Abstract 4270: Induction of autophagic machinery upon oncolytic reovirus treatment in colorectal cancer cells and animal models
Authors: Jiffry Jeeshan , Rao Devika , Thavornwatanayong Thongthai , Saytoo Durvanand , Augustine Titto , Fogel Elisha , Goel Sanjay , Maitra Radhashree 
Published: August 2019

Background: Oncolytic reovirus (REO), a promising player in cancer therapeutics targets transformed cells. The molecular mechanisms at play is yet to be defined. We hypothesize that reovirus implements its oncolytic properties by engaging the autophagic machinery. While autophagy inherently is a survival mechanism, it can also lead to programmed cell death if the autophagic machinery is unable to revive the cell. Based on KRAS status we analyzed the relation between REO infection and autophagy induction in colorectal cancer (CRC) cell lines and syngeneic mouse models.

Methods: Six CRC cell lines HCT116, SW620, SW837 (KRAS Mutant), and HKE3, CACO2, LIM1215 (KRAS WT) were infected at 5 MOI (multiplicity of infection) of REO and harvested at 3, 6, 9, 12, 24, 48 and 72hours for Fluorescence Assisted Cell Sorting (FACS) post FITC labelled LC3 antibody staining, western blotting (WB) and quantitative Polymerase Chain Reaction (qPCR). Allograft animal tissue CT26 (KRAS Mutant) and MC38 (KRAS WT) post REO (1x 106 virus particles daily intra-tumoral) treatment were analyzed by WB and qPCR.

Results: WB demonstrated increased LC3A/B expression in REO treated KRAS mutant cells as compared to the wild type at 6 hours (2.9 fold) and 12hrs (1.98 fold) (p&lt;0.05). FACS indicated FITC positive cells expressing LC3A/B were 1.81-fold more in REO treated KRAS mutant cells at 6 to 9 hrs as compared to KRAS wild type (p=0.037). Kinetics of LC3A and LC3B are well studied in autophagy and induction is marked by an increase in total protein with conversion from LC3A to LC3B. WB on protein isolated from CT26 mice indicated significant decrease in LC3A on treatment with REO (p=0.04). qPCR analysis of REO treated CT26 and MC38 indicated similar trend with significant decrease in LC3A in CT26 (0.069-fold, p=0.05) compared to MC38 (0.93-fold, p=0.92). Further, 12 genes from a panel of 88 autophagy related genes namely APOL8, ATG4B, ATG4C, ATG4D, ATG5, BCL2, EIF4EBP2, FKBP12, LAMP3, RAPTOR, SNX30, ULK3 showed significant reversal of expression (p&lt;0.05) between the two groups.

Discussion: Our preclinical models confirmed an overall induction in expression of LC3, a protein integral to the autophagic pathways, upon REO infection with significant upregulation under KRAS mutant condition. Further analysis of different genes involved in autophagy is underway to understand shifts in autophagy’s dual role at different time points and/or conditions when subject to REO exposure.

Citation Format: Jeeshan Jiffry, Devika Rao, Thongthai Thavornwatanayong, Durvanand Saytoo, Titto Augustine, Sanjay Goel, Radhashree Maitra. Induction of autophagic machinery upon oncolytic reovirus treatment in colorectal cancer cells and animal models [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 4270.