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Dynamic interplay between DNA methylation, histone modifications and super enhancer activity in normal T cells and during malignant T cell transformation

Periodic Reporting for period 4 - EpiTALL (Dynamic interplay between DNA methylation, histone modifications and super enhancer activity in normal T cells and during malignant T cell transformation)

Reporting period: 2020-01-01 to 2020-06-30

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that is diagnosed in children, adolescents and adults. Although originally associated with high relapse rates, the prognosis of T-ALL has gradually improved with the introduction of intensified chemotherapy, with cure rates in modern protocols reaching over 80% in children and about 50% in adults with this disease. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains poor. Therefore, current research efforts are focused on the search for targets for the development of more effective and less toxic antileukemic drugs, which will likely require a greater degree of specificity and an improved understanding of the molecular events that lead to the disease.

In contrast to the wealth of information on gene expression profiles and genetic alterations in T-ALL, information on the DNA methylome is relatively scarce. Indeed, only very few studies have reported on genome-wide DNA methylation patterns in human T-ALL and none of these reports have directly compared, using the same technological platform, T-ALLs with different subsets of normal T cells as their putative cell-of-origin. Therefore, it has been largely unclear if the DNA methylome of human T-ALLs is uniformly perturbed as compared to normal T cells. Furthermore, the molecular mechanisms that drive aberrant DNA methylation profiles in T-ALL have remained unknown so far.

In this research project, we have been able to show that human T-ALLs acquire their aberrant DNA methylation signature as a result of their replicative history, cell-of-origin and leukemia-specific gene expression profile that is driven by patient-specific genetic abnormalities. This epigenetic history of T-cell transformation can be traced back by using 3 main categories of CpG islands or Open Sea CpG sites which show differential methylation between normal and malignant T cells. Notably, these novel insights pave the way towards novel or improved epigenetic therapies for the treatment of human leukemia.
Cancer cells display DNA hypermethylation at specific CpG islands in comparison to their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype remains poorly understood.

In this project, we compared DNA methylation profiles from a large cohort of human T-cell acute lymphoblastic leukemia (T-ALL) patients with a large series of normal T cell counterparts (Figure 1). Notably, we showed that CpG island methylation in human T-ALL mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T-cells and which display a reciprocal association with H3K27me3 binding. In addition, we revealed that this aberrant methylation profile shows a strong correlation with the epigenetic age of the leukemic T cells and elucidate that a similar CpG island methylation signature is gradually established in aging pre-leukemic thymocytes obtained from the CD2-Lmo2 transgenic T-ALL mouse model. Finally, we unexpectedly uncovered that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent Decitabine, a drug currently used in the clinic for other hematological malignancies. However, decitabine shows uniform anti-leukemics effect in preclinical models of human T-ALL, suggesting that DNA hypomethylating agents could serve as a uniform novel therapeutic strategy for the treatment of human T-ALL bot at diagnosis as well as relapse (Figure 2).

Altogether, our work demonstrates that DNA methylation reflects the epigenetic history of leukemic T cells and suggests that methylation-based subtypes of human T-ALL have followed a different trajectory towards T-cell transformation, possibly mediated by differences in the self-renewing capacity of the putative T-ALL cell-of-origin.

Given that the concept of preleukemic thymocytes has only been reported in T-ALL mouse models so far, we here provide, for the first time, conceptual evidence that a pre-leukemic phase might also be involved in the pathogenesis of the human disease.
Within this project, we directly compared the genome-wide DNA methylation pattern in human T-ALL with different subsets of human thymocyte populations covering the complete trajectory of normal T cell development. This simultaneous DNA methylome analysis of T-cell leukemias with their putative cell-of-origin empowered us to distinguish cell-of-origin methylation profiles from leukemia-specific aberrations at CpG islands and Open Sea areas. Integration of DNA methylation profiles with gene expression signatures and ChIP sequencing datasets allowed us to obtain novel insights in the potential relevance of the differentially methylated CpG sites that we identified between normal and malignant T cells.

Moving forward, future research should further focus on obtaining functional insights into the biological processes that drive the 3 main categories of differentially methylated CpG which we established in T-ALL cells during the process of malignant T cell transformation. This aspect of T-ALL disease biology has been significantly understudied over the past decade and therefore will provide an immediate impact on the field of normal and malignant T cell development. Furthermore, our work will also result in the identification of novel conceptual advances that might also be applicable for other disease entities.

This broader impact includes:

1. Long-lived pre-leukemic stem cells (pre-LSCs) have been uncovered as an initiating event in various blood-born cancers. Therefore, our mechanistic insights into the role of thymic aging in CpG island hypermethylation in T-ALL might also be relevant to improve our understanding on the cell-of-origin and the multistep pathogenesis of other haematological malignancies, such as Juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

2. The CpG Island Methylator Phenotype (CIMP), which was originally identified in the context of prostate cancer, has now also been recognized in a variety of other solid tumors. Therefore, improved understanding on the functional drivers of CIMP status in T-ALL might also have implications on solid tumor disease biology.

3. The PRC2 complex is a key player that is broadly involved in a variety of tumour entities. Interestingly, it can both act as a tumour suppressor or an oncogene depending on its cellular context. Therefore, our insights into how PRC2 might affect mitotic aging and DNA methylation might also be relevant for other neoplasms, such as AML/MDS or specific subtypes of human lymphoma. Furthermore, increased expression of EZH2 has also been described in some solid tumours, including prostate and breast cancer.

4. PU.1 is a master regulator and pioneering factor that is involved in normal hematopoiesis and has been extensively studied in the context of AML. Therefore, our results on the potential association between PU.1 activity and DNA methylation might also be relevant for myeloid leukemia disease biology or the field of normal hematopoietic development.
Figure 1. DNA methylation profiling in normal and malignant T-cells
Figure 2. DNA methylation profiling of T-ALL Xenografts treated with Decitabine in vivo