Abstract 4618: A novel Pik3ca-driven mouse model and syngeneic cancer cell line for the preclinical testing of targeted and immune therapies for anal squamous cell carcinoma (ASCC)
Authors: Guerra Glen R. , Roth Sara , Kong Joseph C. , Millen Rosemary M. , Liu David S. , Sampurno Shienny , Narasimhan Vignesh , Pham Toan D. , Montgomery Karen G. , Heriot Alexander G. , Ramsay Robert G. , Phillips Wayne A. 
Published: August 2019

Abstract
Although many patients with localised anal squamous cell carcinoma (ASCC) initially achieve a complete response with standard chemoradiotherapy, those with persistent, relapsed or metastatic disease (35% of all patients) have limited treatment options and poor outcomes. Recent genomic profiling studies have identified PIK3CA as the most frequently mutated gene in anal cancers. Amplification of the PIK3CA gene and mutations in other PI3K pathway genes, have also been detected providing a strong rationale for targeting the PI3K pathway in these tumours. Similarly, the finding that many ASCC tumours express immune checkpoint receptors including PD-L1, has focussed attention on the potential use of checkpoint blockade in anal cancer. However, the use of targeted and/or immune therapies in the management of ASCC has been hampered by a lack of representative preclinical models for in vitro and in vivo testing of potential new therapeutic approaches. We have used mice with a Cre recombinase (Cre)-conditional knock-in of the Pik3caH1047R mutation and deletion of PTEN, crossed with mice expressing a tamoxifen-inducible Cre under the control of the ubiquitin C promoter, to generate a novel model of ASCC. By applying 4-hydroxy-tamoxifen topically to the anal canal we simultaneously induce expression of Pik3caH1047R and deletion of PTEN specifically in the anal epithelium. This results in anal tumors within 3 weeks with 100% penetrance. As all mice are on a C57Bl/6 background we are able to transplant the tumors subcutaneously into wild type C57Bl/6 mice as a syngeneic graft. To improve the utility of the model, we used tumors from these mice to establish a syngeneic mouse ASCC cell line. This line was then transduced with a human papilloma virus 16 E6/7 lentivirus to recapitulate the human virally-driven form of the disease. The cell line expresses both the Pik3ca mutation and E6/7 oncogenes and is positive for the squamous markers p63 and CK5/6. It had a similar response to 5-fluorouracil, Mitomycin C and radiotherapy, as a panel of human ASCC cell lines (also derived in our laboratory) and was sensitive to a PI3K inhibitor (BYL719). It was tumorigenic in both immunocompetent C57Bl/6 and immunodeficient NSG mice, additionally developing lung metastases in the NSG mice. The syngeneic C57Bl/6 tumors induce a peri-tumoral infiltrate, consisting of a heavy myeloid population, with high PDL1 expression, and a T-cell population expressing PD1; similar to human patients with treatment resistant disease. A major barrier to identifying new treatment options and improving overall survival in anal cancer has been the lack of preclinical models. We have now generated and characterised a novel, relevant Pik3ca-driven mouse model and syngeneic cancer cell line that will facilitate the in vitro and in vivo preclinical testing of targeted and immune therapies for ASCC.
Citation Format: Glen R. Guerra, Sara Roth, Joseph C. Kong, Rosemary M. Millen, David S. Liu, Shienny Sampurno, Vignesh Narasimhan, Toan D. Pham, Karen G. Montgomery, Alexander G. Heriot, Robert G. Ramsay, Wayne A. Phillips. A novel Pik3ca-driven mouse model and syngeneic cancer cell line for the preclinical testing of targeted and immune therapies for anal squamous cell carcinoma (ASCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4618.