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Abstract

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer characterized by excessive proliferation of malignant T-lymphocyte progenitors. Current treatment regimens achieve cure rates of more than 80 % but include aggressive high-dose, multiagent chemotherapies causing severe adverse effects. Further, T-ALL represents a particular challenge in pediatric oncology, as the prognosis in the event of a relapse is poor and only 10 – 20 % of patients survive. Therefore, the search for new targets that impact on T-ALL and the development of new treatment options with less toxicity and a reduced risk of relapse is of high clinical importance. To date, research on T-ALL has mainly provided insights on the transcriptional level of the disease. However, an increasing number of studies report posttranscriptional dysregulation; a level of regulation governed by RNA-binding proteins (RBPs). Hence, this work aimed at uncovering RBPs that may be involved in the origin and development of T-ALL and have so far remained unknown. For this purpose, the method "enhanced RNA interactome capture" (eRIC) was carefully developed further and for the first time an RNA interactome was obtained from freshly isolated human tissue, the thymus. This normal thymocyte RNA interactome served as a counterpart to RNA interactomes derived from cell lines representing T-ALL. The RNA interactomes of normal thymocytes and T-ALL cells differ profoundly in size, but present typical characteristics of T-cells such as low metabolic activity, a remarkably high number of cytoskeletal proteins, and proteins related to immune or T-cell development, activation and function. Interestingly, a number of previously not captured RBPs and proteins so far not known to bind RNA were identified (e.g. SASH3, CAPZB, MIA3). As a possible model for the dynamic alteration of an RNA interactome after chemotherapy, the T-ALL RNA interactome was also analyzed upon treatment with the histone deacetylase (HDAC) inhibitor SAHA. Several RBPs with altered RNA binding activity were found, including some eukaryotic initiation factors, in particular EIF4A1. An ultimate and direct comparative analysis of the RNA interactomes indicates a globally increased RNA-binding activity in T-ALL compared to normal thymocytes. In conclusion, this work comprises the first comprehensive investigation of the RNA-binding proteomes of T-ALL and normal T-cell progenitors, and thus, offers unprecedented insights into alterations in the RBP-landscape upon leukemogenesis.