Lower risk (LR) myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem and progenitor disorders caused by the accumulation of somatic mutations in various genes including epigenetic regulators that may produce convergent DNA methylation patterns driving specific gene expression profiles. The integration of genomic, epigenomic, and transcriptomic profiling has the potential to spotlight distinct LR-MDS categories on the basis of pathophysiological mechanisms. We performed a comprehensive study of somatic mutations and DNA methylation in a large and clinically well-annotated cohort of treatment-naive patients with LR-MDS at diagnosis from the EUMDS registry (ClinicalTrials.gov.NCT00600860). Unsupervised clustering analyses identified six clusters based on genetic profiling that concentrate into four clusters on the basis of genome-wide methylation profiling with significant overlap between the two clustering modes. The four methylation clusters showed distinct clinical and genetic features and distinct methylation landscape. All clusters shared hypermethylated enhancers enriched in binding motifs for ETS and bZIP (C/EBP) transcription factor families, involved in the regulation of myeloid cell differentiation. By contrast, one cluster gathering patients with early leukemic evolution exhibited a specific pattern of hypermethylated promoters and, distinctly from other clusters, the upregulation of AP-1 complex members FOS/FOSL2 together with the absence of hypermethylation of their binding motif at target gene enhancers, which is of relevance for leukemic initiation. Among MDS patients with lower-risk IPSS-M, this cluster displayed a significantly inferior overall survival (p < 0.0001). Our study showed that genetic and DNA methylation features of LR-MDS at early stages may refine risk stratification, therefore offering the frame for a precocious therapeutic intervention.

• Publikační typ
• časopisecké články MeSH

The transcription factor PU.1 (Purine-rich DNA binding, SPI1) is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.

• Klíčová slova
• 5-Azacytidine, Celastrol, microRNA miR-155, transcription factor PU.1,
• MeSH
• akutní myeloidní leukemie * farmakoterapie   genetika   MeSH
• buněčná diferenciace genetika   MeSH
• lidé MeSH
• mikro RNA * genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• regulace genové exprese u leukemie MeSH
• trans-aktivátory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA * MeSH
• MIRN155 microRNA, human MeSH Prohlížeč
• proto-oncogene protein Spi-1 MeSH Prohlížeč
• protoonkogenní proteiny MeSH
• trans-aktivátory MeSH

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.

• Klíčová slova
• Noncoding RNAs, acute myeloid leukemia, azacytidine, circular RNAs, myelodysplastic syndrome, transposable elements,
• MeSH
• akutní myeloidní leukemie * farmakoterapie   genetika   MeSH
• azacytidin farmakologie   terapeutické užití   MeSH
• epigeneze genetická MeSH
• lidé MeSH
• myelodysplastické syndromy * farmakoterapie   genetika   MeSH
• RNA dlouhá nekódující * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azacytidin MeSH
• RNA dlouhá nekódující * MeSH

PURPOSE: Treatment options are limited for patients with lower-risk myelodysplastic syndromes (LR-MDS). This phase III, placebo-controlled trial evaluated CC-486 (oral azacitidine), a hypomethylating agent, in patients with International Prognostic Scoring System LR-MDS and RBC transfusion-dependent anemia and thrombocytopenia. METHODS: Patients were randomly assigned 1:1 to CC-486 300-mg or placebo for 21 days/28-day cycle. The primary end point was RBC transfusion independence (TI). RESULTS: Two hundred sixteen patients received CC-486 (n = 107) or placebo (n = 109). The median age was 74 years, median platelet count was 25 × 109/L, and absolute neutrophil count was 1.3 × 109/L. In the CC-486 and placebo arms, 31% and 11% of patients, respectively, achieved RBC-TI (P = .0002), with median durations of 11.1 and 5.0 months. Reductions of ≥ 4 RBC units were attained by 42.1% and 30.6% of patients, respectively, with median durations of 10.0 and 2.3 months, and more CC-486 patients had ≥ 1.5 g/dL hemoglobin increases from baseline (23.4% v 4.6%). Platelet hematologic improvement rate was higher with CC-486 (24.3% v 6.5%). Underpowered interim overall survival analysis showed no difference between CC-486 and placebo (median, 17.3 v 16.2 months; P = .96). Low-grade GI events were the most common adverse events in both arms. In the CC-486 and placebo arms, 90% and 73% of patients experienced a grade 3-4 adverse event. Overall death rate was similar between arms, but there was an imbalance in deaths during the first 56 days (CC-486, n = 16; placebo, n = 6), most related to infections; the median pretreatment absolute neutrophil count for the 16 CC-486 patients was 0.57 × 109/L. CONCLUSION: CC-486 significantly improved RBC-TI rate and induced durable bilineage improvements in patients with LR-MDS and high-risk disease features. More early deaths occurred in the CC-486 arm, most related to infections in patients with significant pretreatment neutropenia. Further evaluation of CC-486 in MDS is needed.

