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The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most critical gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative experimental platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. The complex map of genotype-specific treatment responses uncovered that over 20% of possible interactions show significant resistance or sensitivity. Known and novel interactions were identified, and one of these interactions, the resistance of KEAP1-mutant lung tumors to platinum therapy, was validated using a large patient response data set. These results highlight the broad impact of tumor suppressor genotype on treatment responses and define a strategy to identify the determinants of precision therapies. Significance: An experimental and analytical framework to generate in vivo pharmacogenomic maps that relate tumor genotypes to therapeutic responses reveals a surprisingly complex map of genotype-specific resistance and sensitivity.

The lack of knowledge about the relationship between tumor genotypes and therapeutic responses remains one of the most critical gaps in enabling the effective use of cancer therapies. Here, we couple a multiplexed and quantitative experimental platform with robust statistical methods to enable pharmacogenomic mapping of lung cancer treatment responses in vivo. The complex map of genotype-specific treatment responses uncovered that over 20% of possible interactions show significant resistance or sensitivity. Known and novel interactions were identified, and one of these interactions, the resistance of KEAP1-mutant lung tumors to platinum therapy, was validated using a large patient response data set. These results highlight the broad impact of tumor suppressor genotype on treatment responses and define a strategy to identify the determinants of precision therapies. SIGNIFICANCE: An experimental and analytical framework to generate in vivo pharmacogenomic maps that relate tumor genotypes to therapeutic responses reveals a surprisingly complex map of genotype-specific resistance and sensitivity.

The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.

Cancer growth is a multi-stage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remains largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor suppressor genes. Here, we present a method that integrates tumor barcoding with ultra-deep barcode sequencing (Tuba-seq) to interrogate tumor suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes with great precision. By combining Tuba-seq with multiplexed CRISPR/Cas9-mediated genome editing, we quantified the effects of eleven tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. With unprecedented resolution, parallelization, and precision Tuba-seq enables broad quantification of tumor suppressor gene function.

The kinase LKB1 is a critical tumor suppressor in sporadic and familial human cancers, yet the mechanisms by which it suppresses tumor growth remain poorly understood. To investigate the tumor-suppressive capacity of four canonical families of LKB1 substrates in vivo, we used CRISPR/Cas9-mediated combinatorial genome editing in a mouse model of oncogenic KRAS-driven lung adenocarcinoma. We demonstrate that members of the SIK family are critical for constraining tumor development. Histologic and gene-expression similarities between LKB1- and SIK-deficient tumors suggest that SIKs and LKB1 operate within the same axis. Furthermore, a gene-expression signature reflecting SIK deficiency is enriched in LKB1-mutant human lung adenocarcinomas and is regulated by LKB1 in human cancer cell lines. Together, these findings reveal a key LKB1–SIK tumor-suppressive axis and underscore the need to redirect efforts to elucidate the mechanisms through which LKB1 mediates tumor suppression. Significance: Uncovering the effectors of frequently altered tumor suppressor genes is critical for understanding the fundamental driving forces of cancer growth. Our identification of the SIK family of kinases as effectors of LKB1-mediated tumor suppression will refocus future mechanistic studies and may lead to new avenues for genotype-specific therapeutic interventions. This article is highlighted in the In This Issue feature, p. 1469

Large-scale sequencing of human tumours has uncovered a vast array of genomic alterations. Genetically engineered mouse models recapitulate many features of human cancer and have been instrumental in assigning biological meaning to specific cancer-associated alterations. However, their time, cost and labour-intensive nature limits their broad utility; thus, the functional importance of the majority of genomic aberrations in cancer remains unknown. Recent advances have accelerated the functional interrogation of cancer-associated alterations within in vivo models. Specifically, the past few years have seen the emergence of CRISPR-Cas9-based strategies to rapidly generate increasingly complex somatic alterations and the development of multiplexed and quantitative approaches to ascertain gene function in vivo.

The Nkx2-1 transcription factor promotes differentiation of lung epithelial lineages and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium-binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic inactivation of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate that Selenbp1 is a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo. Implications: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome. Mol Cancer Res; 16(11); 1737–49. ©2018 AACR.

The originally published article failed to acknowledge the equal first authorship contribution of I. P. Winters and C. W. Murray. The article has now been corrected online. The editors apologize for this error.

Cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors. However, the functional importance of the vast majority of these alterations, both alone and in combination, remains unknown. I will describe methods that integrate tumor barcoding with CRISPR/Cas9-mediated genome editing and ultradeep barcode sequencing to interrogate multiple tumor genotypes simultaneously in autochthonous mouse models of human cancer. We initially used this method to quantify the effects of eleven of the most frequently inactivated genes in human lung adenocarcinoma on tumor growth in vivo. We also investigated the in vivo fitness advantage conferred by inactivation of each of these eleven genes in combination with inactivation of p53 or Lkb1. This map of tumor-suppressive effects for >30 common lung adenocarcinoma genotypes revealed a complex landscape of context dependence and variability in effect strength. I will also describe a platform that integrates multiplexed Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple oncogenic alterations in parallel. Using this approach, we introduced a library of nonsynonymous mutations into endogenous Kras in adult somatic cells to initiate tumors. High-throughput sequencing of barcoded Kras HDR alleles from bulk tumor-bearing lung uncovered surprising diversity in Kras variant oncogenicity. These in vivo approaches may redefine our ability to understand how diverse genomic alterations impact tumor initiation, growth, malignant transformation, and therapy responses. Citation Format: Ian P. Winters, Zoë N. Rogers, Christopher D. McFarland, Pranav V. Lalgudi, Shin-Heng Chiou, Mark A. Kay, Dmitri Petrov, Monte M. Winslow. Functional lung cancer genomics through in vivo genome editing [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA03.

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.