ItemInvestigating the granule forming properties of SMAUG1 and the consequence of interaction with the 14-3-3 protein family(University College Cork, 2023-03-01) Fehilly, John Denis; Dean, KellieSMAUG1 is an intrinsically disordered RNA binding protein that forms granules in cells containing RNA. SMAUG1 has been linked to several diseases such as Alzheimers, muscular dystrophy, and cancer. SMAUG1 represents a potentially distinct class of RNA granules and thus is a very interesting target for study. This work furthers our understanding of the SMAUG1 protein by showing that it undergoes rapid recovery following photobleaching indicative of liquid-liquid phase separated granules. Here we also report partial colocalization of SMAUG1 with P-body component Enhancer Of MRNA Decapping 4 (EDC4) and stress granule component Ras GTPase-activating protein-binding protein 1 (G3BP1). Previous work from the Dean lab identified interaction between the 14-3-3 family of proteins and SMAUG1. Here this interaction is validated via co-immunoprecipitation western blot analysis. Further to this potential 14-3-3 binding motifs within SMAUG were mutated and shown to be functional for 14-3-3 binding. Finally, the consequences of 14-3-3 interaction with SMAUG1 granules were assessed. 14-3-3 binding mutant form of SMAUG1 showed a higher propensity for granule formation within cells. Mutant forms of SMAUG1 also showed impaired recovery following photobleaching. This taken together suggests that the 14-3-3 family proteins are negative regulators of SMAUG1 granule formation. ItemInvestigation of mechanisms underpinning cytokine-induced cell death of colon cancer cells(University College Cork, 2023) Linehan, Eva; Nally, Ken; Lee, CiaranInflammatory cytokines induce regulated cell death as part of a robust immune response. Many unique forms of regulated cell death have been described that are critically dependent on specific signal transduction molecules or cellular events. Often, molecules or events canonically associated with cell death signalling pathways, while contributing factors, are not essential for cell death. Given the extensive emerging crosstalk and plasticity between modes of cell death, determining dependency is crucial for categorising cell death and for downstream clinical applications. IFN-γ and TNF-α are inflammatory, Th1-type cytokines elevated in immune-mediated inflammatory diseases and infectious diseases such as COVID-19, that synergistically induce cell death in diverse cell types. Recently IFN-γ-induced JAK/STAT signalling has emerged as an important pathway for triggering cell death, but the downstream mechanisms underpinning JAK/STAT-mediated cell death are incompletely understood. Here, IFN-γ+TNF-α-induced synergistic cell death was confirmed to be dependent on JAK1, JAK2, STAT1, and partially dependent on CASP8, using knockout colon cancer cell lines. IFN-γ+TNF-α-treated cells displayed biochemical hallmarks of multiple cell death pathways. Use of JAK1/2 inhibitors highlighted the unique kinetics of IFN-γ+TNF-α-induced cell death, suggesting that an accumulation of intracellular event/s, reaching a threshold or point of no return, may be responsible for cell death. The inconsistent effects of p300/CBP inhibitors on IFN-γ+TNF-α-mediated cell death suggest that STAT1-dependent transcription is not the main effector of cell death downstream of JAK1 and JAK2. The STAT1-dependency observed may just be due to its effects on JAK2 expression in cytokine-treated cells over time. Flow cytometry assays were used to investigate the kinetics of several biomarkers of mitochondrial and cellular stress in IFN-γ+TNF-α-treated cells, compared to cells undergoing canonical cell death pathways. Loss of mitochondrial integrity coincided with the crucial turning point observed in JAK1/2 inhibitor chase experiments, followed by severe increase in superoxide levels. Though these events cannot be confirmed to be the main drivers of IFN-γ+TNF-α-induced cell death, they are hallmarks of death and may be contributing factors. IFN-γ+TNF-α-induced cell death occurs by a non-canonical and redundant cell death mechanism that is dependent on JAK activity and potentially involves induction of components of multiple cell death pathways. ItemGeneration of a CRISPR-Cas9 mediated knock-in reporter for the GRIA3 candidate gene for schizophrenia(University College Cork, 2022-09-27) Breen, Lisa; McCarthy, Tommie V.Glutamatergic neurotransmission impairment is considered a major feature of the neurobiology of Schizophrenia (SZ) and implicates genes