Kinome-wide CRISPR-Cas9 screen to identify kinases involved in Taxol resistance in breast cancer

Breast cancer is the most frequently diagnosed cancer in women, accounting for over 25% of cancer diagnoses and 13% of cancer-related deaths in Canadian women. There are many types of therapies for treatment or management of breast cancer, with chemotherapy being one of the most widely used. Taxol (paclitaxel) is one of the most extensively used chemotherapeutic agents for treating cancers of the breast and numerous other sites. Taxol stabilizes microtubules during mitosis, causing the cell cycle to arrest until eventually the cell undergoes apoptosis. Although Taxol has had significant benefits in many patients, response rates range from only 25-69%, and over half of Taxol-treated patients eventually acquire resistance to the drug. Drug resistance remains one of the greatest barriers to effective cancer treatment, yet little has been discerned regarding resistance to Taxol, despite its widespread clinical use. Kinases are known to be heavily involved in cancer development and progression, and several kinases have been linked to resistance of Taxol and other chemotherapeutic agents. However, a systematic screen for kinases regulating Taxol resistance is lacking. Thus, in this study, a set of kinome-wide screens was conducted to interrogate the involvement of kinases in the Taxol response. Positive-selection and negative-selection CRISPR-Cas9 screens were conducted, whereby a pooled library of 5070 sgRNAs targeted 507 kinase-encoding genes in MCF-7 breast cancer cells that were Taxol-sensitive (WT) or Taxol-resistant (TxR) which were then treated with Taxol. Next generation sequencing (NGS) was performed on cells that survived Taxol treatment, allowing identification and quantitation of sgRNAs. STK38, Blk, FASTK and Nek3 stand out as potentially critical kinases for Taxol-induced apoptosis to occur. Furthermore, kinases CDKL1 and FRK may have a role in Taxol resistance. Further validation of these candidate kinases will provide novel pre-clinical data about potential predictive biomarkers or therapeutic targets for breast cancer patients in the future.

EXAMINATION OF VALPROIC ACID-INDUCED PERTURBATIONS TO DNA REPAIR, NF-ΚB SIGNALING AND CBP/P300 TRANSCRIPTIONAL CO-ACTIVATORS

Exposure to the antiepileptic drug valproic acid (VPA) is associated with an increased risk of congenital malformations including heart, skeletal and most frequently neural tube defects. Although the mechanisms contributing to its teratogenesis are not well understood, VPA was previously shown to increase homologous recombination (HR)-mediated DNA repair and decrease protein expression of the transcription factor NF-κB/p65. The studies in this thesis utilized in vivo and in vitro models to evaluate the expression of HR mediators, investigate the implications of decreased p65 including DNA binding and transcriptional activation, and the expression and histone acetyltransferase activity of Cbp/p300 with an aim to provide mechanistic insight into VPA-mediated alterations. The first study demonstrated that following maternal administration of VPA, mouse embryonic mRNA expression of HR mediators Rad51, Brca1 and Brca2 exhibited temporal and tissue-specific alterations. Protein expression of Rad51 was similarly altered and preceded increased cleavage of caspase-3 and PARP; indicative of apoptosis. The second study confirms previous findings of decreased total cellular p65 protein using P19 cells, but is the first to demonstrate that nuclear p65 protein is unchanged. NF-κB DNA binding was decreased following VPA exposure and maybe mediated by decreased p50 protein, which dimerizes with p65 prior to DNA binding. Transcriptional activity of NF-κB was also increased with VPA exposure which was not due to increased p65 phosphorylation at Ser276. Furthermore, the transcriptional activation capacity was unaffected by VPA exposure as combined exposure to VPA and TNFα additively increased NF-κB activity. The third study demonstrated that VPA exposure in P19 cells decreased Cbp/p300 total cellular and nuclear protein attributed primarily to ubiquitin proteasome-mediated degradation. Histone acetyltransferase (HAT) activity of p300 was decreased proportionately to nuclear protein following VPA exposure. Inhibition of Cbp/p300 HAT activity decreased p65 total cellular protein, increased caspase-3 cleavage and ROS similar to VPA exposures. Furthermore, pre-treatment with the antioxidant enzyme catalase attenuated the increase in caspase-3 cleavage, but not p65 protein. Overall, this thesis demonstrates that VPA exposure impacts the expression and activity of the transcription factor NF-κB and transcriptional co-activators/HATs Cbp/p300, which has implications for downstream VPA targets including Rad51, Brca1 and Brca2.