Functional precision medicine approaches to identify novel therapeutic strategies in ovarian cancer

<p dir="ltr">Ovarian cancer (OC) is a rare yet highly aggressive disease, representing the most lethal gynecological malignancy. Despite covering multiple histological subtypes with different genetic alterations this disease is primarily treated through surgery followed by platinum-taxol based chemotherapy (Carboplatin and Paclitaxel in Sweden). Although this regimen initially leads to favorable responses, approximately 80% of patients relapse within 18 months and have limited further treatment options, leading to poor overall survival. Precision medicine (PM) has transformed oncology by leveraging genomic insights to stratify patients based on actionable mutations, enabling targeted, personalized therapies. In OC, this approach has led to the introduction of PARP inhibitors for patients with BRCA1/2 mutations or exhibiting homologous recombination deficiency. However, a high molecular heterogeneity of OC and lack of actionable alterations limit the efficacy of genomics-driven approaches. Increasing evidence also highlights the critical role of tumor microenvironment (TME) in driving therapeutic resistance and modulating drug response, emphasizing the need for diagnostic and treatment approaches that capture both tumor-intrinsic and extrinsic factors. Functional PM (fPM) and drug response profiling approaches, offers a powerful, phenotype- driven strategy to uncover clinically relevant vulnerabilities. Although its implementation in solid tumors remains technically challenging, fPM has already shown considerable success in hematological malignancies, highlighting its potential to guide individualized treatment strategies.</p><p dir="ltr">In this thesis, we applied fPM and drug response profiling approaches to identify novel therapeutic strategies to overcome drug resistance in OC and integrating components of the TME.</p><p dir="ltr">Study I aimed to establish a fPM platform compatible with OC tissue and ascites samples with a clinically relevant timeframe. We developed DET3CT (Drug Efficacy Testing in 3D Cultures), a scalable platform capable of generating patient-specific drug response profiles from 3D cultures of fresh patient material within 10 days. Compared to 2D cultures, 3D systems showed higher proliferation and were well suited for image-based profiling. Drug testing on 20 samples from 16 patients using a 58-drug library revealed strong correlation between Carboplatin sensitivity and clinical response, distinguishing patients with short (≤12 months) and long (>12 months) progression-free intervals. The Bcl-xL inhibitor A-1331852 and EGFR inhibitor Afatinib emerged as effective drugs in these patients, showing additive effects when combined with Carboplatin.</p><p dir="ltr">Study II aimed to explore the suitability of DET3CT platform for cryopreserved high-grade serous OC (HGS-OC) samples. A total of 83 samples from 58 patients were screened across 16 drugs and two drug combinations, achieving an 84% assay success rate. Carboplatin combination with A-1331852 or Trametinib were among the most effective treatments. Targeted therapies generally showed greater cytotoxicity than standard chemotherapies, complicating correlation of clinical outcomes for standard-of-care. Notably, sensitivity to the SMAC mimetic Birinapant, as well as higher proportion of EpCam+CD24+ cancer-stem cells, were associated with shorter progression-free interval. In addition, spheroid morphology correlated with HRD and BRCA1/2 mutation status, suggesting its potential as predictive biomarker.</p><p dir="ltr">Study III sought to systematically profile the drug response of a living biobank of patient-derived models from OC patients to identify effective drugs and rational strategies to overcome resistance. We established a collection of patient-derived models (PDMs) from 22 OC patients, including 21 cancer and 14 fibroblast models across seven OC subtypes, predominantly representing HGS-OC, the most common OC subtype. Panel sequencing confirmed that PDMs retained key tumor-specific aberrations. High-throughput drug screening revealed individualized response profiles, with 85% of compounds showing low toxicity in healthy bone marrow cells. EGFR/ERBB2 inhibitors, rapalogs, and BH3 mimetics emerged as active drug subclasses. The Bcl-xL inhibitor A-1331852 showed variable efficacy among models derived from the same patient, prompting proteomics analysis to understand determinants of drug response. The analysis revealed NOTCH pathway upregulation in resistant cells. Combination of Y- secretase inhibitors, A-1331852, and Carboplatin produced a durable cytotoxic effect in long-term treatment and ex vivo cultures, suggesting its potential as promising candidate for future clinical studies.</p><p dir="ltr">Study IV aimed to characterize how fibroblasts influence OC cell behavior and drug response. Using both direct and indirect co-culture systems, we observed that fibroblasts significantly altered OC cell proliferation, morphology and cytokine secretion profiles. An imaging based high-throughput drug screen of 528 oncology compounds revealed that fibroblast presence broadly reduced drug efficacy, highlighting their role in mediating therapy resistance. Notably, combinations of Carboplatin with either Birinapant or Vorinostat effectively restored cancer cell sensitivity, exploiting fibroblast-induced phenotypic changes. These findings were further validated in ex vivo patient samples, where both combinations exhibited enhanced cytotoxicity.</p><p dir="ltr">Together, these studies demonstrate the power of fPM and drug response profiling to uncover patient-specific vulnerabilities and novel therapeutic strategies for OC. By integrating short-term ex vivo cultures, PDMs, and methods incorporating components of the TME, this work identified several drug candidates and synergistic combinations. In particular, inhibition of the Bcl-xL and NOTCH pathways, as well as Birinapant or Vorinostat in combination with Carboplatin, showed potential to overcome resistance. Overall, these finding open the door for fPM in guiding individualized treatment strategies and advancing our understanding of OC biology.</p><h3>List of scientific papers</h3><p dir="ltr">I. Åkerlund E, <b>Gudoityte G,</b> Moussaud-Lamodière E, Lind O, Bwanika HC, Lehti K, Salehi S, Carlson J, Wallin E, Fernebro J, Östling P, Kallioniemi O, Joneborg U, Seashore-Ludlow B The drug efficacy testing in 3D cultures platform identifies effective drugs for ovarian cancer patients NPJ Precis Oncol. 2023 Oct 31;7(1):111<br><a href="https://doi.org/10.1038/s41698-023-00463-z" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41698-023-00463-z</a><br><br></p><p dir="ltr">II. <b>Gudoityte G,</b> Hutyra-Gram Östvos R, Haffa M, Juhani K, Wikström S, Kallioniemi O, Fernebro J, Joneborg U, Seashore-Ludlow B Ex vivo drug screening of biobanked high-grade serous ovarian cancer samples as a scalable approach to precision medicine [Manuscript]</p><p dir="ltr">III. <b>Gudoityte G,</b> Berkovska O, Orre L, Moussaud-Lamodière E, Bergström R, Lindberg J, Haraldsson M, Bou Nafeh S, Louhaur M, Kallioniemi O, Fernebro J, Joneborg U, Seashore-Ludlow B Functional drug screening of patient-derived models uncovers combination therapy to overcome platinum resistance in ovarian cancer [Manuscript]</p><p dir="ltr">IV. <b>Gudoityte G,</b> Sharma O, Leuenberger L, Wallin, E Fernebro J, Östling P, Bergström R, Lindberg J, Joneborg U, Kallioniemi O, Seashore-Ludlow B Systematic profiling of cancer-fibroblast interactions reveals drug combinations in ovarian cancer Mol Oncol. 2025 May 24<br><a href="https://doi.org/10.1002/1878-0261.70051" rel="noreferrer" target="_blank">https://doi.org/10.1002/1878-0261.70051</a></p>