Oncolytic reovirus (pelareorep) induces autophagy in KRAS-mutated colorectal cancer

Colorectal Cancer
10/11/2020

Clin Cancer Res. 2020 Nov 9:clincanres.2385.2020. doi: 10.1158/1078-0432.CCR-20-2385. Online ahead of print.

ABSTRACT

PURPOSE: To explore the effects of pelareorep on autophagy in multiple models of CRC, including patient derived PBMC.

EXPERIMENTAL DESIGN: HCT116 (KRAS-Mut) and Hke3 (KRAS-WT) cells were treated with pelareorep (5MOI) and harvested at 6 and 9 hours. LC3A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by western blot (WB). The expression of 88 autophagy-genes were determined by qPCR. Syngeneic mouse models; CT26/Balb-C (KRAS-mut) and MC38/C57B6 (KRAS-WT) were developed; and treated with pelareorep (10x106 PFU/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMC from 5 experimental and 3 control patients were sampled at 0 (pre) and 48 hours, days 8 and 15. The gene expression normalized to "Pre" was determined using 2-ΔΔCT method.

RESULTS: Pelareorep induced significant upregulation of LC3A/B in HCT116 as compared to Hke3 cells by immunofluorescence (3.24X and 8.67X); flow cytometry (2.37X and 2.58X); autophagosome formation (2.02X and 1.57X), at 6 and 9 hours, respectively; all p<0.05. WB analysis showed increase in LC3A/B (2.38X and 6.82X), Beclin1 (1.17X and 1.24X) at 6 and 9 hours; ATG5 (2.4X), P-62 (1.52X) at 6 hours; and VPS-34 (1.39X) at 9 hours (all p<0.05). Induction of 13 transcripts in cell lines (>4X; 6 and 9 hours; p<0.05), 12 transcripts in CT26 (qPCR), and 14 transcripts in human PBMC (p<0.05) was observed. LC3A/B, RICTOR and RASD1 expression was upregulated in all 3 model systems.

CONCLUSIONS: Pelareorep hijacks host autophagic machinery in KRAS-Mutant conditions to augment its propagation and preferential oncolysis of the cancer cells.