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−ΔΔCt 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.

Riluzole, a glutamate release inhibitor, has been in use for the treatment of amyotrophic lateral sclerosis for over two decades since its approval by the Food and Drug Administration. Recently, riluzole has been evaluated in cancer cells and indicated to block cell proliferation and/or induce cell death. Riluzole has been proven effective as an anti-neoplastic drug in cancers of various tissue origins, including the skin, breast, pancreas, colon, liver, bone, brain, lung and nasopharynx. While cancer cells expressing glutamate receptors frequently respond to riluzole treatment, numerous types of cancer cell lacking glutamate receptors unexpectedly responded to riluzole treatment as well. Riluzole was demonstrated to interfere with glutamate secretion, growth signaling pathways, Ca 2+ homeostasis, glutathione synthesis, reactive oxygen species generation and integrity of DNA, as well as autophagic and apoptotic pathways. Of note, riluzole is highly effective in inducing cell death in cisplatin-resistant lung cancer cells. Furthermore, riluzole pretreatment sensitizes glioma and melanoma to radiation therapy. In addition, in triple-negative breast cancer, colorectal cancer, melanoma and glioblastoma, riluzole has synergistic effects in combination with select drugs. In an effort to highlight the therapeutic potential of riluzole, the current study reviewed the effect and outcome of riluzole treatment on numerous cancer types investigated thus far. The mechanism of action and the various molecular pathways affected by riluzole are discussed. Contents 1. Introduction 2. Mechanisms of action of riluzole 3. Riluzole in cancer treatment 4. Conclusion

Patients with KRAS mutated colorectal cancer (CRC) represent a cohort with unmet medical needs, with limited options of FDA-approved therapies. Representing 40–45% of all CRC patients, they are considered ineligible to receive anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Although several mouse models of CRC have been developed during the past decade, one genetically resembling the KRAS mutated CRC is yet to be established. In this study C57 BL/6 mice with truncated adenomatous polyposis coli (APC) floxed allele was crossed with heterozygous KRAS floxed outbred mice to generate an APCf/f KRAS+/f mouse colony. In another set of breeding, APC floxed mice were crossed with CDX2-Cre-ERT2 mice and selected for APCf/f CDX2-Cre-ERT2 after the second round of inbreeding. The final model of the disease was generated by the cross of the two parental colonies and viable APC f/f KRAS +/f CDX2-Cre-ERT2 (KPC: APC) were genotyped and characterized. The model animals were tamoxifen (TAM) induced to generate tumors. Micro-positron emission tomography (PET) scan was used to detect and measure tumor volume and standard uptake value (SUV). Hematoxylin and eosin (H&E) staining was performed to establish neoplasm and immunohistochemistry (IHC) was performed to determine histological similarities with human FFPE biopsies. The MSI/microsatellite stable (MSS) status was determined. Finally, the tumors were extensively characterized at the molecular level to establish similarities with human CRC tumors. The model KPC: APC animals are conditional mutants that developed colonic tumors upon induction with tamoxifen in a dose-dependent manner. The tumors were confirmed to be malignant within four weeks of induction by H&E staining and higher radioactive [18F] fluoro-2-deoxyglucose (FDG) uptake (SUV) in micro-PET scan. Furthermore, the tumors histologically and molecularly resembled human colorectal carcinoma. Post tumor generation, the KPC: APC animals died of cachexia and rectal bleeding. Implications: This model is an excellent preclinical platform to molecularly characterize the KRAS mutated colorectal tumors and discern appropriate therapeutic strategies to improve disease management and overall survival.