Abstract 1587: Integrated renal cancer post-GWAS functional studies implicates the SWI/SNF DPF3 gene on 14q24 risk locus
Authors: Leandro Machado Colli, Lea Jessop, Sabrina Y. Camp, Timothy Myers, Mitchell J. Machiela, Jiyeon Choi, Kai Yu, Mark Purdue, Kevin Brown, Stephen J. Chanock
Journal: Cancer Research (2019)

Abstract We investigated regulatory elements involved in20 renal cancer (RCC) susceptibility regions identified by Genome-wide Association Study (GWAS) by an integrated approach. In parallel, we conducted Massively Parallel Reporter Assay (MPRA), Assay for Transposase-Accessible Chromatin (ATAC-seq), RCC eQTL analysis, and Capture-HiC. We identified at least one functional variant in 18 of 20 RCC regions. Cis-eQTL linked 16 RCC regions to as many as 198 genes. The integrated approach underscores the spectrum of effects of genetic variation on regulation of genes critical for RCC. We further pursued the region on 14q24 and confirmed the tag SNP, rs4903064, by luciferase assay. Using an EMSA, the rs4903064 T-allele, associated with lower risk, showed higher binding than the C-risk allele. The T-allele creates a binding site for IRX2/IRX5, which can act as a transcriptional repressor. On the other hand, the C-allele creates a HIF1a binding site. The C-risk allele was associated with higher DPF3 expression, a 14q24 region gene, in the TCGA RCC and in normal kidney eQTL. We created four stable renal cell lines that included a DOX-dependency for overexpression of DPF3 isoforms (DPF3a or DPF3b) and performed RNA-seq analysis; we observed over 500 genes deregulated when either isoform is overexpressed. To investigate chromatin accessibility changes due to DPF3 overexpression, we used ATAC-seq and found 86 regions with altered accessibility and 15% of the RNA-seq deregulated genes are located within 2 MB of one of these regions, including the promoter of the top deregulated gene (CEMIP). In growth curve assays, DPF3 increased the growth rate of renal cell lines. When we knocked down the top two DPF3-deregulated genes (CEMIP and IL23R) using siRNA, DPF3’s growth effect was reduced, suggesting that CEMIP and IL23R are important cofactors for growth along with DPF3. Since CEMIP is important for apoptosis, we evaluated apoptosis in our dox-inducible-DPF3 cell lines. DPF3 overexpression decreased the percentage of apoptotic cells, measured by flow cytometry, and reduced Caspase-3 and PARP cleavage, measured by Western blot (WB). The second deregulated gene, IL23R, is connected to STAT3, an important RCC oncogene. By WB, DPF3 overexpression leads to increased STAT3 phosphorylation. Furthermore, we saw evidence of increased activation of the STAT3 signaling pathway by RNA-seq pathway analysis. Lastly, SWI/SNF complex genes have been shown to influence response to immunotherapy. Using co-culture of tumor cells and stimulated PBMCs, we found that DPF3 overexpression in RRC cells leads to an increased PBMC-cytotoxicity, due to an increased proportion of T CD8+ cells. In summary, we integrated RCC post-GWAS functional studies to pinpoint notable variants on 14q24 and suggest that altering DPF3 regulation can perturb the SWI/SNF complex by reduction of apoptosis and STAT3 pathway activation. Citation Format: Leandro Machado Colli, Lea Jessop, Sabrina Y. Camp, Timothy Myers, Mitchell J. Machiela, Jiyeon Choi, Kai Yu, Mark Purdue, Kevin Brown, Stephen J. Chanock. Integrated renal cancer post-GWAS functional studies implicates the SWI/SNF DPF3 gene on 14q24 risk locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1587.