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DNA repair mechanisms and therapy resistance of BRCA2-deficient cancers

Periodic Reporting for period 1 - DREMATURE (DNA repair mechanisms and therapy resistance of BRCA2-deficient cancers)

Reporting period: 2018-01-01 to 2019-12-31

Despite the current advances in cancer therapy, most patients with disseminated cancer still die because their tumors become resistant to the available drugs. Thus, drug resistance remains a major challenge in clinical oncology.
Because tumor cells often have alterations in DNA repair mechanisms, therapies that target DNA are usually effective and quite selective for this type of cells. Examples of these malignancies are ovarian and breast cancer with deficiency in the repair of DNA double strand breaks due to mutations in BRCA1 or BRCA2 genes. They are especially sensitive to PARP inhibitors (PARPi), a recently approved targeted therapy that leads to DNA double strand breaks in BRCA mutated cells. However, resistance is an inevitable fact also in the context of those type of cancers. The precise mechanisms underlying resistance to novel targeted drugs such as PARPi are poorly understood.
The overall goal of the DREMATURE project was to identify new mechanisms by which BRCA2-deficient breast cancer cells develop drug resistance to PARPi and thereby gain novel insights into the basic DNA damage response processes. Based on the results of this action, I expect that, eventually, new tools can be implemented in the clinic to predict and explain patient resistance to PARPi. Moreover in the case of patients who do not respond to PARPi, I predict that based on these results, that a personalized strategy can be developed to increase the sensitivity of the cancer cells to this treatment.
To accomplish the goal, I used a genetically engineered mouse model of BRCA2-deficient breast cancer, which closely mimics the human disease, and combined the next generation sequencing analysis of spontaneous resistant tumors in this model (Figure 1A) with functional in vitro genetic screens using the CRISPR/Cas9 technology (Figure 1B). Among other hits, the overlap of these two approaches yielded a candidate gene MDC1 (Mediator of DNA Damage Checkpoint protein 1). MDC1 is an important protein in the repair of DNA double strand breaks, which binds the damage site and helps in the recruitment of downstream repair factors.
I confirmed that MDC1 deficiency confers a survival advantage in the presence of PARPi, leading to resistance both in vitro (Figure 1C) and in vivo in mouse models mimicking BRCA2-deficient breast cancer (Figure 1D). Moreover, I investigated further the molecular mechanisms of how this loss in MDC1 function leads to resistance (Figure 1E). I observed that MDC1-deficient cancer cells replicate their DNA more slowly than MDC1-proficient cells, due to a delay in DNA replication restart upon replication blockage. I hypothesize that this delays give the cells more time to deal with PARPi-induced DNA damage and therefore contribute to their survival advantage and resistance (Figure 1F).
This work revealed a new way how BRCA2-deficient breast cancer cells become resistant to the targeted therapy inhibiting PARP that is used in the clinic against ovarian and breast cancer. I showed that loss of a DNA repair factor, MDC1, slows down the rate of DNA replication and contributes to resistance to the treatment. Therefore, I uncovered a new role for a DNA repair protein in another cellular process: DNA replication. These observations open new doors to the management of resistant cancers with defects in BRCA (Figure 1G):
o First, it can help predict if the patients will benefit from the therapy or not, depending on MDC1 status (normal or defective); in parallel, loss of MDC1 in these tumors can be used as one explanation for cases of acquired PARPi resistance
o Second, new investigations based on these results should be performed in order to find ways to sensitize MDC1-deficient cells to PARPi treatment.
Overview of the project, including preliminary data, methods, results, model and future directions