Discovering new therapeutic combinations for hepatocellular carcinoma by CombiGEM

Abstract

Hepatocellular carcinoma (HCC) is the most general subtype of liver cancer and ranks as the second leading cause of cancer-related morbidity and mortality worldwide. Limited therapeutic options are available, and tumor recurrence and drug resistance often occur. There remains a critical need to discover new actionable targets and improve the treatment for this disease. Co-treatment with drugs can broaden the way to improve the treatment efficacy compared to monotherapy. With the development of the high-throughput screening platform, combinatorial genetics en masse (CombiGEM), rapid assembly of libraries with unique barcodes are now enabled for functional characterization of genetic combinations. In this study, two CombiGEM-based screens have been performed for the identification of potential therapeutic combinations against HCC: 1) CombiGEM screening of 1,849 miRNA overexpression pairs encoded by combinations of 43 miRNA precursor sequences; 2) CombiGEM-CRISPR screening of 8,649 sgRNA pairs against combinations among 30 selected druggable targets. From the screen results, five miRNA precursor combinations were identified: miR-125a/miR125b-1, let-7a/miR-193b, miR-125b-1/miR-199a-1, miR-101-1/miR-125a, and miR-142/miR-200a. Validation assays confirmed the enhanced anti-proliferative effect brought by these miRNA combinations when compared to the overexpression of their single-miRNA constituents. Eleven sgRNA combinations were identified, including ANXA3/FGFR3, FLT4/FGFR2, GRIN1/FGFR3, GRIN1/FLT4, ITGB7/ANXA3, ITGB7/CFTR, PDGFRA/CFTR, PDGFRA/FLT4, PTK7/NPR1, PTK7/GRIN3B, and SERPINE1/KCND3. In the view of the clinical relevance of GRIN1 in HCC (i.e., the observation that high GRIN1 expression being correlated with poor prognosis), two of the eleven sgRNA combinations, GRIN1/FLT4 and GRIN1/FGFR3 were selected for further characterization. The genetic ablation of GRIN1/FLT4 and GRIN1/FGFR3, as well as treatment with their respective drug inhibitor combinations ifenprodil/sorafenib and ifenprodil/lenvatinib, markedly inhibited the proliferation and cell stemness in multiple HCC cell lines and patient-derived organoids. Co-treatment of ifenprodil and sorafenib suppressed the tumorigenicity and reduced cell-renewal ability of both MHCC97L- and patient- derived xenografts in mice. Notably, the anti-cancer effects were much less significant when either gene within the pair was knocked out or either inhibitor within the drug combination was applied. We further demonstrated that the combination of ifenprodil and sorafenib triggered the endoplasmic reticulum (ER) stress response and down-regulated the Wnt signaling pathway. Together, our study validated the CombiGEM approach for identifying potential therapeutic combinations against HCC. Furthermore, our findings reveal ifenprodil as a potential drug candidate in combination with sorafenib for enhancing the treatment efficacy of HCC.