Distinct molecular pathways regulated by activated AKT and YAP signaling during intrahepatic cholangiocarcinoma progression.

Activated AKT and YAP signaling have been implicated in the pathogenesis of intrahepatic cholangiocarcinoma (iCCA), but their specific roles in tumor progression and regulation of the tumor microenvironment remain unclear. We developed two doxycycline-inducible iCCA mouse models, Akt/TRE-YAP and TRE-Akt/YAP, allowing selective inhibition of YAP or AKT in established tumors upon doxycycline withdrawal. Histological and molecular analyses were then performed to assess phenotypic changes and pathway regulation. YAP suppression in the Akt/TRE-YAP model initially induced tumor regression but ultimately led to transformation into steatosis-associated HCC due to persistent AKT signaling. Conversely, AKT suppression in the TRE-Akt/YAP model induced more profound iCCA regression with minimal tumor burden. Mechanistically, both AKT and YAP regulate tumor cell proliferation, while RNF125 acts as a tumor suppressor inhibited by YAP during iCCA pathogenesis. AKT uniquely regulates tumor metabolic pathways, whereas YAP controls iCCA differentiation and the immune microenvironment. YAP inhibition depleted neutrophils and increased CD4+ and CD8+ T-cell infiltration. The immunosuppressive role of YAP was confirmed by ectopic YAP activation in mouse iCCA and validated in human iCCA, where YAP/TAZ activation correlated with immunosuppressive features. Although YAP inhibition increased T-cell infiltration, these T cells expressed PD-1. Combined YAP suppression and anti-PD-L1 treatment further enhanced tumor regression. We define distinct molecular roles for AKT and YAP in iCCA progression and identify YAP as a key regulator of the tumor immune microenvironment. These findings support combining YAP inhibition with immune checkpoint blockade for iCCA treatment. Our studies elucidate the distinct contributions of AKT and YAP signaling to intrahepatic cholangiocarcinoma (iCCA) progression. Using innovative doxycycline-inducible models, we show that both pathways regulate tumor cell proliferation - YAP by repressing the tumor suppressor RNF125 - while AKT primarily governs metabolism and YAP controls differentiation and suppresses the immune microenvironment. Importantly, YAP inhibition reshapes the tumor immune landscape, and its combination with PD-L1 blockade induces profound tumor regression. These findings establish YAP as a key regulator of the iCCA immune microenvironment and provide a strong preclinical rationale for combining YAP pathway inhibition with immune checkpoint blockade in iCCA therapy.