DNA repair pathway decisions in normal and malignant B cells

Genetic alterations of the DNA are a main cause of cancer. These alterations not only include individual DNA base changes, but also chromosomal re-arrangements of long DNA molecules, all of which can substantially affect gene expression. Eventually, an altered expression of genes that regulate cell growth and proliferation can lead to the development of cancer. The continuous accumulation of genetic alterations is not only responsible for cancerogenesis, but also for an unfavourable course of the disease as well as for resistance to therapy, since it allows the selection of the fittest clones from a heterogeneous pool of genetically different cancer cells. An important cause of genetic alterations is dysregulated DNA repair. While DNA damage occurs constantly in every cell, DNA repair mends this damage and thereby prevents the acquisition of a genetic alteration. All cells can choose from a panel of diverse repair pathways to mend damaged or broken DNA molecules. However, while some repair pathways seem functionally redundant, they differ in their precision and error rate. The aim of this project is to identify factors responsible for DNA repair pathway decisions and for high error rates during DNA repair in some cancer cells. Methodologically, the project utilizes genetic screens as well as gene expression profiling in combination with novel repair-reporter constructs in cell lines and primary normal and leukemic cells. Normal and leukemic B cells are used for this project as a model system, because B cell leukemias have a high incidence and are easily accessible for functional assays in high quantities. Identified factors will subsequently be tested in cell lines upon their selective overexpression, silencing or modification. The knowledge gained by this project on the regulation of DNA repair will help us to better understand the (in)stability of the genome of cancer cells and to find better treatment options for cancer.

One of the main characteristics of cancer is the acquisition of mutations and structural chromosomal alterations, such as translocations or deletions. All these genetic aberrations not only contribute fundamentally to cancer development but also play a crucial role in the development of therapy resistance. Various DNA repair mechanisms play a decisive role in the expression of these genomic alterations, either promoting or preventing them. Therefore, a better understanding of these repair mechanisms is essential to potentially enable targeted interventions in these mechanisms for future therapeutic use. In this research project, we investigated DNA repair mechanisms in context of cancer, particularly with regard to B-cell leukemias. To this end, we analyzed DNA mutations and expression profiles from databases and conducted wet-lab experiments. Our investigations enabled us to demonstrate the influence of a specific genetic profile on the expression and accumulation of somatic mutations in cancer cells of various entities. Furthermore, we gained new insights into the expression of templated sequence insertions (TSIs). TSIs are DNA insertions that exhibit high homology to distant genomic or extrachromosomal regions. The insertion of such sequences occurs regularly in cancer genomes, and we were able to show that they are regulated both by a specific DNA repair factor in combination with the non-homologous end joining (NHEJ) repair pathway and by the structure of the DNA ends to be repaired. This repair pathway also influences the expression of short duplications and inversions of DNA fragments near a repair site of DNA double-strand breaks. To investigate the full complexity of these structural changes at the genome-wide level in cells, we developed a method within this research project based on targeted endonucleolytic DNA cleavage and high-throughput DNA sequencing. This approach allows for the systematic investigation of translocations, insertions/deletions (indels), TSIs, or other alterations depending on individual genetic factors. Finally, the research funding also enabled an in-depth investigation of the stability of a repair enzyme responsible for the excision of deaminated DNA bases (cytosine to uracil alterations). This enzyme plays a crucial role in healthy B lymphocytes during their differentiation into antibody-producing immune cells and regulates the occurrence of cytosine mutation patterns in healthy and malignant cells. We were able to examine proteins that influence the stability of this repair enzyme, thus providing essential data on whether cytosine deaminations are repaired correctly or lead to mutations. As nucleic acid deamination also contributes to RNA editing, the project revealed additional insight into C>U RNA editing in normal and malignant B cells. Summarizing, the results obtained from the project provided important insight into DNA repair, the occurrence of DNA/RNA alterations, and potential targets for future cancer therapies.