Publications by authors named "Monte M Winslow"

68 Publications

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Cell Rep 2021 Nov;37(8):110056

Department of Medical Oncology, West German Cancer Center, University Hospital Essen at the University Duisburg-Essen, Duisburg, Germany; German Cancer Consortium (DKTK) partner site Essen, Essen, Germany. Electronic address:

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.
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http://dx.doi.org/10.1016/j.celrep.2021.110056DOI Listing
November 2021

LKB1 inactivation modulates chromatin accessibility to drive metastatic progression.

Nat Cell Biol 2021 08 2;23(8):915-924. Epub 2021 Aug 2.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Metastasis is the leading cause of cancer-related deaths and enables cancer cells to compromise organ function by expanding in secondary sites. Since primary tumours and metastases often share the same constellation of driver mutations, the mechanisms that drive their distinct phenotypes are unclear. Here we show that inactivation of the frequently mutated tumour suppressor gene LKB1 (encoding liver kinase B1) has evolving effects throughout the progression of lung cancer, which leads to the differential epigenetic re-programming of early-stage primary tumours compared with late-stage metastases. By integrating genome-scale CRISPR-Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumours. Using an in vivo model of metastatic progression, we further show that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumours. The expression of SOX17 is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution.
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http://dx.doi.org/10.1038/s41556-021-00728-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355205PMC
August 2021

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

Genes Dev 2021 Aug 22;35(15-16):1109-1122. Epub 2021 Jul 22.

Division of Cellular and Developmental Biology, Molecular and Cell Biology Department, University of California at Berkeley, Berkeley, California 94705, USA.

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 miRNAs as potent suppressors for lung adenocarcinoma metastasis. expression is specifically repressed in mouse metastatic lung adenocarcinomas, and decrease strongly correlates with poor patient survival. Consistently, deletion of -/ in the ; lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. - 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. 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.
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http://dx.doi.org/10.1101/gad.347344.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336896PMC
August 2021

Quantitative Analyses Reveal a Complex Pharmacogenomic Landscape in Lung Adenocarcinoma.

Cancer Res 2021 Sep 2;81(17):4570-4580. Epub 2021 Jul 2.

Department of Genetics, Stanford University School of Medicine, Stanford, California.

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 . 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 pharmacogenomic maps that relate tumor genotypes to therapeutic responses reveals a surprisingly complex map of genotype-specific resistance and sensitivity.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416777PMC
September 2021

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

Nature 2021 Apr 14;592(7856):794-798. Epub 2021 Apr 14.

Department of Pediatrics, Stanford University, Stanford, CA, USA.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer. However, the mechanisms that regulate levels of cyclin D are incompletely understood. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.
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http://dx.doi.org/10.1038/s41586-021-03474-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246597PMC
April 2021

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

Cancer Discov 2021 Jul 11;11(7):1736-1753. Epub 2021 Mar 11.

Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut.

In lung adenocarcinoma, oncogenic mutations co-occur with many tumor suppressor gene alterations; however, the extent to which these contribute to tumor growth and response to therapy remains largely unknown. By quantifying the effects of inactivating 10 putative tumor suppressor genes in a mouse model of EGFR-driven -deficient lung adenocarcinoma, we found that , or inactivation strongly promoted tumor growth. Unexpectedly, inactivation of or 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, 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 , this study reveals key tumor suppressors that constrain the growth of -mutant tumors. Furthermore, we uncovered that inactivation reduces the sensitivity of these tumors to tyrosine kinase inhibitors. Thus, our approach identifies genotypes of biological and therapeutic importance in this disease..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530463PMC
July 2021

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.

Nucleic Acids Res 2021 04;49(6):e36

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.
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http://dx.doi.org/10.1093/nar/gkaa1264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034634PMC
April 2021

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

Cancer Discov 2021 Jul 19;11(7):1754-1773. Epub 2021 Feb 19.

Department of Genetics, Stanford University School of Medicine, Stanford, California.

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 . 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 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 genetic analyses in autochthonous cancer models..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292166PMC
July 2021

Mechanisms of small cell lung cancer metastasis.

EMBO Mol Med 2021 01 9;13(1):e13122. Epub 2020 Dec 9.

Department of Pediatrics, Stanford University, Stanford, CA, USA.

Metastasis is a major cause of morbidity and mortality in cancer patients. However, the molecular and cellular mechanisms underlying the ability of cancer cells to metastasize remain relatively poorly understood. Among all solid tumors, small cell lung cancer (SCLC) has remarkable metastatic proclivity, with a majority of patients diagnosed with metastatic disease. Our understanding of SCLC metastasis has been hampered for many years by the paucity of material from primary tumors and metastases, as well as the lack of faithful pre-clinical models. Here, we review recent advances that are helping circumvent these limitations. These advances include methods that employ circulating tumor cells from the blood of SCLC patients and the development of diverse genetically engineered mouse models of metastatic SCLC. New insights into the cellular mechanisms of SCLC metastasis include observations of cell fate changes associated with increased metastatic ability. Ongoing studies on cell migration and organ tropism promise to expand our understanding of SCLC metastasis. Ultimately, a better molecular understanding of metastatic phenotypes may be translated into new therapeutic options to limit metastatic spread and treat metastatic SCLC.
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http://dx.doi.org/10.15252/emmm.202013122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799359PMC
January 2021

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

Cancer Res 2021 02 25;81(3):567-579. Epub 2020 Nov 25.

