Abstract 3788: Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer
Authors: Maitra Radhashree , Augustine Titto , Coffey Matt , Goel Sanjay 
Published: July 2016

Introduction

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for patients whose tumors harbor a mutation in the KRAS oncogene (40-45%). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a dsRNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. We hypothesize that effective expression of host Toll Like Receptors 3 (TLR3) will dampen the infection potential of reovirus propagation by mounting an innate immune response. Development of strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved virus productivity to specifically target the KRAS mutated cancer cells.

Methodology: TLR3 expressing HEK293 cells were treated with reovirus to confirm that TLR 3 is the host pattern recognition motif of the host cellular machinery that is responsible for the detection of dsRNA harboring reovirus. TLR 3 was next down regulated by shRNA technology in KRAS mutated HCT116 CRC cell line and treated with reovirus at dose of 5 MOI (multiplicity of infection) for 48 hours. The cell proliferation profile under this treatment condition was measured by MTT assay and corelated with the proliferation pattern of TLR3 expressing HCT116 cells undergoing similar treatment. Cytokine ELISA was performed for interferon (INF) alpha (α)and beta (β) to determine the downstream consequences of the gene silencing. Finally xenograft tumor models of athymic nude mice (Foxn1nu) were developed with TLR3 silenced HCT116 cells and treated with reovirus at a daily intra tumoral (IT) dose of 1×107 TCID (tissue culture infective dose) and compared to tumors generated by HCT116 cells receiving identical treatment.

Results

¬ TLR 3 is confirmed to be the host pattern recognition motif for dsRNA containing reovirus

¬ TLR3 which is constitutively expressed by colon epithelium is also a mediator of virus recognition in CRC cell line HCT116.

¬ Down regulation of TLR3 by shRNA technology enhances viral propagation as measured by MTT assay.

¬ Cytokine ELISA assay of INF α β distinctly reveals that downstream activation post reovirus infection is compromised.

¬ In xenograft models, those of TLR3 down regulated HCT116 cells, showed improved control of tumor growth with reovirus treatment as compared to those with TLR 3 expressing HCT 116 cells (p = 0.04)

Conclusion

TLR3 plays an important role in recognition of reovirus and mounting an innate immune response by inducing the secretion of type I INF. Mitigation of the TLR3 receptor mediated pattern recognition by shRNA down regulates host immune response and thus improves virus mediated cell cytotoxicity. The findings can be therapeutically utilized towards improved and beneficial killing of cancer cells by reovirus.

Citation Format: Radhashree Maitra, Titto Augustine, Matt Coffey, Sanjay Goel. Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3788.