Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

Female-biased embryonic death from inflammation induced by genomic instability.

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation1,2. Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex-the DNA replicative helicase comprising MCM2 to MCM73,4-that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to 'maleness'-XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties5. Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair6. In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4Chaos3 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone.

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.

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.

A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo.

Cancer growth is a multistage, 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 remain 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 ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes. By combining Tuba-seq with multiplexed CRISPR-Cas9-mediated genome editing, we quantified the effects of 11 tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. Tuba-seq enables the broad quantification of the function of tumor-suppressor genes with unprecedented resolution, parallelization, and precision.

An in vivo multiplexed small-molecule screening platform.

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

Post-transcriptional homeostasis and regulation of MCM2-7 in mammalian cells.

The MiniChromosome Maintenance 2-7 (MCM2-7) complex provides essential replicative helicase function. Insufficient MCMs impair the cell cycle and cause genomic instability (GIN), leading to cancer and developmental defects in mice. Remarkably, depletion or mutation of one Mcm can decrease all Mcm levels. Here, we use mice and cells bearing a GIN-causing hypomophic allele of Mcm4 (Chaos3), in conjunction with disruption alleles of other Mcms, to reveal two new mechanisms that regulate MCM protein levels and pre-RC formation. First, the Mcm4(Chaos3) allele, which disrupts MCM4:MCM6 interaction, triggers a Dicer1 and Drosha-dependent ≈ 40% reduction in Mcm2-7 mRNAs. The decreases in Mcm mRNAs coincide with up-regulation of the miR-34 family of microRNAs, which is known to be Trp53-regulated and target Mcms. Second, MCM3 acts as a negative regulator of the MCM2-7 helicase in vivo by complexing with MCM5 in a manner dependent upon a nuclear-export signal-like domain, blocking the recruitment of MCMs onto chromatin. Therefore, the stoichiometry of MCM components and their localization is controlled post-transcriptionally at both the mRNA and protein levels. Alterations to these pathways cause significant defects in cell growth reflected by disease phenotypes in mice.

AMG 193, a Clinical Stage MTA-Cooperative PRMT5 Inhibitor, Drives Antitumor Activity Preclinically and in Patients With MTAP-Deleted Cancers.

One of the most robust synthetic lethal interactions observed in multiple functional genomic screens has been dependency on PRMT5 in cancer cells with MTAP deletion. We report the discovery of the clinical stage MTA-cooperative PRMT5 inhibitor AMG 193, which preferentially binds PRMT5 in the presence of MTA and has potent biochemical and cellular activity in MTAP-deleted cells across multiple cancer lineages. In vitro, PRMT5 inhibition induces DNA damage, cell cycle arrest, and aberrant alternative mRNA splicing in MTAP-deleted cells. In human cell line and patient-derived xenograft models, AMG 193 induces robust antitumor activity and is well tolerated with no impact on normal hematopoietic cell lineages. AMG 193 synergizes with chemotherapies or the KRAS G12C inhibitor sotorasib in vitro, and combination treatment in vivo significantly inhibits tumor growth. AMG 193 is demonstrating promising clinical activity, including confirmed partial responses in patients with MTAP-deleted solid tumors from an ongoing phase 1/2 study.

Incremental genetic perturbations to MCM2-7 expression and subcellular distribution reveal exquisite sensitivity of mice to DNA replication stress.

Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4(Chaos3) cancer susceptibility allele. Mcm4(Chaos3/Chaos3) embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also "backup" origins that are recruited in times of replication stress to ensure complete replication of the genome.

Rova-T enhances the anti-tumor activity of anti-PD1 in a murine model of small cell lung cancer with endogenous Dll3 expression.

Rovalpituzumab tesirine (Rova-T) offers a targeted therapy for ~85% of SCLC patients whose tumors express DLL3, but clinical dosing is limited due to off-target toxicities. We hypothesized that a sub-efficacious dose of Rova-T combined with anti-PD1, which alone shows a clinical benefit to ~15% of SCLC patients, might elicit a novel mechanism of action and extend clinical utility. Using a pre-clinical murine SCLC tumor model that expresses Dll3 and has an intact murine immune system, we found that sub-efficacious doses of Rova-T with anti-PD1 resulted in enhanced anti-tumor activity, compared to either monotherapy. Multiplex immunohistochemistry (IHC) showed CD4 and CD8 T-cells primarily in normal tissue immediately adjacent to the tumor. Combination treatment, but not anti-PD1 alone, increased Ki67+/CD8 T-cells and Granzyme B+/CD8 in tumors by flow cytometry and IHC. Antibody depletion of T-cell populations showed CD8+ T-cells are required for in vivo anti-tumor efficacy. Whole transcriptome analysis as well as flow cytometry and IHC showed that Rova-T activates dendritic cells and increases Ccl5, Il-12, and Icam more than anti-PD1 alone. Increased tumor expression of PDL1 and MHC1 following Rova-T treatment also supports combination with anti-PD1. Mice previously treated with Rova-T + anti-PD1 withstood tumor re-challenge, demonstrating sustained anti-tumor immunity. Collectively our pre-clinical data support clinical combination of sub-efficacious Rova-T with anti-PD1 to extend the benefit of immune checkpoint inhibitors to more SCLC patients.

