Understanding the origin of small tandem duplications and other break-induced mutations in cancer

Principal Investigator: dr. Joost Schimmel (LUMC)

Funding: KWF Young investigator 2020

Description

The aim is to understand the formation of small tandem duplications (TDs) at chromosomal breaks and to study the contribution of this novel end-joining activity to different genome rearrangements observed in cancer.

To realize this, a cell- based assay specific for TD-formation in genetic screens will be used to identify (additional) factors that either stimulate or suppress NHEJ-mediated repair. In parallel, I will take a proteomic approach based on proximity-dependent labeling to identify interactors of end joining proteins at DNA breaks using mass-spectrometry. Hits from both screens as well as the 53BP1- Shieldin-CST axis will be thoroughly investigated for their role in TD-formation. Additionally, I will test their involvement in other mutational outcomes of end-joining, for example by using a novel translocation assay. Recent work (including my own) has established that mutagenic repair outcomes are dictated by the sequence-context of DNA breaks. Therefore, I will perform in-depth bioinformatics analysis of break-induced mutational signatures (including FLT3-ITD mutations derived from databases and AML patient material) and use the obtained knowledge in cellular repair assays, in which I will generate DNA breaks of different configurations (e.g. sequence context, polarity and length of overhang, degree of micro-homology). Another important determinant in end-joining pathway choice and mutational consequence of a double strand break is the moment of introduction. To study this in great detail, I will generate cell-lines that express different end-joining components in a cell-cycle specific manner and I will use modified Cas9-constructs to introduce DSBs in a designated phase of the cell-cycle. Finally, I will study how different end-joining factors contribute to secondary malignancies by analyzing the sensitivity and mutational signatures of knockout cells exposed to radiation induced breaks (photons and protons) and other break-inducing agents used in cancer treatment.