Functional Assessment of Amplification-Associated AKT2 and PTK2 Expression in High-Grade Serous Carcinoma Models

High-grade serous carcinoma (HGSC) is characterised by extensive somatic copy-number alterations, but recurrent amplification alone does not establish that an individual gene modifies treatment response. AKT2 and PTK2 were selected because recurrent amplifications of these genes in patient tumours were associated with increased transcript levels. This thesis tested whether increasing or reducing transcription from the endogenous AKT2 or PTK2 loci altered the responses of COV362 cells to carboplatin, cisplatin or olaparib. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) were used to examine expression-level effects without recreating or reversing the full genomic context of an amplicon. Selected patient-derived HGSC organoids were used to assess whether comparable perturbations could be established in three-dimensional backgrounds. Transcript levels were measured in both systems, whereas protein-level validation and drug-response testing were performed only in COV362 cells. Drug response was assessed using dose–response and crystal-violet colony formation assays. In COV362 cells, CRISPRi produced strong, comparatively target-restricted transcript repression, reducing AKT2 and PTK2 transcript levels to approximately 3% and 5% of their matched parental effector control levels, respectively. The reductions in i.AKT2 and i.PTK2 were qualitatively accompanied by lower total AKT2 and FAK band intensity, respectively. CRISPRa was less target-restricted. In a.PTK2, PTK2 and AKT2 transcript levels were both approximately 265% of a.Control, with visibly increased total FAK, whereas in a.AKT2, AKT2 transcript levels were approximately 290% of a.Control without a clear increase in total AKT2 protein. Exploratory western blots indicated stronger phospho-STAT1 Tyr701 band intensity in both CRISPRa lines and stronger total and phospho-STAT3 Tyr705 band intensity in i.AKT2; however, these single, unquantified observations did not establish altered STAT phosphorylation or pathway activity. Across the three patient-derived organoid models, perturbations varied in efficiency, target restriction and direction, and the intended AKT2 activation was not achieved. No statistically significant overall or condition-specific differences among the perturbation lines were detected in the colony-formation assay. Given the limited biological replication, fitted IC₅₀ estimates were interpreted descriptively. Across both assays, a.AKT2 showed lower carboplatin assay readouts than a.Control, whereas i.AKT2 showed higher carboplatin assay readouts than i.Control. The a.PTK2 line showed lower carboplatin and olaparib assay readouts than a.Control in both assays. Cisplatin findings were weak and inconsistent. Overall, these exploratory perturbation-associated patterns do not establish target-specific effects on drug response and require confirmation using matched non-targeting controls, independent guides, additional biological replicates and additional HGSC models.