Altered Mitochondria Functionality Defines a Metastatic Cell State in Lung Cancer and Creates an Exploitable Vulnerability.

Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for most cancer-related deaths but the cellular changes that enable cancer cells to leave the primary tumor and establish inoperable and lethal metastases remain poorly understood. To uncover genes that are specifically required to sustain metastasis survival or growth, we performed a genome-scale pooled lentiviral-shRNA library screen in cells that represent nonmetastatic and metastatic states of lung adenocarcinoma. Mitochondrial ribosome and mitochondria-associated genes were identified as top gene sets associated with metastasis-specific lethality. Metastasis-derived cell lines in vitro and metastases analyzed ex vivo from an autochthonous lung cancer mouse model had lower mitochondrial membrane potential and reduced mitochondrial functionality than nonmetastatic primary tumors. Electron microscopy of metastases uncovered irregular mitochondria with bridging and loss of normal membrane structure. Consistent with these findings, compounds that inhibit mitochondrial translation or replication had a greater effect on the growth of metastasis-derived cells. Finally, mice with established tumors developed fewer metastases upon treatment with phenformin in vivo. These results suggest that the metastatic cell state in lung adenocarcinoma is associated with a specifically altered mitochondrial functionality that can be therapeutically exploited. SIGNIFICANCE: This study characterizes altered mitochondria functionality of the metastatic cell state in lung cancer and opens new avenues for metastasis-specific therapeutic targeting.

Axon-like protrusions promote small cell lung cancer migration and metastasis.

Metastasis is the main cause of death in cancer patients but remains a poorly understood process. Small cell lung cancer (SCLC) is one of the most lethal and most metastatic cancer types. SCLC cells normally express neuroendocrine and neuronal gene programs but accumulating evidence indicates that these cancer cells become relatively more neuronal and less neuroendocrine as they gain the ability to metastasize. Here we show that mouse and human SCLC cells in culture and in vivo can grow cellular protrusions that resemble axons. The formation of these protrusions is controlled by multiple neuronal factors implicated in axonogenesis, axon guidance, and neuroblast migration. Disruption of these axon-like protrusions impairs cell migration in culture and inhibits metastatic ability in vivo. The co-option of developmental neuronal programs is a novel molecular and cellular mechanism that contributes to the high metastatic ability of SCLC.

Tumor Suppressor Activity of Selenbp1, a Direct Nkx2-1 Target, in Lung Adenocarcinoma.

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.

Molecular definition of a metastatic lung cancer state reveals a targetable CD109-Janus kinase-Stat axis.

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer.

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

An Arntl2-Driven Secretome Enables Lung Adenocarcinoma Metastatic Self-Sufficiency.

The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments.

Obligate progression precedes lung adenocarcinoma dissemination.

UNLABELLED: Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis. SIGNIFICANCE: Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression.

A conditional system to specifically link disruption of protein-coding function with reporter expression in mice.

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans.

An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance-deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.

Comparison of Tir from enterohemorrahgic and enteropathogenic Escherichia coli strains: two homologues with distinct intracellular properties.

Tir of enteropathogenic Escherichia coli (EPEC) or enterohemorrahgic E. coil (EHEC) is translocated by a type III secretion system to the host cell membranes where it serves as a receptor for the binding of a second bacterial membrane protein. In response to the binding, EPEC Tir is phosphorylated at Tyr474, and this phosphorylation is necessary for the signaling of pedestal formation. Tir of EHEC has no equivalent phosphorylation site but it is similarly needed for cytoskeleton rearrangement. How these two Tir molecules achieve their function by apparently different mechanisms is not completely clear. To examine their intrinsic differences, the two Tirs were expressed in HeLa cells and compared. Actin in complexes could be pelleted down from the lysate of cells expressing EHEC Tir but not EPEC Tir. By immunostaining, neither Tir molecule was found in phosphorylated state. In the cytoplasm, EHEC Tir was frequently found in fibrous structures whereas EPEC Tir was observed completely in a diffusive form. The determinant critical for the EHEC Tir fibrous formation was mapped to the C-terminal region of the molecule that deviates from the EPEC counterpart. This region may play a role in taking an alternative route different from Tyr474 phosphorylation to transduce signals.