Abstract LB-195: Identifying cooperative effects of tumor suppressor gene mutations in lung adenocarcinoma using paired CRISPR-Cas9

Abstract

Abstract Cancer is a heterogeneous disease that arises from the accumulation of genetic mutations that lead to a loss of tumor suppressor genes and activation of oncogenes. Mutational inactivation of genes that regulate epigenetic mechanisms, such as post translational modifications to DNA and histones, also contribute to cancer progression but the mechanisms are poorly understood. Trimethylation on histone 3 lysine 36 (H3K36me3) is encoded by the histone lysine methyltransferase (SETD2). SETD2 is recurrently inactivated by missense and frameshift mutations in multiple tumor types. Previously, we identified that Setd2 loss has potent tumor promoting activities in a mouse model of oncogenic Kras-induced lung adenocarcinoma. However, whether Setd2 loss has cooperating effects with other tumor suppressor gene mutations remains unstudied. To simultaneously inactivate distinct tumor suppressor genes, we use a lentiviral-based CRISPR/Cas gene editing approach to express a combination of specific gRNA-pairs along with Cas9 and Cre recombinase. Inhalation of these lentiviral particles leads activation of an oncogenic KrasLSL-G12D allele and simultaneously inactivation two gRNA-targeted genes. We hypothesize that SETD2 will collaborate with other tumor suppressor gene mutations in lung adenocarcinoma. To clone lentivirus vectors that have two different gRNA we use oligonucleotides encoding gRNAs targeting Setd2 and either Lkb1, Apc, or Rb1 to PCR amplify the 3' region of the murine U6 (mU6) promoter and the 5' region of the gRNA scaffold. Overlapping sequences in the 148-bp PCR amplicon, facilitate recombination with a 415-bp donor fragment derived from BbsI-digested pDonor plasmid using the Gibson cloning method. The newly circularized fragment is then re-linearized via BbsI digestion and cloned into LentiCRISPRv2Cre vector through the Golden Gate method using BsmBI type IIs restriction endonuclease and T4 DNA ligase. Our data demonstrate the development and presence of dual gRNA nucleotides through NotI/EcoRI restriction enzymes digestion and DNA Sequencing. Moreover, to study the cooperative effects of these mutations in mice, we created viruses and corroborated through Flow cytometry and Fluorescence microscopy. In conclusion, we generate lentiviral particles expressing pairs of gRNAs targeting distinct tumor suppressor genes, to identify cooperative effects of their mutation in lung adenocarcinoma models. Citation Format: Mabel G. Perez-Oquendo, David M. Feldser, David Walter. Identifying cooperative effects of tumor suppressor gene mutations in lung adenocarcinoma using paired CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-195.