The Mechanism Of KLB In FGF19-driven Hepatocellular Carcinoma

In China and other countries in the world,hepatocellular carcinoma(HCC)is one of the leading malignancies with the highest incidence and lethality.However,current therapeutic options for HCC treatment are limited and their efficacy is not satisfactory.It is thus particularly urgent to identify new therapeutic targets and develop new therapeutic strategies.The traditional methods to identify cancer therapeutic targets focus on oncogenes and tumor suppressor genes mutated in cancers.Oncogenes and tumor suppressor genes drive cancer initiation and development;meanwhile,they render the growth or survival of cancer cells selectively dependent on certain genes(selective essential genes).Identifying selective essential gene is a novel strategy to identify anti-cancer therapeutic targets.DepMap is a database established by Broad Institute and their collaborators to systematically analyze the gene dependence of cancer cell lines.By mining the DepMap database,we identified ?-klotho(KLB)as a selective essential gene in liver cancers.Previous studies have shown that under physiological conditions,KLB acts as the co-receptor of FGF19 to bind and activate the FGFR4 signaling pathway in hepatocytes to regulate bile acid synthesis and hepatocyte proliferation.When FGF19 is amplified or over-expressed in the liver,this pathway also promotes the initiation and development of liver cancer.Using in-vitro cell competitive growth and in-vivo xenograft experiments,we verified KLB as a selective vulnerability in FGF19-driven HCC.However,our results showed that loss-of-function of KLB has a greater impact on cell growth than FGFR4 inactivation.Combining biochemistry and genetics,we revealed that FGF19/KLB binds to and activates both FGFR4 and FGFR3;dual inactivation of FGFR3 and FGFR4 mimics the loss of KLB in suppressing FGF19 signaling and cancer cell growth.Therefore,targeting KLB is more efficacious than targeting FGFR4 for treating FGF19-driven HCC.Next,we identified the binding sites of FGF19 and FGFRs on KLB via structure modeling and protein mutagenesis.KLB with mutations at these sites cannot effectively form hetero ternary complex with FGF19 and FGFRs,thereby blocking FGF19 signaling and leading to the loss of cell viability.Overexpression of the extracellular D1-D3 domains of FGFR4 blocks FGF19 signaling pathway.Based on these results,targeting KLB by disrupting FGF19/KLB/FGFRs ternary complex can be a promising direction for treating FGF19-driven HCC.Numerous FGFR4-selective inhibitors are in various stages of drug development and some of them have shown certain efficacy in clinical trials.We found that FGFR4-specific inhibitor BLU-554 exert its anti-cancer activity in FGF19-driven HCC via three mechanisms:1)FGFR4 kinase activity inhibition;2)dominant negative effect of inactive FGFR4;3)dual inhibition of both FGFR3 and FGFR4.Combination with the pan-FGFR inhibitor BGJ-398 improves the sensitivity of liver cancer cells to BLU-554.These results indicate that inhibitors targeting both FGFR3 and FGFR4 will likely display improved efficacy in treating FGF19-driven HCC.Furthermore,we discovered that FGF19 neutralizing antibodies cannot disrupt the interaction between FGF19 and KLB,and thus they cannot effectively block FGF19 signaling pathway.In summary,in this study we identified KLB as a novel selective essential gene in HCC,and uncovered the distinct mechanisms of KLB action between HCC and normal hepatocytes.As the co-receptor of FGF19,KLB activates both FGFR3 and FGFR4 in FGF19-driven HCC.Therefore,strategies targeting KLB may bypass the redundancy of FGFRs to elicit improved therapeutic efficacy in treating FGF19-driven HCC.