Merkel cell carcinoma (MCC) is a lethal, virus-associated cancer that lacks effective therapies for advanced disease. Agents blocking the PD-1/PD-L1 pathway have demonstrated objective, durable tumor regressions in patients with advanced solid malignancies and efficacy has been linked to PD-L1 expression in the tumor microenvironment. To investigate whether MCC might be a target for PD-1/PD-L1 blockade, we examined MCC PD-L1 expression, its association with tumor-infiltrating lymphocytes (TILs), Merkel cell polyomavirus (MCPyV), and overall survival. Sixty-seven MCC specimens from 49 patients were assessed with immunohistochemistry for PD-L1 expression by tumor cells and TILs, and immune infiltrates were characterized phenotypically. Tumor cell and TIL PD-L1 expression were observed in 49% and 55% of patients, respectively. In specimens with PD-L1(+) tumor cells, 97% (28/29) demonstrated a geographic association with immune infiltrates. Among specimens with moderate-severe TIL intensities, 100% (29/29) demonstrated PD-L1 expression by tumor cells. Significant associations were also observed between the presence of MCPyV DNA, a brisk inflammatory response, and tumor cell PD-L1 expression: MCPyV(−) tumor cells were uniformly PD-L1(−). Taken together, these findings suggest that a local tumor-specific and potentially MCPyV-specific immune response drives tumor PD-L1 expression, similar to previous observations in melanoma and head and neck squamous cell carcinomas. In multivariate analyses, PD-L1(−) MCCs were independently associated with worse overall survival (hazard ratio 3.12; 95% CI, 1.28-7.61; p=0.012). These findings suggest that an endogenous immune response promotes PD-L1 expression in the MCC microenvironment when MCPyV is present, and provide a rationale for investigating therapies blocking PD-1/PD-L1 for patients with MCC.
The nuclear receptor CAR is a xenobiotic responsive transcription factor that plays a central role in the clearance of drugs and bilirubin while promoting cocaine and acetaminophen toxicity. In addition, CAR has established a "reverse" paradigm of nuclear receptor action where the receptor is active in the absence of ligand and inactive when bound to inverse agonists. We now report the crystal structure of murine CAR bound to the inverse agonist androstenol. Androstenol binds within the ligand binding pocket, but unlike many nuclear receptor ligands, it makes no contacts with helix H12/AF2. The transition from constitutive to basal activity (androstenol bound) appears to be associated with a ligand-induced kink between helices H10 and H11. This disrupts the previously predicted salt bridge that locks H12 in the transcriptionally active conformation. This mechanism of inverse agonism is distinct from traditional nuclear receptor antagonists thereby offering a new approach to receptor modulation.
Summary
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339–345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.
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