A Network Landscape of HPVOPC Reveals Methylation Alterations as Significant Drivers of Gene Expression via an Immune-Mediated GPCR Signal

SIMPLE SUMMARY: In this manuscript, we demonstrated a particular chemokine axis (CXCR3/CXCL9,10,11) to be highly expressed in HPV-associated oropharynx carcinoma (HPVOPC). This classically paracrine chemokine axis is thought to play an anti-tumor role through immune surveillance and tumor suppression. More recently, however, the same axis has also been shown to have paradoxically a pro-tumor effect, whereby in an autocrine signaling fashion it increases tumor cell proliferation, angiogenesis, and metastasis in colorectal, breast, cervical, and gastric cancers, but this is the first time in HPVOPC. An athymic murine model demonstrates that CXCR3 antagonism does indeed inhibit tumor growth; however, CXCR3 antagonism in immunocompetent animals shows mixed results. The CXCR3 axis is implicated as a possible driver of malignancy in HPVOPC via tumor growth stimulation, though caution is warranted as inhibition of this axis may also adversely impact immunosurveillance. Further work is indicated to investigate opportunities for targeted therapy. ABSTRACT: HPV-associated oropharynx carcinoma (HPVOPC) tumors have a relatively low mutational burden. Elucidating the relative contributions of other tumor alterations, such as DNA methylation alterations, alternative splicing events (ASE), and copy number variation (CNV), could provide a deeper understanding of carcinogenesis drivers in this disease. We applied network propagation analysis to multiple classes of tumor alterations in a discovery cohort of 46 primary HPVOPC tumors and 25 cancer-unaffected controls and validated our findings with TCGA data. We identified significant overlap between differential gene expression networks and all alteration classes, and this association was highest for methylation and lowest for CNV. Significant overlap was seen for gene clusters of G protein-coupled receptor (GPCR) pathways. HPV16–human protein interaction analysis identified an enriched cluster defined by an immune-mediated GPCR signal, including CXCR3 cytokines CXCL9, CXCL10, and CXCL11. CXCR3 was found to be expressed in primary HPVOPC, and scRNA-seq analysis demonstrated CXCR3 ligands to be highly expressed in M2 macrophages. In vivo models demonstrated decreased tumor growth with antagonism of the CXCR3 receptor in immunodeficient but not immunocompetent mice, suggesting that the CXCR3 axis can drive tumor proliferation in an autocrine fashion, but the effect is tempered by an intact immune system. In conclusion, methylation, ASE, and SNV alterations are highly associated with network gene expression changes in HPVOPC, suggesting that ASE and methylation alterations have an important role in driving the oncogenic phenotype. Network analysis identifies GPCR networks, specifically the CXCR3 chemokine axis, as modulators of tumor–immune interactions that may have proliferative effects on primary tumors as well as a role for immunosurveillance; however, CXCR3 inhibition should be used with caution, as these agents may both inhibit and stimulate tumor growth considering the competing effects of this cytokine axis. Further investigation is needed to explore opportunities for targeted therapy in this setting.