Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.

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Over the last decades, it has become increasingly clear that tumors are characterized by inter-individual heterogeneity, which can account as one of the prominent reasons for treatment failure. To address this problem, personalized precision medicine approaches need to be applied such as comprehensive genomic profiling to identify actionable driver mutations in individual tumors. Unfortunately, genomics alone is not sufficient in tumors that are driven by mutated but otherwise undruggable targets and a typically low mutational burden. This is a characteristic of many pediatric malignancies including rhabdomyosarcoma (RMS), the most common childhood soft-tissue sarcoma. Hence, we aim to develop an in vitro drug-profiling platform to identify and prioritize treatment strategies for RMS patients. To this end, we generated a panel of patient-derived xenografts (PDXs) including some diagnostic and relapse samples. We then screened 18 different culture conditions to identify suitable parameters to establish in vitro primary cultures of PDX tumors. Interestingly, addition of fetal calf serum to cell culture media has a detrimental effect on viability of most primary RMS cell cultures (PRCCs). In contrast, defined serum-free conditions allow to grow primary cultures for several passages that closely preserve the clonal composition and phenotypic characteristics of the parental tumor, as assessed by genomic and copy number analysis. Pharmacologic profiles of PRCCs using a targeted drug library of more than 200 compounds revealed patient-specific vulnerabilities, among them an unexpected sensitivity to AKT inhibitors in some fusion-positive RMS. Interestingly, hierarchical clustering of drug sensitivities clustered PAX3-FOXO1 fusion-positive tumors together and separated them from fusion-negative RMS. Moreover, a screen to establish effective drug combinations in a highly resistant high-risk relapse sample using standard chemotherapeutics (doxorubicin, etoposide, vincristine) together with our targeted compound library revealed the BH3-only mimetic ABT-263 as the top-scoring drug capable of resensitizing recurrent PRCCs to first-line treatment. Resensitizing with ABT-263 was not a patient-specific vulnerability as it was observed in several additional PRCCs. Mechanistically, genetic loss-of-function validation experiments revealed that this occurs via blockade of the BCL-XL-MCL-1 axis and is dependent on upregulation of the BH3-only protein NOXA. Taken together, our study provides an in vitro tool kit to prioritize actionable drug targets or combinatorial options for RMS patients for whom conventional therapies are failing. Citation Format: Beat W. Schaefer, Gabriele Manzella, Michaela Roemmele, Luduo Zhang, Joëlle Tchinda, Felix Niggli, Marco Wachtel. Development of an in vitro drug-profiling platform for functional guidance of treatment decisions and identification of vulnerabilities in chemoresistant relapsed rhabdomyosarcoma tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B01.

The paradigm of cancer therapy currently shifts from broadly used cytotoxic drug cocktails to patient specific precision therapies. These novel therapies are normally directed towards tumor specific driver oncogenes, identified by genomic analysis of individual tumors. In case of tumors with a very low mutational burden and/or currently undruggable driver oncogenes this approach is less suitable. There, direct drug profiling of in vitro cultured tumor cells represents a promising alternative for identification of patient specific therapies. Conditions to maintain primary tumor cells in culture for this purpose however are largely unknown for most tumor entities. Here, we aimed to establish an in vitro drug profiling platform for Rhabdomyosarcoma (RMS), a childhood cancer with above described characteristics. Towards this aim, we isolated cells from 6 alveolar and 8 embryonal RMS patient derived xenografts and systematically tested 18 different culture conditions in order to find suitable parameters for in vitro propagation of primary RMS cells. This approach revealed that cells from most tumors survive and proliferate only in serum-free medium, whereas in serum containing medium tumor cells are progressively lost over time. Importantly, cells preserve the clonal composition and phenotypic characteristics of the primary PDX under these conditions, as assessed by genomic and copy number analysis. Drug profiles established with a library of 204 drugs revealed, beside patient-specific vulnerabilities, a yet unrecognized sensitivity of a subgroup of alveolar RMS towards AKT inhibitors. Furthermore, screening with the same drugs for resensitization of resistant relapse samples identified the BCL-2 family inhibitor ABT-263 as most potent resensitizer towards standard-of-care chemotherapy. Detailed molecular analysis revealed that this effect is based on blockade of the BCL-XL-MCL-1 axis. Overall, our proof of concept study highlights the feasibility of in vitro drug profiling of primary RMS cells for patient-specific treatment selection in a co-clinical setting. Citation Format: Marco Wachtel, Gabriele Manzella, Michaela Römmele, Luduo Zhang, Joelle Tchinda, Felix Niggli, Beat Schäfer. Development of an in vitro drug profiling platform with primary Rhabdomyosarcoma cells for tailoring patient-specific treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3194.

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