Background Immunotherapy resistance has been correlated with epithelial-to-mesenchymal transition (EMT),1 2 however our understanding of tumor-intrinsic mechanisms driving this immune evasive phenotype is lacking. We have previously shown that Wnt ligands are upregulated in anti-PD-1 resistant melanomas,3 and postulated that upstream transcriptional regulation of select EMT pathways may underpin these findings. The hedgehog signaling (HH) transcription factor Gli2 promotes EMT.
Methods Gli2 was constitutively activated (Gli2CA ) in a BRAFV600EPTEN-/- murine cell line via an N-terminal truncating mutation and silenced using CRISPR-Cas9. Multi-parameter flow cytometry and RNAseq was utilized to evaluate the impact of Gli2 on the tumor immune microenvironment. Anti-PD-1 resistance studies were performed in Gli2CA and control tumors. Bioinformatics studies were conducted using the melanoma TCGA and Hugo et al databases.2
Results We found upregulation of Gli2 targets in patients with anti-PD-1-refractory metastatic melanoma as well as in an autochthonous BRAFV600EPTEN-/- melanoma model after escape from anti-PD-1. RNAseq and Western blot studies demonstrated Gli2CA to promote EMT and Wnt ligand production in addition to upregulated COX2 in BRAFV600EPTEN-/- melanoma. This finding was reversed by genetic ablation and pharmacologic inhibition of Gli2, implicating a previously undescribed role for Gli2 in modulating COX2. These data were consistent with a notable correlation between a Gli2 signature and a prostaglandin synthesis signature in human melanoma TCGA database. Flow cytometry analysis showed exclusion of cytolytic T and NK cells, a shift from cDC1s to cDC2s, and enhanced MDSC recruitment in Gli2CA tumors. Consistent with these findings, whole tumor RNAseq of Gli2CA tumors demonstrated a decrease in Cd3e, Prf1, and Xcr1 with a concomitant increase in Cxcl1, Cxcl2, Ccl2, Ptgs2, and Arg1 relative to control tumors. RNAseq of FACS-sorted DCs from Gli2CA tumors demonstrated a loss of cDC1-associated genes including Xcr1, Wdfy4, and Clec9a compared to DCs derived from control tumors. In-line with our previous results showing that Wnt5a promotes MDSC recruitment in a Yap-dependent manner,4 we found that Yap inhibition or Wnt5a deletion in the BRAFV600EPTEN-/-Gli2CA cell line diminished MDSC-recruiting chemokines. Further consistent with these findings, Gli2CA tumors resist anti-PD-1 antibody therapy.
Conclusions Our data demonstrates that the HH transcription factor Gli2 drives the development of a tolerogenic tumor microenvironment unfavorable to anti-PD-1 immunotherapy by coordinating the upregulation of Wnt ligand expression and prostaglandin synthesis (figure 1). We propose that HH gene signatures are worthy of further study as a guide for selecting Wnt ligand and prostaglandin inhibitors in future immunotherapy studies.
Acknowledgements The authors would like to acknowledge the Duke Cancer Institute Flow Cytometry Core.
Bagaev A, et al. Conserved pan-cancer microenvironment subtypes predict response to immunotherapy. Cancer Cell 2021.
Hugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 2016;165(1):35–44.
DeVito NC, et al. Pharmacological Wnt ligand inhibition overcomes key tumor-mediated resistance pathways to anti-PD-1 immunotherapy. Cell Rep 2021;35(5):109071.
Theivanthiran B, et al. A tumor-intrinsic PD-L1/NLRP3 inflammasome signaling pathway drives resistance to anti-PD-1 immunotherapy. J Clin Invest 2020;130(5):2570–2586.