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An Lkb1-Sik axis suppresses tumor growth and controls differentiation [Tumor Barcode Sequencing]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE133698 | GEO | 2019/07/03

REPOSITORIES: GEO

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Project description: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.

Project description: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 ascertain the tumor-suppressive capacity C/EBP family members, including Cebpa, Cebpb, and Cebpd, through CRISPR/Cas9-mediated targeting in 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 each of the three C/ebp paralogs individually, in pairwise combinations, and all three simultaneously. 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: The targeting of Cebpa significantly increased tumor size relative to tumors initiated with vectors encoding inert sgRNAs (oncogenic KRAS only tumors). The targeting of Cebpb and Cebpd had no significant impact on tumor growth. There were no additive effects of knocking out additional paralogs on top of Cebpa. Conclusions: C/EBPa is the dominant tumor-suppressive paralog and there was no evidence of functional redundancy among C/EBPa, C/EBPb, and C/EBPd.

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An Lkb1-Sik axis suppresses tumor growth and controls differentiation [Tumor Barcode Sequencing]

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