in this pathway as potential candidates for the condition. A study on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor genes found strong evidence of association between the Glutamate Ionotropic Receptor AMPA Type Subunit 3 (GRIA3) gene and SZ. Similarly, a recent report has identified a number of genes, including GRIA3, with ultra-rare disabling variants that promote SZ. The association of a rare disabling GRIA3 variant with SZ indicates that reduced expression of the gene predisposes people to SZ and suggests that increasing the expression GRIA3 could be a potential therapeutic avenue for treatment of the condition. The aim of this thesis was to establish a cell model enabling rapid analysis of GRIA3 expression. Such a model would be of high value and in addition to facilitating expression studies on GRIA3, would enable screening for new drugs that increase GRIA3 expression which could have therapeutic potential. This project aimed to modify the cell line using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR associated protein 9 (Cas9) homology directed repair (HDR) approach so that a donor reporter vector containing the Gaussia secreted luciferase gene and linked green fluorescent protein (GFP) or neomycin resistance gene (Neo) gene would be integrated directly under the control of the endogenous promoter of the GRIA3 gene, in a manner that retains intact expression of the GRIA3 protein. This donor reporter vector was successfully constructed and has significant general use as it facilitates cloning of any pair of homology arms and the insertion of a reporter cassette into any target gene via CRISPR-Cas9 HDR. Flanking GRIA3 homology arms were inserted 5’ and 3’ of the reporter cassette for CRISPR-Cas9 HDR mediated insertion into the GRIA3 locus in human U87 glioblastoma cells. Luciferase activity was monitored post-transfection and was present at low levels suggesting successful HDR events. However, the presence of the donor cassette could not be demonstrated at the GRIA3 locus. It was not possible to distinguish if the luciferase activity resulted from read through of the donor plasmid or if a low number of targeted integration events had occurred. Further work involving isolation of individual clones of the targeted U87 cells and checking for the presence of the donor at the GRIA3 locus will be necessary to resolve this question. Overall, this reporter system should be of high value for targeting other loci and can be improved further by modifications to ensure luciferase is only active when inserted into the targeted locus. 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. ItemInhibition of the endosomal recycling pathway to overcome resistance to cancer therapies(University College Cork, 2022) Fletcher, Kelsey; Lindsay, AndrewCancer is a major public health threat with incidence and mortality rates continuing to rise every year. Breast and prostate cancer are the most diagnosed cancers in women and men, respectively. Both cancers account for roughly 30% of all cancers diagnosed in each sex. Despite the continuous development of new therapies, drug resistance is a growing problem and is a major cause of cancer treatment failure, accounting for many cancer recurrences and deaths. Therefore, new drugs and treatment regimens are urgently required to overcome this resistance. Recent findings from our own lab and others have found that inhibition of the endosomal recycling pathway may be a promising strategy to downregulate clinically relevant cell surface proteins and to overcome drug resistance. This thesis focuses on two clinically relevant hormone receptors that are strongly linked to the development and progression of breast and prostate cancer linked to disease development and progression, the estrogen receptor alpha and the androgen receptor. The aim of this project was to confirm data obtained from a reverse-phase protein array (RPPA) study performed by our lab that found that the endosomal recycling inhibitor primaquine downregulates ER-α and AR expression. We used Western blot, quantitative RT-PCR, and immunofluorescence microscopy to confirm the RPPA results. We found that another endosomal recycling inhibitor, monensin, also potently downregulates these hormone receptors and that both inhibitors synergise with tamoxifen and enzalutamide, standard-of-care therapies for breast and prostate cancer. Keywords Cancer, prostate, breast, androgen receptor, estrogen receptor, endosomal recycling pathway, primaquine, monensin, drug resistance, tamoxifen, lapatinib, enzalutamide, synergy.