Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma.

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with the inactivation of additional tumor suppressor genes. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations that drive the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Loss of Drosophila Myb interrupts the progression of chromosome condensation.

Completion of chromosome condensation is required before segregation during the mitotic cell cycle to ensure the transmission of genetic material with high fidelity in a timely fashion. In many eukaryotes this condensation is regulated by phosphorylation of histone H3 on Ser 10 (H3S10). This phosphorylation normally begins in the late-replicating pericentric heterochromatin and then spreads to the earlier replicating euchromatin. Here, we show that these phases of condensation are genetically separable in that the absence of Drosophila Myb causes cells to arrest with H3S10 phosphorylation of heterochromatin but not euchromatin. In addition, we used mosaic analysis to demonstrate that although the Myb protein can be removed in a single cell cycle, the failure of chromosome condensation occurs only after many cell divisions in the absence of Myb protein. The Myb protein is normally located in euchromatic but not heterochromatic regions of the nucleus, suggesting that Myb may be essential for a modification of euchromatin that is required for the efficient spread of chromosome condensation.

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex.

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics.

Combinatorial in vivo genome editing identifies widespread epistasis during lung tumorigenesis.

Lung adenocarcinoma, the most common subtype of lung cancer, is genomically complex, with tumors containing tens to hundreds of non-synonymous mutations. However, little is understood about how genes interact with each other to enable tumorigenesis in vivo , largely due to a lack of methods for investigating genetic interactions in a high-throughput and multiplexed manner. Here, we employed a novel platform to generate tumors with all pairwise inactivation of ten tumor suppressor genes within an autochthonous mouse model of oncogenic KRAS-driven lung cancer. By quantifying the fitness of tumors with every single and double mutant genotype, we show that most tumor suppressor genetic interactions exhibited negative epistasis, with diminishing returns on tumor fitness. In contrast, Apc inactivation showed positive epistasis with the inactivation of several other genes, including dramatically synergistic effects on tumor fitness in combination with Lkb1 or Nf1 inactivation. This approach has the potential to expand the scope of genetic interactions that may be functionally characterized in vivo , which could lead to a better understanding of how complex tumor genotypes impact each step of carcinogenesis.

Aging represses lung tumorigenesis and alters tumor suppression.

Most cancers are diagnosed in persons over the age of sixty, but little is known about how age impacts tumorigenesis. While aging is accompanied by mutation accumulation - widely understood to contribute to cancer risk - it is also associated with numerous other cellular and molecular changes likely to impact tumorigenesis. Moreover, cancer incidence decreases in the oldest part of the population, suggesting that very old age may reduce carcinogenesis. Here we show that aging represses tumor initiation and growth in genetically engineered mouse models of human lung cancer. Moreover, aging dampens the impact of inactivating many, but not all, tumor suppressor genes with the impact of inactivating PTEN, a negative regulator of the PI3K/AKT pathway, weakened to a disproportionate extent. Single-cell transcriptomic analysis revealed that neoplastic cells from tumors in old mice retain many age-related transcriptomic changes, showing that age has an enduring impact that persists through oncogenic transformation. Furthermore, the consequences of PTEN inactivation were strikingly age-dependent, with PTEN deficiency reducing signatures of aging in cancer cells and the tumor microenvironment. Our findings suggest that the relationship between age and lung cancer incidence may reflect an integration of the competing effects of driver mutation accumulation and tumor suppressive effects of aging.

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

Fully accessible fitness landscape of oncogene-negative lung adenocarcinoma.

Cancer genomes are almost invariably complex with genomic alterations cooperating during each step of carcinogenesis. In cancers that lack a single dominant oncogene mutation, cooperation between the inactivation of multiple tumor suppressor genes can drive tumor initiation and growth. Here, we shed light on how the sequential acquisition of genomic alterations generates oncogene-negative lung tumors. We couple tumor barcoding with combinatorial and multiplexed somatic genome editing to characterize the fitness landscapes of three tumor suppressor genes NF1, RASA1, and PTEN, the inactivation of which jointly drives oncogene-negative lung adenocarcinoma initiation and growth. The fitness landscape was surprisingly accessible, with each additional mutation leading to growth advantage. Furthermore, the fitness landscapes remained fully accessible across backgrounds with additional tumor suppressor mutations. These results suggest that while predicting cancer evolution will be challenging, acquiring the multiple alterations required for the growth of oncogene-negative tumors can be facilitated by the lack of constraints on mutational order.

Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth.

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.

A multiplexed in vivo approach to identify driver genes in small cell lung cancer.

Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

Combinatorial Inactivation of Tumor Suppressors Efficiently Initiates Lung Adenocarcinoma with Therapeutic Vulnerabilities.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma being the most common subtype. Many oncogenes and tumor suppressor genes are altered in this cancer type, and the discovery of oncogene mutations has led to the development of targeted therapies that have improved clinical outcomes. However, a large fraction of lung adenocarcinomas lacks mutations in known oncogenes, and the genesis and treatment of these oncogene-negative tumors remain enigmatic. Here, we perform iterative in vivo functional screens using quantitative autochthonous mouse model systems to uncover the genetic and biochemical changes that enable efficient lung tumor initiation in the absence of oncogene alterations. Generation of hundreds of diverse combinations of tumor suppressor alterations demonstrates that inactivation of suppressors of the RAS and PI3K pathways drives the development of oncogene-negative lung adenocarcinoma. Human genomic data and histology identified RAS/MAPK and PI3K pathway activation as a common feature of an event in oncogene-negative human lung adenocarcinomas. These Onc-negativeRAS/PI3K tumors and related cell lines are vulnerable to pharmacologic inhibition of these signaling axes. These results transform our understanding of this prevalent yet understudied subtype of lung adenocarcinoma. SIGNIFICANCE: To address the large fraction of lung adenocarcinomas lacking mutations in proto-oncogenes for which targeted therapies are unavailable, this work uncovers driver pathways of oncogene-negative lung adenocarcinomas and demonstrates their therapeutic vulnerabilities.

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.

Genetic Determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo.

In lung adenocarcinoma, oncogenic EGFR mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy in vivo remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven Trp53-deficient lung adenocarcinoma, we found that Apc, Rb1, or Rbm10 inactivation strongly promoted tumor growth. Unexpectedly, inactivation of Lkb1 or Setd2-the strongest drivers of growth in a KRAS-driven model-reduced EGFR-driven tumor growth. These results are consistent with mutational frequencies in human EGFR- and KRAS-driven lung adenocarcinomas. Furthermore, KEAP1 inactivation reduced the sensitivity of EGFR-driven tumors to the EGFR inhibitor osimertinib, and mutations in genes in the KEAP1 pathway were associated with decreased time on tyrosine kinase inhibitor treatment in patients. Our study highlights how the impact of genetic alterations differs across oncogenic contexts and that the fitness landscape shifts upon treatment. SIGNIFICANCE: By modeling complex genotypes in vivo, this study reveals key tumor suppressors that constrain the growth of EGFR-mutant tumors. Furthermore, we uncovered that KEAP1 inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease.This article is highlighted in the In This Issue feature, p. 1601.

A long lost key opens an ancient lock: Drosophila Myb causes a synthetic multivulval phenotype in nematodes.

The five-protein MuvB core complex is highly conserved in animals. This nuclear complex interacts with RB-family tumor suppressor proteins and E2F-DP transcription factors to form DREAM complexes that repress genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with Myb family oncoproteins to form the Myb-MuvB complexes that activate many of the same genes. We show that animal-type Myb genes are present in Bilateria, Cnidaria and Placozoa, the latter including the simplest known animal species. However, bilaterian nematode worms lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in Caenorhabditiselegans Here, we show that, despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9-LIN52 proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB-class genes including those that encode homologs of the MuvB core, RB, E2F and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt these activities in C. elegans We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.

An LKB1-SIK Axis Suppresses Lung Tumor Growth and Controls Differentiation.

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.

The Drosophila LIN54 homolog Mip120 controls two aspects of oogenesis.

The conserved multi-protein MuvB core associates with the Myb oncoproteins and with the RB-E2F-DP tumor suppressor proteins in complexes that regulate cell proliferation, differentiation, and apoptosis. Drosophila Mip120, a homolog of LIN54, is a sequence-specific DNA-binding protein within the MuvB core. A mutant of Drosophilamip120 was previously shown to cause female and male sterility. We now show that Mip120 regulates two different aspects of oogenesis. First, in the absence of the Mip120 protein, egg chambers arrest during the transition from stage 7 to 8 with a failure of the normal program of chromosomal dynamics in the ovarian nurse cells. Specifically, the decondensation, disassembly and dispersion of the endoreplicated polytene chromosomes fail to occur without Mip120. The conserved carboxy-terminal DNA-binding and protein-protein interaction domains of Mip120 are necessary but not sufficient for this process. Second, we show that a lack of Mip120 causes a dramatic increase in the expression of benign gonial cell neoplasm (bgcn), a gene that is normally expressed in only a small number of cells within the ovary including the germline stem cells.

Epigenetic regulation of gene expression by Drosophila Myb and E2F2-RBF via the Myb-MuvB/dREAM complex.

The Drosophila Myb oncoprotein, the E2F2 transcriptional repressor, and the RBF and Mip130/LIN-9 tumor suppressor proteins reside in a conserved Myb-MuvB (MMB)/dREAM complex. We now show that Myb is required in vivo for the expression of Polo kinase and components of the spindle assembly checkpoint (SAC). Surprisingly, the highly conserved DNA-binding domain was not essential for assembly of Myb into MMB/dREAM, for transcriptional regulation in vivo, or for rescue of Myb-null mutants to adult viability. E2F2, RBF, and Mip130/LIN-9 acted in opposition to Myb by repressing the expression of Polo and SAC genes in vivo. Remarkably, the absence of both Myb and Mip130, or of both Myb and E2F2, caused variegated expression in which high or low levels of Polo were stably inherited through successive cell divisions in imaginal wing discs. Restoration of Myb resulted in a uniformly high level of Polo expression similar to that seen in wild-type tissue, whereas restoration of Mip130 or E2F2 extinguished Polo expression. Our results demonstrate epigenetic regulation of gene expression by Myb, Mip130/LIN-9, and E2F2-RBF in vivo, and also provide an explanation for the ability of Mip130-null mutants to rescue the lethality of Myb-null mutants in vivo.