SRA

Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Inactivation of Lkb1 accelerates the growth and progression of oncogenic KRAS-driven lung tumors in mouse models. However, the molecular mechanisms by which LKB1 constrains lung tumorigenesis and whether the aggressive cancer state that stems from Lkb1 deficiency can be reverted remains unknown. Purpose: To assess the impact of CRISPR/Cas9-mediated targeting of a subset of LKB1-dependent genes and C/EBP factors on tumor size in the context of a genetically engineered mouse model of oncogenic KRAS-driven lung adenocarcinoma. Approach: Measurements of tumor size across genetic perturbations were obtained by tumor barcode sequencing (Tuba-seq; Rogers et al., 2017 Nature Methods). Briefly, lung tumors were initiated in KrasLSL-G12D/+;R26LSL-tdTomato (KT) and KT;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that are each modified with two-component barcodes composed of sgRNA-specific and clonal identifiers. This particular pool of Lenti-sgRNA/Cre vectors was composed of vectors targeting a series of LKB1-dependent genes as well a subset of C/EBP transciription factors. Following tumor development, the two-component lentiviral barcodes integrated within transduced populations were amplified from genomic DNA of whole-lung homogenates. The resulting amplicons were then subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to conversion to absolute numbers of neoplastic cells via normalization to spiked-in samples of known quantities of barcoded neoplastic cells. The resulting tumor size distributions were then analyzed by multiple statistical measures as described previously (Rogers et al., 2017 Nature Methods). Results: Targeting of Lkb1 dramatically increased lung tumor size relative to inert sgRNAs. Little to no impact on tumor size was observed upon targeting LKB1-dependent genes. However, simultaneous targeting of Cebpa/b/d resulted in a significant increase in tumor size. Conclusions: A subset of C/EBP transcription factors constrain oncogenic KRAS-driven lung tumor growth. Overall design: Lung tumors were initiated in 15 KrasLSL-G12D/+;R26LSL-tdTomato;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors targeting a series of LKB1-dependent genes, C/EBP family members Cebpa, Cebpb, and Cebpd in addition to five inert sgRNAs (to generate tumors driven by oncogenic KRAS only). Nine KrasLSL-G12D/+;R26LSL-tdTomato mice (lacking Cas9) were also transduced with the same lentiviral pool to control for differences in virus titer. Lentiviral vectors were diversified with two-component barcodes composed of sgRNA-specific and clonal identifiers to uniquely tag each tumor and label with a genotype-specific identifier. The size of each clonal outgrowth was determined by deep sequencing of amplicons derived from the lentiviral barcodes integrated within the genomic DNA of tumor-bearing lungs.