B9 (new in 2nd funding period): Causes and consequences of the epigenetic perturbances in T-prolymphocytic leukemia
T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)/t(14;14) or t(X;14) leading to activation of either of the highly homologous genes TCL1 or MTCP1, respectively. Only few cases of T-PLL lack either of these aberrations and it is a matter of debate whether these represent true T-PLL. In various aspects, T-PLL can be regarded as T-cell counterpart of B-CLL with what it shares morphologic features, the mature lymphocyte phenotype, the pathogenetic im-portance of TCL1 activation and ATM inactivation and a mutational landscape dominated by recurrent chromosomal imbalances rather than recurrently mutated genes. With current treatment, the outcome of T-PLL is comparably poor though a recent trial showed some activity of epigenetically modifying drugs. In the last decade, we and others have extensively characterized the patterns of chromosomal imbalances and coding gene mutations in T-PLL. Among others, we could show the importance of mutational activation of the JAK-STAT pathway in T-PLL with mutually exclusive changes in JAK3 and STAT5B. Moreover, we identi-fied recurrent mutation of the epigenetic modifier genes EZH2, TET2, and BCOR and recurrent chromoso-mal imbalances which correlate with changes in gene expression as compared to normal B cells. Already in 2009 we showed that T-PLL was a striking exception among a broad range of hematologic malignancies in that it showed low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. In the framework of the International Human Epigenome Consortium (IHEC) project BLUERPINT, we have meanwhile confirmed these findings on a much higher resolution and in an extended set of more than 80 T-PLL samples. Moreover, we have generated his-tone modification maps of reference epigenomes from T-PLL samples and integrated them with those of normal T-cell subsets. Our unpublished findings show a massive DNA hypomethylation in T-PLL at enhancer sites in normal T-cells. Indeed, the chromatin states at these sites shows a gain in H3K4me3 and H3K27ac in T-PLL indicating a switch from enhancer marks to predominantly active promoters . This enhancer hypo-methylation phenotype is remarkably homogenous throughout T-PLL samples and could be shown in a pre-malignant inv(14)-positive T-cell clone.
The aim of the proposed project is, thus, to investigate the causes and consequences of these epigenomic perturbances in T-PLL. To this end, the role of the genes directly involved in the pathogenesis of T-PLL, namely TCL1/MTCP1, ATM, and of epigenetic modifiers like EZH2 and BCOR as well as of candidates to be identified in the course of the project shall be investigated. As prerequisite, we plan to develop yet missing human T-PLL models using Cas9 technologies in human T-cells and precursor cells as well as mouse xeno-grafts. Various types of ChIP-Seq and reverse ChIP experiments shall be performed in normal T-cells, T-PLL cells, and the newly to establish models (or conventional transfection models as back-up if necessary) to identify the factors driving the epigenetic switch. In addition, by integrating genomic sequencing data with epigenomic data we aim at identifying a potential impact of the epigenetic perturbances on the development of the chromosomal imbalance pattern in T-PLL. The evolution of this pattern shall moreover be studied in the novel models to be generated. Finally, pharmacologic inhibition or stimulation of pathways or molecules contributing to the epigenetic switching is aimed at as soon as these are identified. By combining develop-ment of human models with up-to-date genomic and epigenomic technologies and considering the appro-priate fall-back strategies we are confident that these experimental approaches can further elucidate the pathogenetic mechanisms of T-PLL. Considering that the integration in the CRC 1074 moreover allows inter-secting the findings in T-PLL with those of related analyses in B-CLL, there is an added value which might indeed support the rapid translation of the findings of this project in the treatment of this yet still mostly lethal disease.
For a current list of project-related publications, please go to this page