GSE133698
An Lkb1-Sik axis suppresses tumor growth and controls differentiation [Tumor Barcode Sequencing]
Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Despite being implicated in the regulation of a variety of cellular processes, the mechanisms by which LKB1 constrains lung tumor growth and progression remains an area of intense investigation. Purpose: To assess the impact of CRISPR/Cas9-mediated targeting of the canonical substrates of Lkb1 on tumor size within the context of a genetically engineered mouse model of oncogenic Kras-driven lung adenocarcinoma. Approach: Comparisons of tumor size across genetic perturbations were conducting by measuring tumor sizes via tumor barcode sequencing (Rogers, et al. 2017 Nature Methods). Briefly, tumors were initiated in KrasLSL-G12D/+;R26LSL-Tomato, KrasLSL-G12D/+;Lkb1flox/flox;R26LSL-Tomato;H11LSL-Cas9, and KrasLSL-G12D/+;R26LSL-Tomato;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that encode a cassette comprised of an sgRNA-specific ID along with clonal identifier. Following, tumor development/progression, the two-component lentiviral cassettes integrated within transduced populations were amplified from genomic DNA extracted from whole lungs homogenates and subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to translation to absolute numbers of cancer cells via normalization to spiked-in samples of known quantities of cancer cells. The resultant tumor size distribution were then analyzed by multiple statistical measures as described by Rogers, et al. Results: Lkb1 targeting dramatically increased tumor size in the Kras-only setting relative to functionally inert sgRNAs. Unlike other families of Lkb1 substrates, the targeting of the salt inducible kinase (Sik) family increased tumor growth comparable to Lkb1 targeting. In contrast to targeting the paralogs Sik1 and Sik3 individually, dual targeting of Sik1 and Sik3 enhanced tumor growth. Sik targeting in the Lkb1-deficient setting also modestly increased tumor size whereas Lkb1 targeting had no effect. Conclusions: Siks are potent tumor suppressors belonging the family of canonical Lkb1 substrates. Siks retain some tumor-suppressive activity in the absence of Lkb1. Overall design: 23 mice total. Eight replicates for KrasLSL-G12D/+;Lkb1flox/flox;R26LSL-Tomato;H11LSL-Cas9, 5 replicates for KrasLSL-G12D/+;R26LSL-Tomato, and ten KrasLSL-G12D/+;R26LSL-Tomato;H11LSL-Cas9.