An RNA sequencing approach for isoform detection of DPEP1 in colorectal cancer and in vitro characterization of a novel isoform.

Abstract

e15734 Background: Microsatellite stable colorectal cancer (CRC) is historically poorly responsive to immunotherapy, due in part to low neoantigen burden, an immunosuppressive tumor microenvironment, and poor T-cell infiltration. Dipeptidase-1 (DPEP1) is a GPI-anchored protein involved in glutathione and leukotriene metabolism and was identified as a part of a 4-gene immune cell exclusion signature associated with worse overall and progression free survival. Here we investigated the incidence and distribution of DPEP1 isoforms in data from The Cancer Genome Atlas (TCGA) and the in vitro properties of both isoforms. Methods: To determine the prevalence of DPEP1 Isoform B in human CRCs, we aligned TCGA Colon Adenocarcinoma (COAD) and Rectal Adenocarcinoma (READ) sample transcripts to the RefSeq (NCBI) genome database which has annotation of both DPEP1 isoforms. Murine CRC MC38 cells overexpressing human DPEP1 isoform A or isoform B were generated via lentiviral infection. The resulting cells underwent RNA sequencing followed by genome ontology and KEGG pathway over-representation analysis using the WebGestalt R package with FDR q-values ≤ 0.05. These DPEP1-expressing cells were injected into the tail veins of host mice to determine the effect of each isoform on metastasis, differences in means determined via Welch’s T-test. Results: Bulk RNA sequencing of 640 samples from 615 patients revealed DPEP1 A was found in 61% of CRC samples, while DPEP1 B was found in 91% of CRC samples, making DPEP1 B the predominant isoform. Furthermore, samples expressing high levels of DPEP1 B as compared to low or no expression, were associated with left-sided primary tumor location, MSS status, younger age at diagnosis, and no prior history of colon polyps. Transcriptional profiling revealed three distinct gene expression clusters that were correlated with DPEP1 isoform expression. Cluster one was associated with low to no DPEP1 A but high DPEP1 B. This cluster was enriched for non-coding RNAs and alternative spicing genes including RUNX1T1 and the small nuclear spliceosome RNAs. Cluster two was associated with high levels of both DPEP1 A and B expression. This was enriched for multiple G2/M cell cycle and Myc-associated genes. RNA sequencing revealed an upregulation in Myc-targets, EMT, and TNFα pathways in MC38 cells expressing isoform B. This expression of isoform B led to an increased metastatic burden in a mouse model compared to isoform A (p < 0.05). Conclusions: This marks the discovery of a novel isoform of DPEP1 that is upregulated in colorectal cancer patients. These data support the continued exploration of DPEP1 as a predictive biomarker for response to immune checkpoint inhibitors in MSS CRC as well as an emerging therapeutic target given its association with Myc and increased metastatic burden in preclinical models.