Dissecting a novel tumor-promoting axis in cholangiocarcinoma

There is an urgent need to identify effective treatments for intrahepatic cholangiocarcinoma (CCA), a highly desmoplastic tumor of the bile ducts with a five-year survival rate of 5%. Over half of iCCA cases are diagnosed after metastases are present, when treatment choices are limited and minimally effective. The long-term goal of our work is to identify novel therapeutic interventions to improve outcomes for this devastating malignancy. This proposal leverages our exciting preliminary data suggesting that the immune checkpoint protein B7-H4 is over- expressed in more than 60% of iCCA cases where, in addition to its known T cell immunosuppressive role mediated by its membrane-bound form, it exerts a critical role in the control of tumor growth and stromal deposition through a previously unknown cooperation of its intracellular form with the TGFβ pathway. Genetic ablation of B7-H4 in murine models generated by the co-expression of Notch1 intracellular domain and activated Akt1 (Nicd1/Akt1) using sleeping beauty transposon/transposase and hydrodynamic tail vein injection (HTVI) significantly prolongs survival and profoundly alters the surrounding desmoplasia. Mechanistically, B7-H4 over- expression increases the levels of the docking receptor for latent-TGβ1, GARP, and thus promotes the release of active TGFβ1 and activation of the downstream SMAD2/3-dependent pathway. We herein propose three aims to validate our hypothesis that, through activation of the TGFβ pathway, B7-H4 shapes a unique desmoplastic response and promotes tumor growth of iCCA, so that pharmacologic inhibition of this cross-talk will contribute to tumor inhibition through several complementary ways. In Aim 1, we will dissect the molecular mechanisms through which B7-H4 promotes cancer cell growth and motility by performing in vitro functional studies in iCCA cell lines (murine and human) and patient-derived organoids upon genetic manipulation of the B7-H4/TGFβ axis. Furthermore, we will unveil the full interactome of intracellular B7-H4 and its exact subcellular localization. In Aim 2, we will clarify how the newly identified axis shapes the desmoplastic reaction, immune infiltration and composition, and instructs other cell types, particularly cancer-associated fibroblasts, to deposit and organize collagen, in the genetically versatile HTVI-based Nicd1/Akt1 mouse model, which - as supported by our preliminary data - highly resembles human iCCA with endogenous expression of B7-H4. In Aim 3, we will assess the efficacy of pharmacologically modulating the B7-H4 axis using clinically-relevant drugs blocking TGFβ1 and other identified vulnerabilities in combination with a monoclonal antibody directed against B7-H4 in genetic and orthotopic murine models . Here, by combining our large collection of cell lines/organoids, human samples, mouse models with single cell-based approaches and a single-fiber artificial intelligence-based platform, we will dissect a previously unknown B7-H4/TGFβ cross-talk and yield new insights into iCCA biology. The proposed research is highly significant since its successful completion would establish the B7-H4 axis as a novel target for iCCA therapy that could improve the clinical outcome of this deadly cancer.