- ItemTargeting transcription-regulating cyclin dependent kinase 12 for the treatment of breast cancer(University College Cork, 2022-11-27) Burns, Martha; Krajewska, MalgorzataCyclin-dependent Kinase 12 (CDK12) with its binding partner Cyclin K regulates transcription through phosphorylation of RNA Polymerase II (RNA Pol II) at serine 2. Previous work showed that inhibition of CDK12 leads to decreased expression of DNA damage response (DDR) genes and sensitizes cancer cells to DNA damage-inducing agents. However, the exact mechanism by which this is achieved remains unclear. This study aimed to investigate CDK12 as a therapeutic target in breast cancer and to identify new drug combinations involving the inhibition of CDK12 and clinically relevant inhibitors of DNA repair. We demonstrated that CDK12 inhibitor SR-4835 is cytotoxic in MCF-7 breast cancer cells at a low nanomolar concentration. The observed toxicity was associated with G2-M cell cycle arrest, increased apoptosis, and DNA damage. We showed that CDK12 inhibition had a minor effect on the phosphorylation of RNA Pol II at serine 2 indicating that global transcription was not affected. Interestingly, we observed that CDK12 inhibition resulted in decreased expression of chromodomain helicase DNA binding protein 2 (CHD2), a potentially new target of CDK12. Next, we tested the combination of CDK12 inhibition with inhibitors of DNA repair including inhibitors of PARP (olaparib) and CHK1 (AZD7762) using a colony formation assay. The preliminary results indicate that combining CDK12 and CHK1 inhibition will likely have greater therapeutic potential than the combination of CDK12 and PARP inhibition. Future studies are required to establish the exact role of CDK12 in transcription and DDR as well as to investigate further the potential of combining CDK12 and CHK1 inhibition in breast cancer.
- ItemEvaluation of selective γ-secretase inhibitors as novel modulators of TNF-α-mediated apoptosis(University College Cork, 2021-05-04) O'Brien, Ciara; McCarthy, Justin V.; Science Foundation IrelandThe presenilin proteins (presenilin 1 and presenilin 2) were identified in mutagenesis screens causing the early onset forms of familial Alzheimer’s disease (FAD) in 1995. Subsequently characterized as the catalytic subunits of the γ-secretase protease complexes, the presenilins are responsible for the cleavage of the amyloid precursor protein (APP) and generation of amyloid beta (Aβ). To date, γ-secretase proteases have over 120 substrates which demonstrate contribution to a diverse range of cellular processes and signalling events. However, recent findings have revealed several γ-secretase-independent presenilin functions, including calcium signalling, autophagy and apoptosis. We and others have previously reported members of the TNF receptor super-family as substrates for γ-secretase proteolysis, and that presenilin-deficient cells have increased resistance to TNFα-induced apoptosis. In this study, we sought to determine whether loss of presenilin expression or loss of γ-secretase protease activity is associated with increased resistance to TNFα-induced apoptosis. Utilizing the cleavage of caspase 3 and Poly ADP ribose polymerase (PARP) in target cells as a readout, we tested the anti-apoptotic characteristics of a panel of well characterized γ-secretase inhibitors. In this study, we show that loss of presenilin expression is associated with increased resistance to TNFα-induced apoptosis, and that loss of γ-secretase protease activity does not affect sensitivity to TNFα-induced apoptosis. These observations suggest a γ-secretase-independent role of presenilins in the regulation of TNFα-induced apoptosis.