Abstract 2152: Oncolytic virus (RT3D) administration in combination with cetuximab in head and neck squamous cell cancer (HNSCC) models harboring active EGFR/RAS/PI3K signaling
Authors: Psyrri Amanda , Economopoulou Panagiota , Kotsantis Ioannis , Ramfidis Vassilios , Vagia Elena , Koutsontodis George , Sasaki Clarence , Rampias Theodoros 
Published: August 2015

Abstract
Introduction: Reovirus type 3 Dearing (RT3D) exerts selective toxicity against cells with an activated MAPK pathway. In reovirus-resistant cells, viral protein transcripts phosphorylate and activate the double-stranded RNA activated protein kinase (PKR) which subsequently phosphorylates the translation initiation factor (elF-2) which in turn inhibits viral gene translation. We sought to study the proliferation and oncolytic function of RT3D in HNSCC cells in relation to constitutively active RAS/PI3K pathway. We also sought to determine whether the combination of cetuximab(C) treatment and RT3D infection in HNSCC cells and animal models bearing constitutively active EGFR/RAS/PI3K pathway is more efficient compared to treatment only with C.
Methods: We used a eEF1a1 KrasG12D transgenic mouse to test the therapeutic effect of combination of C treatment and RT3D infection in a mouse model of ras-driven oral SCC. Mice tumors were either treated with C or RT3D alone, or with a combination of RT3D and C. Tumor proliferation and apoptosis were measured by Ki67 and caspase 5,9 immunostaining before and after treatment.
We also infected C resistant BB49 (HRAS mutant) and CAL33 (PIK3CA mutant) cell lines with RT3D and we compared the growth rates of RT3D infected and non-infected cells using MTT viability assay. We subsequently silenced HRAS expression using a lentivirus expressing shRNA and we compared the growth rates of parental and HRAS depleted cells that were either RT3D- infected or C treated. Protein extracts were subjected to immunobloting for elF2,p-elF2, PKR, p-PKR.
Results: Combinatorial C and RT3D treatment decreased proliferation and enhanced apoptosis more efficiently compared to C or RT3D monotherapy in oral cancers that were developed in eEF1a1 KrasG12D transgenic mice.
Our experiments also showed that RT3D infection suppressed the growth of CAL33 and BB49 cells more efficiently, compared to C treatment. Interestingly, RT3D infection also exerted an equipotent antiproliferative effect on HRAS silenced BB49 and Cal-33 cells. Immunoblot analysis revealed that PKR does not phosphorylate elF2 in HRAS depleted BB49 and CAL33 cells, upon RT3D infection providing evidence that reovirus proliferation is not exclusively dependent on HRAS signaling in HNSCC cells.
Conclusions: RT3D infection exhibits a strong oncolytic effect in C resistant HNSCC with activated EGFR/RAS/MAPK signaling. Our findings provide evidence that RAS independent molecular mechanisms can also support the RT3D proliferation in this subset of HNSCC. Interestingly, combinatorial C and RT3D treatment was more effective compared to C or RT3D monotherapy in oral cancers developed in eEF1a1 KrasG12D transgenic mice, possibly due to the inhibitory effect of C in EGFR-dependent signaling pathways that promote resistance to RT3D infection and proliferation.
Citation Format: Amanda Psyrri, Panagiota Economopoulou, Ioannis Kotsantis, Vassilios Ramfidis, Elena Vagia, George Koutsontodis, Clarence Sasaki, Theodoros Rampias. Oncolytic virus (RT3D) administration in combination with cetuximab in head and neck squamous cell cancer (HNSCC) models harboring active EGFR/RAS/PI3K signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2152. doi:10.1158/1538-7445.AM2015-2152