Abstract 1325: Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency
Authors: Deycmar Simon , Johnson Brendan , Ryan Declan , Sills Shane , Caudell David , Dugan Greg , Schaaf George , Whitlow Christopher , Sai Kiran Kumar Solingapuram , Ferguson Betsy , Bimber Benjamin , Ray Karina , Cullin Cassandra , Dozier Brandy , Burgos Armando , Hettich Michael , Gomes Bruno , Charo Jehad , Ceppi Maurizio , Cline Mark 
Published: June 2022

Background Non-human primates (NHP) such as rhesus macaques with naturally occurring cancers are a proposed model for translational cancer immunotherapy (CIT) research and have generated relevant proof-of-mechanism evidence for 3 different CIT agents. NHP spontaneously develop cancers with progression patterns, histology, and clinical symptoms similar to humans. Gene suppression by DNA hypermethylation in the promoter region is the major characteristics of the CpG-island-methylator-phenotype (CIMP) described in human CRC patients but information in rhesus macaques is scarce. To further validate these animals as translational models for CIT, we conducted a deep molecular characterization of NHP colorectal cancers and established novel qPCR panels to assess DNA methylation of marker genes published for humans.

Methods Our cohort (n=16) consisted of Indian-origin rhesus macaques (Macaca mulatta) with naturally occurring CRC (n=16, female=11). Clinical examination, imaging (contrast-enhanced CT, FDG-PET) and biopsy to confirm cancer histology were performed. Molecular characterization was done by IHC for CRC-associated mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 and by PCR/electrophoresis for microsatellite instability. Ultimately, we designed DNA methylation- and rhesus-specific qPCR probes (Methylight) targeting corresponding regions as published in human patients, including CACNA1G, CDKN2A, CRABP1, MLH1, and NEUROG1 as parts of the CIMP panel and BMP3, NDRG4, and SEPTIN9 as probed for human CRC-screening.

Results MLH1 deficiency by IHC, in conjunction with PMS2 absence, is observed in all NHP CRC cases, clearly exceeding frequencies reported in human CRCs (ranging from 2-15%). Moreover, we have documented microsatellite instable cases in some NHP CRCs, analog to human CRCs. DNA methylation of the MLH1 promoter region was significantly elevated in CRCs (100% of CRCs &gt;2-fold, p&lt;0.0001) compared to healthy colon. We hypothesized that this elevation would suppress MLH1 mRNA expression. This hypothesis of epigenetic suppression is corroborated by both qPCR and RNA sequencing which demonstrate significantly downregulated levels of MLH1 mRNA. DNA methylation of the other markers is less consistent but revealed CIMP positive and CIMP negative cases in our NHP CRC cohort.

Conclusions Transcriptional suppression of MLH1 by promoter hypermethylation is a major and widespread driver of genetic instability and carcinogenesis in rhesus macaque colorectal cancer. Differential DNA methylation in the promoter regions as observed in NHP CRCs can provide a screening target for liquid biopsies. This work highlights the possible translatability of naturally occurring NHP cancers for human cancer immunotherapy research and can be further explored in future tumor-bearing monkey trials.

Citation Format: Simon Deycmar, Brendan Johnson, Declan Ryan, Shane Sills, David Caudell, Greg Dugan, George Schaaf, Christopher Whitlow, Kiran Kumar Solingapuram Sai, Betsy Ferguson, Benjamin Bimber, Karina Ray, Cassandra Cullin, Brandy Dozier, Armando Burgos, Michael Hettich, Bruno Gomes, Jehad Charo, Maurizio Ceppi, Mark Cline. Naturally occurring colorectal cancer in nonhuman primates used to study human immunotherapeutic agents confirms a link between DNA methylation and mismatch repair deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1325.