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Abstract

Thymopoiesis is a process by which bone-marrow-derived lymphoid progenitor cells migrate to the thymus and undergo multi-step differentiation into mature CD4+ or CD8+ Tlymphocytes. The entire process is tightly regulated and governed by the transcriptional/epigenetic changes necessary for lineage commitment and cellular identity. Genetic lesions such as somatically acquired point mutations or chromosomal rearrangements lead to differentiation blockade resulting in hematological malignancy known as T-cell acute lymphoblastic leukemia (T-ALL). While the thymopoiesis and T-ALL are well characterized by transcriptional studies, high-resolution mapping of the epigenetic changes is still lacking. DNA methylation (DNAm) changes involving the addition of de-novo, or the erasure of existing methyl groups from the cytosine nucleotide, are dynamic during cellular differentiation and form the cell-type-specific signatures. In this doctoral thesis, DNAm dynamics during the human thymopoiesis is studied by whole-genome bisulfite sequencing of seven distinct intra-thymic cell types. DNAm changes during the thymopoiesis are characterized by the uni-directional and irreversible loss methylation primarily occurring at the transcription factor binding sites critical for T-cell lineage commitment (e.g., NOTCH1 and MYB) and T-cell receptor rearrangements. A DNAm atlas of thymopoiesis is established by identifying 381 de-novo differentially methylated regions (tDMRs) that are highly conserved across cell-types originating from the thymic lineage. The tDMRs can recapitulate the in-silico ontogeny of T-cell differentiation and are validated in an independent dataset. Remarkably, combined analysis with bone-marrow-derived hematopoietic progenitors and peripheral derived mature blood cells shows the hypermethylation of tDMRs among non-lymphoid cell types suggesting the epigenetic silencing of pathways necessary for thymic lineage commitment. To further highlight the role of tDMRs in disease development, a combined array-centric analysis of intra-thymic cell types and a well-defined cohort of 143 primary adult T-ALLs was performed. Interestingly, DNAm classified the T-ALL cohort into five distinct subtypes (C1-C5) with characteristic levels of DNAm (C1 lowest level and C5 the highest). Moreover, each II subtype is correlated with a specific somatic event, including a novel adult T-ALL specific subtype with co-occurring DNMT3A/IDH2 mutations (C1), and well known transcriptionally deregulated subtypes resulting from TAL1 (C2), TLX3 (C3), TLX1/in cis-HOXA9 (C4), or in trans- HOXA9 (C5) overexpression. Utilizing tDMRs as the blueprint, maturation arrest stages of TALL subtypes are established, revealing a hierarchical ordering, with C1 and C5 arising earlier during the T-cell development followed by TLX3/1 overexpression (C3, C4) and TAL1 deregulation (C5). Although tDMRs highlight the ontogeny of T-ALL subtypes, global DNAm levels did not correlate with the maturation arrest stages suggesting a non-linear association of DNAm and differentiation blockade. Subsequent integrative analysis with epigenetic marks associated with active transcription (H3K27ac and H3K4me1) revealed the hypomethylation of pathogenic enhancer elements. Importantly, careful survival analysis identified an unexpected, clinically aggressive hypermethylated subtype (C5) that can be targeted with DNA hypomethylating agents. Finally, using machine learning models, a 79 CpG classifier was developed for de-novo classification of newly diagnosed adult T-ALLs. In summary, results from the comprehensive analysis of DNAm changes during human thymopoiesis and the subsequent modifications leading to T-ALL provide meaningful insights into the role of DNAm in maintaining the cellular identity and disease development. Furthermore, the identification of clinically actionable hypermethylated T-ALL subtype paves the way for targeted epigenetic therapies.