Department of Medical Oncology, West German Cancer Center, University Hospital Essen at the University of Duisburg-Essen, Essen, Germany.

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 and metastases analyzed 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 . 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.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137518PMC
February 2021

Zmat3 Is a Key Splicing Regulator in the p53 Tumor Suppression Program.

Mol Cell 2020 11;80(3):452-469.e9

Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse Kras-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.
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http://dx.doi.org/10.1016/j.molcel.2020.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654708PMC
November 2020

A versatile system to record cell-cell interactions.

Elife 2020 10 7;9. Epub 2020 Oct 7.

Department of Genetics, Stanford University School of Medicine, Stanford, United States.

Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of the cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.
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http://dx.doi.org/10.7554/eLife.61080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682987PMC
October 2020

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Nature 2020 04 11;580(7801):136-141. Epub 2020 Mar 11.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

Cancer genomics studies have identified thousands of putative cancer driver genes. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
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http://dx.doi.org/10.1038/s41586-020-2099-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368463PMC
April 2020

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

Elife 2019 12 13;8. Epub 2019 Dec 13.

Cancer Biology Program, Stanford University School of Medicine, Stanford, United States.

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.
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http://dx.doi.org/10.7554/eLife.50616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940020PMC
December 2019

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

Cancer Discov 2019 11 26;9(11):1590-1605. Epub 2019 Jul 26.

Cancer Biology Program, Stanford University School of Medicine, Stanford, California.

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 , 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 -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..
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http://dx.doi.org/10.1158/2159-8290.CD-18-1237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825558PMC
November 2019

Publisher Correction: Towards quantitative and multiplexed in vivo functional cancer genomics.

Nat Rev Genet 2018 Dec;19(12):801

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

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.
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http://dx.doi.org/10.1038/s41576-018-0062-6DOI Listing
December 2018

Towards quantitative and multiplexed in vivo functional cancer genomics.

Nat Rev Genet 2018 12;19(12):741-755

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

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.
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http://dx.doi.org/10.1038/s41576-018-0053-7DOI Listing
December 2018

Hmga2 is dispensable for pancreatic cancer development, metastasis, and therapy resistance.

Sci Rep 2018 09 18;8(1):14008. Epub 2018 Sep 18.

Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence of disseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.
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http://dx.doi.org/10.1038/s41598-018-32159-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143627PMC
September 2018

Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin.

Cancer Discov 2018 10 18;8(10):1316-1331. Epub 2018 Sep 18.

Cancer Biology Program, Stanford University, Stanford, California.

The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms. We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195211PMC
October 2018

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

Mol Cancer Res 2018 11 12;16(11):1737-1749. Epub 2018 Jul 12.

Cancer Biology Program, Stanford University School of Medicine, Stanford, California.

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 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 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. .
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541221PMC
November 2018

Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice.

Nat Genet 2018 04 2;50(4):483-486. Epub 2018 Apr 2.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

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.
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http://dx.doi.org/10.1038/s41588-018-0083-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061949PMC
April 2018

Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity.

Nat Commun 2017 12 12;8(1):2053. Epub 2017 Dec 12.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
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http://dx.doi.org/10.1038/s41467-017-01519-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727199PMC
December 2017

BLIMP1 Induces Transient Metastatic Heterogeneity in Pancreatic Cancer.

Cancer Discov 2017 10 8;7(10):1184-1199. Epub 2017 Aug 8.

Department of Genetics, Stanford University School of Medicine, Stanford, California.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character. PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628145PMC
October 2017

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival.

Proc Natl Acad Sci U S A 2017 07 26;114(28):E5625-E5634. Epub 2017 Jun 26.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139;

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising , , and is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.
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http://dx.doi.org/10.1073/pnas.1707054114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514763PMC
July 2017

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

Nat Methods 2017 Jul 22;14(7):737-742. Epub 2017 May 22.

Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1038/nmeth.4297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495136PMC
July 2017

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

Nat Med 2017 Mar 13;23(3):291-300. Epub 2017 Feb 13.

Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1038/nm.4285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453542PMC
March 2017

An in vivo multiplexed small-molecule screening platform.

Nat Methods 2016 10 12;13(10):883-889. Epub 2016 Sep 12.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088491PMC
http://dx.doi.org/10.1038/nmeth.3992DOI Listing
October 2016

Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Cell 2016 Jul 30;166(2):328-342. Epub 2016 Jun 30.

Cancer Biology Program, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2016.05.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004630PMC
July 2016

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

J Clin Invest 2016 07 13;126(7):2610-20. Epub 2016 Jun 13.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.
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http://dx.doi.org/10.1172/JCI81603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922696PMC
July 2016

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

Cancer Cell 2016 05 14;29(5):697-710. Epub 2016 Apr 14.

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; Cancer Biology Program, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ccell.2016.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4864124PMC
May 2016
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