DNA methylation in myelodysplastic syndromes and aging: novel approaches and new perspectives

Myelodysplastic syndromes (MDS) are hematopoietic neoplasms primarily affecting older individuals, characterized by clonal hematopoiesis and frequent epigenetic dysregulation, mainly alterations in DNA methylation. We aimed to investigate whether DNA methylation patterns in low risk as well as in high risk MDS could give information on epigenetic aging, and possibly correlate the profiles in the different settings to somatic mutations and inflammatory profiles. To implement the methylation data and achieve a composite prognostic marker we tried to evaluate it with different approaches. Very few publications succeeded in producing informative and consistent data of DNA methylation modulation in MDS and their correlation with disease biology. This thesis has 2 aims: 1) assess DNA methylation in LR-MDS with an EPICv2 Illumina methylation array to identify clusters of cases characterized by specific methylation pattern and compare them with methylation of a healthy population. To achieve this goal, epigenetic age was evaluated by epigenetic clocks and correlated with proinflammatory cytokines and somatic mutations; 2) assess DNA methylation in HR-MDS with Oxford Nanopore Technology to verify the modulation of methylation during treatment with azacitidine by a relatively straightforward method, applicable in a bed-to-bench approach. In our setting, modulation of methylation was evaluated during treatment with two formulations of azacitidine, i.e. subcutaneous and oral. There have been a few studies on methylation in MDS, hampered by the intrinsic technical issues in handling scarce material of a complex disease as well as by the almost exclusive focus on the analysis of CpG island promoters, disregarding intergenic regions. Although aging is considered a major risk factor in the development of MDS, scarce evidence is available regarding alterations of epigenetic aging in MDS. 1) Epigenetic age was assessed by epigenetic clocks (Horvath1, Hannum, DNAmPhenoAge, GrimAgeV1/2, DNAmTL and DunedinPACE) with data obtained from DNA methylation at ≥ 900.000 CpG sites. LR-MDS cases were age- and sex-matched with healthy-aged individuals from a geriatric longitudinal study (InCHIANTI Study of Aging). LR-MDS cases showed significantly accelerated epigenetic aging. This observation supports the hypothesis that LR-MDS results in alterations in aging trajectory. We implemented a novel mathematical method to enable direct comparison of InCHIANTI data produced by an Illumina 450K methylation array with our cohort data produced with EPICv2. Unsupervised clustering analysis of epigenetic clock values identified three subgroups of LR-MDS cases with specific epigenetic age acceleration. We defined fast agers, slow agers, and an intermediate heterogeneous agers group. Even using epigenetic clocks that are generated with a limited number of CpGs, we were able to identify subgroups of differentially methylated LR-MDS that were characterized by distinct levels of pro-inflammatory cytokines, co-mutational profiles, response to erythropoiesis-stimulating agents (ESAs) and risk of disease progression. 2) We applied a modified Oxford Nanopore Technologies protocol as a novel approach for evaluating DNA methylation in primary samples of CD34+ cells obtained from HR-MDS patients with low-input DNA. We evaluated in a controlled clinical study whether by such method we could demonstrate with sufficient sensitivity modulation of DNA methylation by oral azacitidine vs subcutaneous azacitidine. We thought that a rapid method to demonstrate equivalence between the two formulations would allow a prompt assessment of the activity of the oral treatment. Preliminary analyses confirm previous findings by other Authors, indicating that we could well evaluate with ONT the effect of therapy. We confirmed methylation modulation at single chromosome level at a genome-wide level. When we analyzed DNA methylation at the time of disease progression or loss of response to AZA, ONT was able to capture subtle modifications. In conclusion, specific DNA methylation patterns may be identified in MDS that stratify cases in various risk subtypes differing from those identified by IPSS-R or IPSS-M prognostic scores. Such subgroups present with distinct molecular and inflammatory profiles, thus capturing disease phenotypes. 1) We identified LR-MDS cases with significantly different pace of aging (the three subgroups). Moreover, we identified a “CMML-like” LR-MDS subtype characterized by increased biological aging, high levels of M-CSF, and increased absolute monocyte counts. This subgroup had poor response to ESA therapy and increased progression to AML. 2) We demonstrated that ONT may measure modulation of DNA methylation to rapidly assess DNMTi-related in vivo effects, and correlate them with clinical response and/or disease progression. In this work, we used two different effective methods to assess DNA methylation that, although limited in their genome-wide coverage of methylation sites, were able to capture specific biological characteristics of MDS.