRNAs show specific patterns in MDS and that their expression changes during disease progression, providing a rationale for the potential clinical usefulness of circulating sncRNAs in MDS prognosis. However, monitoring sncRNA levels in total plasma or in the EV fraction does not reflect one another, instead, they seem to represent distinctive snapshots of the disease and the data should be interpreted circumspectly with respect to the type of material analyzed.

Background and Aims A part of lower-risk myelodysplastic (LR-MDS) patients progress to higher-risk MDS or acute myeloid leukemia (AML). The progression may be predicted in some of these patients by determining mutations associated with myeloid malignancies at the time of diagnosis. We focused to find out dysregulated pathways caused by a well-defined mutation associated with disease progression. Methods We examined 158 samples from LR-MDS patients at diagnosis using TruSight Myeloid Sequencing Panel containing 54 genes (Illumina) to identify the mutational profile. We applied RNA-seq (NEB; Illumina) on bone marrow CD34+ cells of 5 LR-MDS patients with RUNX1 mutation and 6 LR-MDS patients without mutations. We performed differential gene expression analysis, gene set enrichment analysis (GSEA), Reactome biological pathway and Gene Ontology (GO) annotations. Results Forty out of 158 patients (25%) progressed during the follow-up period (median: 45.1 months). Mutation in RUNX1 gene was detected in 10 patients. Using RNA-seq, significantly dysregulated expression was observed in 986 genes (FDR2). Patients with mutation in RUNX1 gene had significant down regulation of expression genes involved in cellular senescence induced by DNA Damage/Telomere Stress (FDR=1.4e-11) and Oxidative Stress (FDR=3.6e-9), HDACs deacetylate histones (FDR =1.1e-13) and Senescence Associated Secretory Phenotype (FDR=1.4e-11) according to Reactome pathway database. There were 21 genes involved in the regulation of these pathways, of which 10 were part of all described pathways and belonged to the Nucleosome Assembly according to GO biological process with 33 down regulated genes (FDR=6.3e-28). According to GSEA, GO chromatin silencing gene set (Figure 1) was down regulated in the patients with RUNX1 mutation (p2); however, they did not form a specific pathway. The up-regulated genes were classified as so-called leukemia-associated antigens (LAAs) such as PRAME (adjusted p=6.9e-09; logFC=6.8), WT1AS (adjusted p=4.4e-07; logFC=5.7), BAALC (adjusted p=3.5e-06; logFC=2.1), WT1 (adjusted p=6.8e-07; logFC=3.2), FLT3 (adjusted p=0.004; logFC=1.3) and LEF1 (adjusted p=5.3e-08; logFC=-4.2). Conclusions To study the transcriptome of the hematopoietic stem cells of LR-MDS patients, we used gene expression profiling and identified biological processes in relation to mutations in RUNX1 gene that were detected in patients with disease progression. According to our results, we suppose that cells of LR-MDS patients with no mutations are maintained in senescent state or apoptosis in opposite to RUNX1 mutated cells. Finding out of dysregulated senescence was reinforced by other dysregulated pathways such as HDACs deacetylate histones, chromatin structural changes and senescence-associated secretory phenotype. About cellular senescence is known that may act as a strong tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. We suppose that in precancerous cells of LR-MDS patients apoptosis or cellular senescence limits the replicative capacity of cells, thus preventing the proliferation. The disturbed state of cellular senescence in RUNX1 gene mutated hematopoietic stem cell can escape proliferation control and then can contribute to the disease progression in these patients. Supported by grants NV18-03-00227 and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a tendency to transform to acute myeloid leukemia. Several prognostic systems based almost entirely on clinicopathological characteristics have been developed. However, considering large heterogeneity of MDS, there is an ongoing substantial effort to identify new molecular markers, which would improve patient stratification. It has recently been discovered that various types of small noncoding RNAs (sncRNAs) are exported into blood circulation. Encapsulated in extracellular vesicles (EVs) or as a part of various molecular complexes, they play important roles in long distance cell-to-cell communication. Their secretion appears to be controlled and specific and thus may reflect (patho)physiological processes occurring in different cells. As diagnostic procedures move from bone marrow biopsies towards less invasive techniques, circulating sncRNAs have become of particular interest as potential novel semi-invasive biomarkers. Aims: We investigated circulating sncRNAs in MDS on the genome-wide level to better understand MDS pathogenesis and to search specific RNAs applicable as potential biomarkers. We analyzed paired samples from the whole plasma and EV fractions to define which of these two materials is better source of relevant sncRNA-based markers of MDS. Methods: We performed small RNA-seq in paired samples (plasma and EVs) in 42 patients and 17 healthy controls. To associate sncRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Overall survival (OS)‐associated sncRNAs were identified by performing univariate Cox regression along with a permutation test using BRB‐ArrayTools. Multivariate Cox regression analysis with backward variable selection was done to identify independent variables associated with patient survival. Results: Data analysis showed that the most abundant category of RNAs were miRNAs (51 %), followed by rRNAs, piRNAs, tRNAs, and mRNAs. Sample clustering revealed that RNA content of EV cargo is more homogenous than of plasma. Differential analysis identified a striking disbalance in RNA content between plasma and EVs in MDS patients (> 400 significantly deregulated sncRNAs) whereas these sncRNA profiles remained similar in controls. Such disproportion may suggest that mechanisms of sncRNA export into blood circulation may be specifically affected in MDS. As a result, these changes might substantially affect cell-to-cell communication of blood cells, contributing to the disease development. Further, we analyzed levels of individual sncRNAs and found that many hematopoiesis-related miRNAs were significantly increased in MDS patients compared to healthy controls, mostly both in plasma and EVs (e.g., miR-34a-5p, miR-125a-5p, and miR-150). Several miRNAs (e.g., miR-103b, miR-107, and miR-221) showed different levels between early and advanced MDS. Interestingly, the majority of miRNAs from the 14q32 miRNA cluster were specifically increased in early MDS. Additionally, we investigated possible impacts of somatic mutations on expression of circulating sncRNAs. However, only low numbers of circulating sncRNAs significantly associated with mutational status, suggesting no fundamental effects of somatic mutations on sncRNA export from MDS cells. Survival analysis identified sncRNAs with the highest level of association with OS (plasma: miR-1260b, miR-3191-3p, and miR-328-3p; EVs: miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p). To better stratify MDS patients, we defined risk prediction scores (separately for plasma and EVs) that combined effects of the above mentioned sncRNAs. The results showed that EV-combined score significantly increased the predictive power of the survival risk model (Figure 1). Moreover, Cox multivariate analysis revealed that this score is an independent variable the most significantly associated with OS (HR = 2.942, 95% CI 1.786 to 4.849, p < 0.001). Conclusions: Our data demonstrate that profile of circulating sncRNAs is specific in MDS and changes with the disease progression. Monitoring of sncRNA levels in EVs provides better prognostic performance for patient survival (compared to whole plasma), probably due to higher homogeneity of the EV fraction. Supported by grants 16-33617A and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.