Mechanisms of resistance to the hypomethylating agent azacitidine in Myelodysplastic Syndromes

MECHANISMS OF RESISTANCE TO THE HYPOMETHYLATING AGENT AZACITIDINE IN MYELODYSPLASTIC SYNDROMES. GenOMeC_XXXII cycle. PhD Candidate: ALICE BROGI Tutor: PROF. VALERIA SANTINI Myelodysplastic syndromes (MDS) are a clinically heterogeneous group of clonal disorders, characterized by ineffective erythropoiesis, dysplastic changes of hematopoietic marrow precursors, genetic and epigenetic abnormalities, which share a high frequency of progression to acute myeloid leukemia. Hypomethylating agents (HMA) are standard of care for MDS and may improve survival of high risk MDS, but predictive factors for clinical response and optimal therapeutic duration remain to be defined. The great majority of MDS patients present with profound epigenetic abnormalities and it is believed that the clinical activity of HMAs is due to reversal of aberrant DNA methylation. However, still only ~50% of patients with MDS have a clinical response to HMAs and all of them will invariably lose response after variable length of time. Despite extensive studies of promoter DNA methylation over the last decade, the identification of specific epigenetic markers that may identify at diagnosis those patients who will benefit from HMA therapy remains elusive. For this reason, in this thesis 22 patients affected by high-risk MDS, treated with the hypomethylating agent azacitidine (75 mg/m2/d for 7 days every 28 days) were included. Bone marrow aspirate were collected before and after treatment with the drug. For some of those patients who responded to the treatment, we had specimens during remission and at relapse, when they lost response. All samples were screened for methylation, expression and somatic mutational profile. Methylation profile analysis demonstrated that methylation could be an indicator of response and loss of response. After treatment with azacitidine, the global genome methylation decreased while at relapse increased in specific genomic regions. The analysis of differentially methylated regions (DMRs) in samples at relapse showed an enrichment in biological processes related to neutrophil and granulocyte pathways correlated with a block of maturation of these cells. At baseline, methylation pattern was different between responder and non-responder patients. Moreover, the comparison between primary resistant and secondary resistant cases identified 7 different methylated clusters. Further analysis of these regions could help to understand why some patients did not respond to the therapy while others initially responded and developed the resistance later. Then, RNA-Seq was performed and expression and methylation results were combined. Since mechanisms of resistance to HMA in MDS cannot be correlated with a unique factor given the complexity of this disease, topologically associated domains (TADs) were studied in our primary and secondary resistant cases. TADs analysis allowed to understand the correlation and the spatial organization of differentially expressed genes associated with differentially methylated regions. In our cohort, 75 TADs were found and this result reflected the complexity of 3D-genome organization. By using Phenolyzer, several genes more associated with leukemia and myeloid disorders were identified. However, by using this bioinformatic tool, we discovered an important role of MAP2K1 as a seed gene. MAP2K1 was linked with many of other genes in the 75 TADs and it is strongly considered a key gene in the development of leukemia’s disease. These studies would improve our understanding of the mechanisms behind HMA response, ultimately allowing us to better select patients who will benefit from this kind of therapy and avoid exposure to anyhow toxic drugs like HMA that require long treatment (at least 6 cycles) before their activity is evident.