Project Details
Projekt
Splicing and epigenomic analyses in high-risk CLL for the development of novel therapeutic strategies.
Applicant
Professorin Dr. Michal-Ruth Schweiger
Subject Area
Hematology, Oncology
Term
from 2016 to 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 226262100
Besides ATM and TP53 the splicing factor SF3B1 and epigenetic modulators (e.g. CHD2, ARID1A, TET1,2,3) are the most frequently mutated genes in chronic lymphocytic leukemia (CLL), affecting 20% and 15% of CLL patients, respectively. In particular, SF3B1 and TP53 mutations are associated with poor clinical outcome. However, exact modalities of their functions and how they may participate in poor prognosis for CLL are only slowly emerging. During the startup funding period, we performed strand-specific RNA sequencing of 13 patients with wild type SF3B1 and 14 patients with mutations in SF3B1 and detected a significant decrease in intron retention (IR) among the mutated patients. Furthermore, we found a strong decrease in mRNA levels of genes with IR, most likely due to premature stop codons introduced by the retained intron and non-sense mediated decay of mRNAs. Interestingly, our previous work on BRD4, revealed an increase in IR after BRD4 depletion under heat shock conditions. Moreover, BRD4 was recruited to the nuclear stress bodies after heat shock and co-localized with HSF1. Since during the heat shock response and the proteotoxic stress response in cancer cells similar mechanisms are utilized , and since SF3B1 participates in the heat shock response, it is tempting to speculate that BRD4 and SF3B1 function together in the splicing process during stress response. Moreover, a chemical inhibition of these mechanisms may lead to a selective inhibition of the proliferation of SF3B1 mutated cells. Here, we intend to extend our findings and, in addition, try to identify epigenetic regulators of SF3B1. We will analyze the functional consequences of SF3B1 mutations using minigene assays and in vitro splicing systems. Since splicing and epigenetic regulation is highly interconnected, e.g. SF3B1 is associated with nucleosomes and we could show that the epigenetic reader BRD4 is implicated in the splicing process following heat shock, we will use an siRNA screen to identify chromatin modulators which transfer SF3B1 functions. This will be complemented with epigenomic profiles of 5-methylcytosine, 5-hydroxymethylcytosine and of histone modifications of H3K79me3, H3K4me3 and H3K36me3 of CLL patients with mild disease versus refractory/relapsed disease (relapse after FCR/BR-treatment within 3 years). These analyses will help to identify candidate biomarkers suitable to identify patients with poor prognosis. Finally, we will test drugs targeting the splicing - epigenetic axis in vitro and in two in vivo mouse models of poor prognosis CLL for their suitability for therapeutic treatments of CLL.
DFG Programme
Clinical Research Units
