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theses

DDR is often considered as a critical barrier during tumor initiation with most pre-malignant cells accumulate endogenous DNA damage before acquiring additional genetic alterations that provides a survival advantage. The work in this thesis focused on delineating the biological roles of BRCA1-PALB2 coiled-coil domain interaction, particularly on utilizing patient-derived VUS as means to identify novel BRCA1 function and possibility therapeutic targets in BRCA-associated cancers. No known structural information of the BRCA1-PALB2 interaction is available to date. The BRCA1-PALB2 coiled-coil domain is still potentially targetable due to the different post-translational modifications flanking this domain; protein modifications such as phosphorylation and ubiquitination can be explored further to modulate the BRCA1-PALB2 interaction. The BRCA1-PALB2 interaction is important for HR and suppression of error prone repair pathways. This protein-protein interaction is also required for efficient cell cycle checkpoint to ensure optimal repair timing when DNA damage occurs. We first investigated several PALB2 N-terminal coiled-coil domain mutants to not only determine the critical residues involved in PALB2 function but also to assess if varied repair ability would translate equally to chemotherapy sensitivity. Although PALB2 c.104T>C, p.L35P behaves as expectedly as a bona fide pathogenic missense variant, our results on other hypomorphic mutations such as c.83A>G , p.Y28C raised an interesting question on the complexity of VUS. Unlike pathogenic mutations that often resulted from frameshift mutations, hypomorphic PALB2 missense mutation that still retained HR activity above a poorly understood threshold is a challenge for personalized treatment of cancer patients. We next examine the direct relationship between known protein kinases such as ATM/ATR, CDK and PLK1 on BRCA1 function. BRCA1, a critical DDR protein, can function in various component of DDR network of responses. Several S/TQ sites on BRCA1 were widely reported based on either in vitro biochemical reactions or mass spectrometric analysis. The HR ability of cells often correlate well with drug resistance as genotoxic stress are often alleviated by HR. However, our experimental results focusing on the HR and SSA suppression activity of widely reported SQ BRCA1 mutants suggest that most of these phosphorylation events may not have as strong of a role in DDR than previously thought. S1387, T1394 and S1423 are phosphorylatable residues flanking the PALB2 binding coiled-coil domain. Here, we report that abolishing phosphorylation of both S1387 and S1423 leads to partial sensitization of cells to both cisplatin and olaparib despite similar to wt HR activity. T1394 phosphorylation, although undetectable in most mass spectrometry studies to date, appears to be a critical event as single amino acid alteration on this site is sufficient to partially reduce HR activity, increase the more erroneous SSA activity and completely sensitized cells towards cisplatin and olaparib. Both artificial and patient-derived VUS affecting T1394 phosphorylation do not affect PALB2 binding. Phosphospecific antibody against pT1394 confirms that this residue is potentially a ATM/ATR target. Results and observations of this study will hopefully contribute to improvement in our knowledge of DDR by targeting BRCA1 function, while assessing the potential of ATM/ATR inhibition for cancer therapy. In conclusion, this thesis offers new insights in understanding the biological relevance of the BRCA1-PALB2 coiled-coil domain interaction, particularly, as therapeutic targets in BRCA-associated cancers. My observations established that BRCA1-PALB2 interaction is critical for maintenance of genome stability and suppression of cancer development. Moreover, our data offers support for better risk assessment and clinical decision making for carriers of BRCA1 and PALB2 mutations affecting the BRCA1-PALB2 interaction.

ETD_9352

Ph.D.

Includes bibliographical references

by Tzeh Keong Foo

doi:10.7282/t3-8a3r-av49

ETD doctoral

The author owns the copyright to this work.