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Abstract
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide with poor prognosis and limited therapeutic options. Wnt/b-catenin signaling is active in ~50% of all HCC, making this pathway a prime target for precision-medicine therapeutics. In this thesis, I investigated the relevance of RNAi-mediated b-catenin inhibition in CTNNB1-mutated HCC models, and addressed underlying mechanisms of response. These studies utilized a novel siRNA that targets CTNNB1, with high mouse and human specificity, encapsulated in a lipid nanoparticle (referred henceforth as LNP-CTNNB1). Through in vivo models coupled with single-cell and spatial transcriptomics, I showed that LNP-CTNNB1 treatment reprograms both tumor metabolism and immunity to promote anti-HCC effect. Early-stage disease LNP-CTNNB1 treatment resulted in significant and durable tumor responses in multiple immunocompetent CTNNB1-mutated HCC mouse models. Response included zonal metabolic rewiring and restoration of global immune surveillance facilitated by re-engaged interferon (IFN) signaling. Through unbiased transcriptomic analysis, I revealed that re-engaged IFN signaling was mediated via activation of an immune regulatory module of transcription factors, including IRF2 and POU2F1, which are active when mutant-β-catenin is absent from the nucleus. Re-expression of either IRF2 or POU2F1 in background of CTNNB1-mutated HCC mouse models delayed tumor development and restored adaptive immunity. Advanced-stage disease LNP-CTNNB1 treatment in the CTNNB1-mutated HCC mouse models resulted in heterogenous responses, yet in combination with a-PD-1, an immune checkpoint inhibitor, enhanced the overall response.
I also developed a mutated-β-catenin gene signature (MBGS), which accurately predicts CTNNB1 mutational status in HCC patients from whole and spatial transcriptomics. Through unbiased whole transcriptomics from multiple β-catenin-mutated and non-mutated HCC mouse models, I developed 10- and 13-gene MBGS panels with high sensitivity to detect CTNNB1-mutated HCC. I also demonstrated that MBGS defined parenchymal regions with strong Wnt/β-catenin activation, with spatial colocalization of MBGS and HCC immune signatures illustrating the immune excluded nature of CTNNB1-mutated HCC. Overall, this thesis presents new insights into the underlying tumor cell-intrinsic and -extrinsic biology of CTNNB1-mutated HCC, highlights the relevance and translatability of RNAi-based therapeutics to target this unique subclass, and identifies a unique transcriptomic biomarker to stratify patients most likely to be susceptible to anti-b-catenin therapies.
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