Abstract 2575: Concurrent mutation of CDKN2A and TP53 loci in primary keratinocytes
Authors: Li Bin , Mroz Edmund A. , Rocco James W. 
Published: July 2017

Abstract
Concurrent inactivation of the p16Ink4a and p53 tumor suppressors, often through mutation of CDKN2A and TP53, is frequent in early development of head and neck squamous cell carcinoma (HNSCC) and other cancers. The role of this early combined inactivation, and the oncogenic drivers that subsequently lead to tumor development, are poorly defined. To characterize the precancerous role of concurrently mutated CDKN2A and TP53, we combined a number of recent advances to disrupt both loci in primary cells. First, we grew primary keratinocytes in medium conditioned by irradiated J2-3T3 cells and containing an inhibitor of ROCK kinases, conditions that greatly extend their normally limited proliferative potential. Second, we transfected primary keratinocytes with plasmid vectors expressing both Cas9 and sets of guide RNAs targeting specific regions of CDKN2A and TP53. We targeted either or both of the p14Arf and p16Ink4a protein products of CDKN2A for knockout (KO), together with either TP53 KO or a TP53 point mutation (R248Q protein product), two major categories of genetic alterations of p53 in HNSCC. Third, we evaluated use of nutlin-3, an inhibitor of p53-MDM2 interaction, to try to select against cells that still contained wild-type p53. We assessed KO efficiency by PCR of genomic DNA; the TP53 point mutation introduced a SauA3i site into TP53. Two weeks of nutlin-3 treatment led to a 3 fold increase in p53 KO efficiency versus untreated cells (50% versus 14%). p16Ink4a KO efficiencies were enhanced 2 to 14 fold after nutlin-3 treatment, depending on the specific Cas9/guide-RNA constructs used. In cells transfected to produce p53 R248Q, 14% of PCR products amplifying the mutated region after nutlin-3 exposure showed Sau3Ai digestion, whereas no Sau3Ai digestion was detected in untreated cells. This result suggests that up to 14% of nutlin-3 treated keratinocytes had integrated the mutated donor oligo sequence. We similarly enrich for mutated keratinocytes having p14Arf-specific or combined CDKN2A KO along with TP53 KO or mutation. Combined transfection to generate concurrent CDKN2A and TP53 mutations under conditions that allow extended proliferation in culture, followed by selection for successful TP53 mutation with nutlin-3, will facilitate identification of keratinocyte clones with bi-allelic knockout genotypes. This panel of keratinocytes with concurrent CDKN2A and TP53 mutations will help delineate the roles of these genes in early cancer development and will allow screening for the oncogenic drivers required for tumor development and metastasis, with emphasis on discovering more effective and specific druggable targets for improving treatment.
Citation Format: Bin Li, Edmund A. Mroz, James W. Rocco. Concurrent mutation of CDKN2A and TP53 loci in primary keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2575. doi:10.1158/1538-7445.AM2017-2575