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Final Report Summary - CHROMSTAB (Mammalian Chromosome Stability)

DNA rearrangements are hallmarks of cancer cells and of several genetic disorders. The DNA damage response acts as a “genome guardian” mechanism that prevents formation of aberrant DNA configurations. The broad objective of the project was to gain insight into mechanisms of normal and pathological DNA repair and recombination in primary mammalian cells. The Fellow is studying these processes in both somatic and germline cells.

We originally put forward the following two hypotheses:
1. Tumors are hotbeds of ongoing genome evolution
There may be substantial variation in the frequency and location of ongoing DNA rearrangements when different patients and/or different cells from the same tumor are compared
2. DNA repeats are a risk factor for germline rearrangements
Developmentally programmed (meiotic) DNA double-strand breaks that occur in repeat sequences are more likely to produce germline rearrangements than those that occur in unique sequences

Research on the first hypothesis has resulted thus far in the publication of proof-of-principle experiments of our newly developed long-range inverse PCR method (Pradhan et al., Genes Chromosomes Cancer 2016). This technique allows the detection and molecular characterization of in principle any de novo genome rearrangements. Our pilot study was performed on DNA extracted from leiomyomas, benign tumors of the uterus. We have since expanded and further refined this technique to analyse a particular class of genome instability, that associated with a particular class repetitive DNA elements of the human genome (long interspersed nuclear element (LINE)-1 retrotransposition). In this approach, we combined long-range inverse PCR with single-molecule sequencing technology, and unraveled mechanistic details of de novo insertions of mobile LINE-1s in the genome of one colon cancer sample. A manuscript on these findings is currently in preparation. Ongoing experiments are extending these analyses to recurrent rearrangement regions in other tumor types, with a particular focus on high-grade serous ovarian cancer.

To address the second hypothesis, we have focused our attention on the instability of the coding minisatellite segment within the mouse Prdm9 gene. This gene is of particular interest, since it encodes a protein crucial for the correct distribution and formation of meiotic double-stranded breaks that initiate germline recombination. Thus, PRMD9 dictates how mammalian genomes are reshuffled from one generation to the next and any alterations in its structure will have profound consequences for genome evolution. We are exploring repeat instability at the Prdm9 minisatellite during different stages of mouse spermatogenesis, using small-pool PCR followed by Southern blotting. We discovered that germline instability is remarkably frequent but transient at this locus, and have reproducibly obtained mutant Prdm9 molecules from cells undergoing meiosis I. The mutant molecules were predominantly deletions, implying single-strand annealing as the underlying mechanism. So far we have found no evidence for somatic instability.

Unraveling molecular mechanisms that underlie DNA rearrangements should in the long-term lead to safer, more personalized cancer therapies. In the past year, we have focused our attention on chromosomal aberrations in high-grade serous ovarian cancer, which has very poor prognosis. Future work will focus on identifying and characterizing DNA rearrangements with functional relevance for tumorigenesis and/or drug response/resistance in this deadly cancer type, thereby providing improved patient stratification. The aim is to establish molecular read-outs predictive of each tumor’s chemotherapy responses that can guide drug choices. Choosing the right drugs for the right patients (and omitting ”wrong”, i.e. inefficient or unsuitable, drugs or drug combinations) will be enormously beneficial for both patients and the public health care system. For germline DNA, little is known about the causes of genome instability; our work is anticipated to lead to insights into recombination-related male infertility.

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