Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

Ovarian cancer (OC) is a leading cause of death of gynecological cancers in women. Poor patient response to treatment highlights the need to better understand how the tumor microenvironment affects OC progression. Growing evidence indicates the crucial role of non‐cancerous components, such as cancer‐associated fibroblasts, in establishing a complex network of cellular and molecular interactions, influencing cancer progression and response to treatment. Therefore, in this study, we sought to characterize the impact of fibroblasts on OC cell behavior and drug response. Using both direct and indirect cell co‐culture systems, we observed distinct changes in cancer cell proliferation, morphology, and secretome in the presence of fibroblasts. Furthermore, an imaging‐based high‐throughput drug screen of 528 oncology compounds revealed multiple drugs that showed altered efficacy in the co‐culture conditions, demonstrating the role of fibroblasts in driving cancer cell resistance to treatment. Most importantly, our data identified the two drug combinations of Birinapant or Vorinostat with Carboplatin as promising treatments, exploiting the altered cancer cell phenotype in co‐cultures. These findings were supported by the increased sensitivity of ex vivo cultures to these combinations.

Background: Tumors of the pleura, such as metastatic lung cancer and mesothelioma, are amongst the most lethal and therapy-resistant tumors. The first manifestation of the disease is often pleural effusion, the first available material for diagnosis. The five-year survival rate is exceptionally low, around 10–20%, and only a small proportion of patients harbor mutations that allow targeted treatments. Almost all patients develop resistance to treatment, which is often palliative. There is therefore an urgent need to refine the selection of drugs and patients for personalized treatment. Methods: We isolated and cultured cells from pleural effusions in 3D cell aggregates and compared their drug sensitivity ex vivo to the clinical response to the same chemotherapeutic agents, combined with targeted sequencing and network analysis. Results: The ex vivo drug response showed a positive correlation with the treatment response and survival of patients in the clinic, with a stronger link to overall survival than to progression-free survival. Cryopreserved cells showed a similar response to freshly collected cells from the clinic. Conclusions: The findings advance the field of ex vivo screening and present an opportunity to combine strategies for functional precision medicine with comprehensive characterization of disease for improved treatment and future management of lung cancer.

Many ovarian cancer (OC) patients receiving chemotherapy relapse with acquired resistance, hence novel treatment allocation strategies are urgently needed. 3D cell culture models and organoid technologies have emerged as exciting tools to study cancer. Despite the advances, there is a need for protocols that can quickly generate patient-relevant 3D models for high-throughput drug screening and for diagnostic drug sensitivity testing. Ideally, such methods should be cost-effective, provide multi-parametric data to reveal distinct phenotypes, have single cell resolution to reveal heterogeneity and cell interactions, meet diagnostic timeframes and be miniaturized to allow the analysis of many drugs and combinations with small numbers of primary tumor cells. Here, we aimed to develop a robust technology for high-throughput ex vivo drug sensitivity testing for functional precision medicine using OC tissue and ascites samples (mostly high-grade serous carcinoma) from patients. We present a scalable drug screening platform: DETECT (Drug Efficacy Testing in Ex-vivo 3D Cultures), where tumor-derived patient cells can be rapidly screened within one week from sampling. Using this information patient-specific drug combinations are subsequently designed and tested with these data available within 9 days. This is much faster than with most screening protocols for organoids and PDC models where many weeks or months are required for establishment of the models and expansion of the required numbers of cells. We have currently tested the efficacy of 58 selected chemo- and targeted drugs in 5 doses using high content imaging and then a complete dose-matrix of 3 combinations at step 2. We report robust drug screening data in 15 out of 18 OC patient samples, which has resulted in a functional taxonomy of patient samples and a taxonomy of drugs based on their efficacy across patient samples. Some of the most common responses were seen for the BCL-XL inhibitor A-1331852 (9/15 patients), Topotecan (7), Dactinomycin (7), Omipalisib (6) and Omacetaxine (6). All but 2 drugs showed efficacy in at least one OC sample, suggesting heterogeneity and opportunities to make use of unique drug response patterns. Combination screening revealed that Carboplatin and A-1331852, a BCL-XL inhibitor, showed increased efficacy in 3 of the 5 tested patient samples. In conclusion, our 3D HT-drug testing assay DETECT with a combination screening capability could in the future be useful for guiding individualized treatment in a clinical setting as well as for identifying existing and emerging drugs and drug combinations for repurposing in OC. Citation Format: Emma Åkerlund, Greta Gudoityte, Elisabeth Moussaud Lamodiere, Joseph Carlson, Emelie Wallin, Josefin Fernebro, Olli Kallioniemi, Päivi Östling, Ulrika Joneborg, Brinton Seashore-Ludlow. Development of the drug efficacy testing in ex vivo 3D cultures (DETECT) platform and its application to functional precision medicine in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1890.

Platinum resistance remains a major therapeutic challenge in ovarian cancer (OC) and is one of the main causes leading to disease relapse and mortality. Although PARP inhibitors have improved outcomes for a subset of patients, most women continue to rely on platinum-taxol based chemotherapy and ultimately develop recurrent, treatment-resistant disease with limited further therapy options. Therefore, there is a critical need to identify actionable, patient-specific vulnerabilities that would help as an alternative for chemoresistant patients. To identify such therapeutic opportunities, we established 35 patient-derived models from 22 OC patients, representing seven OC subtypes and preserving key molecular features of individual patients. High-throughput drug profiling across a library of 528 oncology-focused compounds generated over 29,000 drug response measurements, revealing inter-patient heterogeneity and sensitivity patterns. Among these, a subset of models exhibited a pronounced dependency on the anti-apoptotic protein Bcl-xL with minimal effects observed on patient-derived fibroblasts and healthy bone marrow, suggesting a therapeutic window. Proteomics-based comparison of Bcl-xL-sensitive and -resistant subclones identified activation of NOTCH signaling as a determinant to reduced response to Bcl-xL inhibition. Blocking of NOTCH signaling with gamma-secretase inhibitors restored sensitivity to Bcl-xL targeting and resensitized resistant cells to Carboplatin, resulting in sustained cytotoxicity in long-term washout assays and ex vivo cultures. Similar effects were observed with both Bcl-xL protein degrader and small molecule inhibitor, supporting robust targeting of Bcl-xL through different modalities. Together, these findings define a NOTCH-modulated Bcl-xL survival axis as a therapeutic vulnerability in platinum resistant OC. More broadly, this study demonstrates how translational drug profiling of physiologically relevant disease models can generate insights with clinical potential and provide precision oncology framework for identifying rational combination strategies to overcome chemoresistance.