Oncolytic Reovirus (pelareorep) Induces Autophagy in KRAS-mutated Colorectal Cancer

Clin Cancer Res. 2021 Feb 1;27(3):865-876. doi: 10.1158/1078-0432.CCR-20-2385. Epub 2020 Nov 9.

Abstract

Purpose: To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs).

Experimental design: HCT116 [KRAS mutant (mut)] and Hke3 [KRAS wild-type (WT)] cells were treated with pelareorep (multiplicity of infection, 5) and harvested at 6 and 9 hours. LC3 A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by Western blotting. The expression of 88 autophagy genes was determined by qRT-PCR. Syngeneic mouse models, CT26/Balb-C (KRAS mut) and MC38/C57B6 (KRAS WT), were developed and treated with pelareorep (10 × 106 plaque-forming unit/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMCs from five experimental and three control patients were sampled at 0 (pre) and 48 hours, and on days 8 and 15. The gene expression normalized to "pre" was determined using 2-ΔΔC t method.

Results: Pelareorep induced significant upregulation of LC3 A/B in HCT116 as compared with Hke3 cells by immunofluorescence (3.24 × and 8.67 ×), flow cytometry (2.37 × and 2.58 ×), and autophagosome formation (2.02 × and 1.57 ×), at 6 and 9 hours, respectively; all P < 0.05. Western blot analysis showed an increase in LC3 A/B (2.38 × and 6.82 ×) and Beclin1 (1.17 × and 1.24 ×) at 6 and 9 hours, ATG5 (2.4 ×) and P-62 (1.52 ×) at 6 hours, and VPS-34 (1.39 ×) at 9 hours (all P < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; P < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs (P < 0.05) was observed. LC3 A/B, RICTOR, and RASD1 expression was upregulated in all three model systems.

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

Publication types

  • Clinical Trial, Phase I
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antineoplastic Combined Chemotherapy Protocols / administration & dosage
  • Autophagy / genetics
  • Autophagy / immunology*
  • Camptothecin / administration & dosage
  • Camptothecin / analogs & derivatives
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / immunology
  • Colorectal Neoplasms / therapy*
  • Combined Modality Therapy
  • Disease Models, Animal
  • Female
  • Fluorouracil / administration & dosage
  • Gene Expression Regulation, Neoplastic / immunology
  • HCT116 Cells
  • Humans
  • Infusions, Intravenous
  • Injections, Intralesional
  • Leucovorin / administration & dosage
  • Male
  • Mice
  • Microtubule-Associated Proteins / genetics
  • Middle Aged
  • Oncolytic Virotherapy / methods*
  • Oncolytic Viruses / immunology*
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • Rapamycin-Insensitive Companion of mTOR Protein / genetics
  • Up-Regulation
  • ras Proteins / genetics

Substances

  • KRAS protein, human
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • RASD1 protein, human
  • RICTOR protein, human
  • Rapamycin-Insensitive Companion of mTOR Protein
  • reolysin
  • Hras protein, mouse
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins
  • Leucovorin
  • Fluorouracil
  • Camptothecin

Supplementary concepts

  • IFL protocol