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Synergistic combination therapies involving regorafenib in experimental glioma based on genome-wide CRISPR-Cas9 functional screens

ABSTRACT: Registered systemic treatment options for glioblastoma patients are limited, particularly for progressive glioblastoma, including bevacizumab in the US and some other countries as well as lomustine. The phase II REGOMA trial suggested an improvement of overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE, however. Of note, regorafenib has been administered as monotherapy in progressive glioblastoma or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib, instead of entirely discontinuing any consideration of this compound, might be therefore a reasonable strategy. We addressed this research gap and used an innovative discovery and validation approach to identify functionally-instructed combination therapies. We applied a genome-wide CRISPR-Cas9-based functional genomics experimental approach including activation and knockout screens for the discovery of potential molecular targets modifying the therapeutic efficacy of regorafenib in experimental glioma. Selected functionally instructed molecular hits were then validated regarding synergistic interaction in vitro, ex vivo and in two orthotopic mouse models in vivo.This functional genomics approach led to the discovery of several molecular targets. Based on subsequent validation experiments including a customized compound library, we identified novel combination therapies. Particularly, the combination of regorafenib with Bcl-2/Bcl-xL inhibition was among the most promising strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272737 | GEO | 2024/08/21

REPOSITORIES: GEO

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Project description:Radiotherapy has a critical role in the treatment of small cell lung cancer (SCLC). Effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using a Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 induce apoptosis in SCLC cells by inducing hallmarks of apoptosis (cleaved caspase-3, sub-G1 fraction induction and induction of the mitochondrial caspase activation pathway). S44563 markedly enhanced sensitivity of H146 cells and H69 cells to radiation in clonogenic assay. In addition, the combination S44563 and cisplatin-based chemo-radiation showed a dramatic tumor growth delay and increased overall survival in mouse xenograft models. In vitro experiments demonstrated a greater induction of apoptosis (sub-G1 fraction and cleaved caspase-3) with the combination as well as in vivo caspase-3 immunostaining. This positive interaction between S44563 and radiation was greater when S44563 was given after the completion of the radiation, which may be explained by the radiation-induced over expression of anti-apoptotic proteins (Bcl-2 and Bcl-XL) through the NF-?B pathway activation. Taken together, these data underline the critical role of sequence administration of targeted therapies with conventional therapies. SCLC cells which survive to IR highly rely on the induction of anti apoptotic proteins in part through NF- ?B activation for their survival yielding to the concept of 'contextual oncogene addiction' to the Bcl-2/Bcl-XL pathway providing rational for targeting survival pathways to potentiate IR. For transcriptome analysis, 106 cells were seeded in T25 flasks, allowed to growth for 24 h, and then left untreated or treated with 2 Gy radiation. After 24 hours, cells were harvested, lysed for the extraction of RNA, and processed to analyze gene expression.The design of this study consists to 6 hybridizations in dual color with Agilent Human Genome 4x44K (design 014850). The reference is always the untreated condition.

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Synergistic combination therapies involving regorafenib in experimental glioma based on genome-wide CRISPR-Cas9 functional screens

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