Vismodegib regulates microenvironment of basal cell carcinoma via BRD9-mediated Hedgehog and PD-L1 signaling

To investigate how vismodegib (Vis) influences the pathogenesis of basal cell carcinoma (BCC) via a chromatin remodeling factor, bromodomain containing protein 9 (BRD9), and to analyze the expression profile of BRD9 in BCC and its relationship with the immune checkpoint, programmed cell death-1 ligand 1 (PD-L1) and the Hedgehog (Hh) signaling pathway.

① A UVB-induced BCC model was established in SKH-1 hairless background Ptch1^+/-; LacZ reporter mice. Then the mice were treated with Vis, and those without treatment served as control. X-gal staining, immunohistochemistry (IHC) staining, immunofluorescence (IF) assay, and Western blotting were used to assess the expression and localization of BRD9 and PD-L1 in tumor tissues and to evaluate immune-cell infiltration. ② In vitro, mouse BCC cell line ASZ001 (ASZ cells) were treated with Vis or a BRD9 degrader (dBRD9), and BRD9-overexpressing cells were generated. Cell viability and the protein and mRNA levels of BRD9, PD-L1, Gli1, and cyclin D1 (Ccnd1) were measured. ChIP-qPCR was performed to examine BRD9 and H3 K27 ac enrichment at the PD-L1 promoter, including the promoter-proximal site (P1) and an upstream active segment (P2).

① At the tissue level, BRD9 was highly expressed in BCC, and co-localization of BRD9 and PD-L1 was observed within tumor regions, with evident immune-cell infiltration. Vis markedly suppressed UVB-induced BCC formation, reduced the probability of large-volume tumors (by probability-density analysis), decreased the X-gal-positive lesion area (P<0.000 1), down-regulated BRD9 (P=0.024 9), and attenuated immune-cell infiltration. ② At the cellular level, Vis treatment reduced cell viability and down-regulated BRD9, Gli1, and Ccnd1 in ASZ cells (P<0.000 1). dBRD9 inhibited ASZ cell viability in a dose-dependent manner and decreased PD-L1, Gli1, and Ccnd1 (P<0.000 1), whereas its overexpression increased the expression of these molecules (P<0.000 1). In ASZ cells, BRD9 and H3 K27 ac were enriched at the PD-L1 promoter P1/P2 regions. Treatment with dBRD9 or Vis reduced BRD9 and H3 K27 ac enrichment at P1/P2 regions (P<0.000 1).

In BCC, BRD9 maintains chromatin activation at the proximal PD-L1 promoter and modulates Hh/Gli1 signaling, thereby promoting immune evasion. Vis remodels the tumor immune microenvironment by inhibiting the Hh-BRD9-PD-L1 axis.