Abstract Background: Nuclear receptor related 1 protein (NURR1/NR4A2) is a transcription factor that mediates numerous functions in differentiation/development, maintenance and metabolism. In cancer cells, NR4A2 promotes proliferation, migration, transformation and chemo-resistance in many cancers. Here, we explore the role of NR4A2 in head and neck squamous cell cancers (HNC) tumorigenesis and define its target genes. Methods: NR4A2 expression was determined by immunohistochemistry (IHC) and in publically available databases (Oncomine, TCGA). NR4A2 was knockedout/knockdown by CRISPR or shRNA. Coimmunoprepitation and reverse chromatin immunoprecipitation were done to study the interaction between EGFR/STAT3. ChIPSeq analysis was done by using Illumina NextSeq 500 Genome Analyzer and integrated with microarray (HPV+/- patients) to identify the true target genes of NR4A2 using BETA Software and validated by PCR/qPCR. Results: IHC and database analysis demonstrated overexpression of NR4A2 in HNC patients. NR4A2 was significantly overexpressed in HPV+ HNC patients, SCC47 and SCC104 cell lines. Functionally, CRISPR/shRNA knockout/knockdown of NR4A2 in cell lines significantly decreased colonogenicity, proliferation and migration. Expression of NR4A2 was positively correlated with EGFR expression (R2=0.92), and EGF treatment (100ng/ml; 24-48h) induced NR4A2 (~2 fold) expression coupled with epithelial to mesenchymal transition in SCC1 and SCC10B cells (HPV-). We also observed EGF mediated NR4A2 induction was abrogated by panEGFR inhibitor (afatinib) with concomitant decrease in STAT3 expression. Physical interaction between EGFR and STAT3 with EGF treatment regulated NR4A2 expression. Whole genome integration of ChIPseq and microarray demonstrates 9% (2015) and 23% (3002) of the enriched up and down-regulated target genes, respectively were canonically regulated in HPV+ HNC. ChIP PCR validation indicates enrichment of NR4A2 in some of the target genes, wnt10, p53, cxcl13, cln10 and hoxb8 in HPV+ cells. Conclusions: Our data indicate overexpression of NR4A2 in HNC, and its positive correlation with EGFR expression. We also noted the physical interaction between EGFR and STAT3 which functionally attribute the NR4A2 expression. Integration and validation data showed the differential enrichment of NR4A2 target genes in HPV+/- cell lines which suggest its possible discriminatory role in HNC pathogenesis which needs to be confirmed. Citation Format: Sanjib Chaudhary, Ramesh Pothuraju, Pranita Atri, Satyanarayana Rachagani, Surinder K. Batra, Muzafar A. Macha. NR4A2 role in head and neck cancer: Mechanistic and functional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1909.