• MeSH
• aplikace orální MeSH
• azacytidin aplikace a dávkování   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• myelodysplastické syndromy farmakoterapie   patologie   MeSH
• následné studie MeSH
• prognóza MeSH
• prospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze III MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• azacytidin MeSH
• cc-486 MeSH Prohlížeč

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.

• Klíčová slova
• IDH2, azacitidine therapy, histone acetylation, metabolic signature, myelodysplastic syndromes,
• Publikační typ
• časopisecké články MeSH

Azacitidine (AZA) for higher risk MDS patients is a standard therapy with limited durability. To monitor mutation dynamics during AZA therapy we utilized massive parallel sequencing of 54 genes previously associated with MDS/AML pathogenesis. Serial sampling before and during AZA therapy of 38 patients (reaching median overall survival 24 months (Mo) with 60% clinical responses) identified 116 somatic pathogenic variants with allele frequency (VAF) exceeding 5%. High accuracy of data was achieved via duplicate libraries from myeloid cells and T-cell controls. We observed that nearly half of the variants were stable while other variants were highly dynamic. Patients with marked decrease of allelic burden upon AZA therapy achieved clinical responses. In contrast, early-progressing patients on AZA displayed minimal changes of the mutation pattern. We modeled the VAF dynamics on AZA and utilized a joint model for the overall survival and response duration. While the presence of certain variants associated with clinical outcomes, such as the mutations of CDKN2A were adverse predictors while KDM6A mutations yield lower risk of dying, the data also indicate that allelic burden volatility represents additional important prognostic variable. In addition, preceding 5q- syndrome represents strong positive predictor of longer overall survival and response duration in high risk MDS patients treated with AZA. In conclusion, variants dynamics detected via serial sampling represents another parameter to consider when evaluating AZA efficacy and predicting outcome.

• Klíčová slova
• AML, MDS, NGS, azacitidine, somatic mutation,
• Publikační typ
• časopisecké články MeSH

Azacitidine (AZA) is a hypomethylating drug used to treat disorders associated with myelodysplasia and related neoplasms. Approximately 50 % of patients do not respond to AZA and have very poor outcomes. There is thus great interest in identifying predictive biomarkers for AZA responsiveness. We searched for specific genes whose expression level was associated with response status. Using microarrays, we analyzed gene expression patterns in bone marrow CD34+ cells in serial samples from 32 patients with myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia with myelodysplasia-related changes before and during the AZA therapy. At baseline, a comparison of the responders and non-responders showed 52 differentially expressed genes (P < 0.01). Functional annotation of the deregulated genes revealed categories primarily related to ribosomes and pathways associated with proliferation. The expression level of RPL28 correlated with overall survival. We identified altered expression in 167 genes in responders, 26 genes in non-responders with stable disease, and 13 genes in non-responders with disease progression using paired t test of expression levels in patients before and during treatment. Our data indicate that AZA treatment failure is associated with the up-regulation of ribosomal genes/pathways that are likely related to intensive proteosynthesis in proliferative/neoplastic cells of non-responders.

• Klíčová slova
• Azacitidine, Gene expression profiling, Myelodysplastic syndromes, Ribosomal genes,
• MeSH
• akutní myeloidní leukemie genetika   MeSH
• azacytidin farmakologie   terapeutické užití   MeSH
• buňky kostní dřeně metabolismus   MeSH
• chronická myelomonocytární leukemie genetika   MeSH
• lidé MeSH
• myelodysplastické syndromy farmakoterapie   genetika   MeSH
• neúspěšná terapie MeSH
• proliferace buněk genetika   MeSH
• ribozomální proteiny genetika   MeSH
• transkriptom účinky léků   MeSH
• upregulace účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• azacytidin MeSH
• ribozomální proteiny MeSH