Publications by authors named "Daniel Schramek"

46 Publications

Pan-cancer analysis of non-coding transcripts reveals the prognostic onco-lncRNA HOXA10-AS in gliomas.

Cell Rep 2021 Oct;37(3):109873

Ontario Institute for Cancer Research, Toronto, ON, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. Electronic address:

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer and powerful biomarkers; however, most remain uncharacterized. Here, we analyze 5,592 prognostic lncRNAs in 9,446 cancers of 30 types using machine learning. We identify 166 lncRNAs whose expression correlates with survival and improves the accuracy of common clinical variables, molecular features, and cancer subtypes. Prognostic lncRNAs are often characterized by switch-like expression patterns. In low-grade gliomas, HOXA10-AS activation is a robust marker of poor prognosis that complements IDH1/2 mutations, as validated in another retrospective cohort, and correlates with developmental pathways in tumor transcriptomes. Loss- and gain-of-function studies in patient-derived glioma cells, organoids, and xenograft models identify HOXA10-AS as a potent onco-lncRNA that regulates cell proliferation, contact inhibition, invasion, Hippo signaling, and mitotic and neuro-developmental pathways. Our study underscores the pan-cancer potential of the non-coding transcriptome for identifying biomarkers and regulators of cancer progression.
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http://dx.doi.org/10.1016/j.celrep.2021.109873DOI Listing
October 2021

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer Cell 2021 Aug 21;39(8):1115-1134.e12. Epub 2021 Jul 21.

Lunenfeld Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, ON M5G 1X5, Canada; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, ON M5T 3A9, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAP or YAP cancer classes that express or silence YAP, respectively. YAP solid cancers are neural/neuroendocrine and frequently RB1, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAP or YAP cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.
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http://dx.doi.org/10.1016/j.ccell.2021.06.016DOI Listing
August 2021

Prevalence of the Brazilian Founder c.1010G>A (p.Arg337His) in Lung Adenocarcinoma: Is Genotyping Warranted in All Brazilian Patients?

Front Genet 2021 2;12:606537. Epub 2021 Feb 2.

Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of , c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic mutations were more frequent in c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of and germline c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if c.1010G>A screening in all Brazilian LUAD patients is justified.
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http://dx.doi.org/10.3389/fgene.2021.606537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885268PMC
February 2021

AJUBA: A regulator of epidermal homeostasis and cancer.

Exp Dermatol 2021 Apr 26;30(4):546-559. Epub 2021 Jan 26.

Molecular, Structural and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.

The epidermis, outermost layer of the skin, is constantly renewing itself through proliferative and differentiation processes. These processes are vital to maintain proper epidermal integrity during skin development and homeostasis and for preventing skin diseases and cancers. The biological mechanisms that permit this balancing act are vast, where individual pathway regulators are known, but the exact regulatory control and cross-talk between simultaneously turning one biological pathway on and an opposing one off remain elusive. This review explores the diverse roles the scaffolding protein AJUBA plays during epidermal homeostasis and cancer. Initially identified for its role in promoting meiotic progression in oocytes through Grb2 and MAP kinase activity, AJUBA also maintains cytoskeletal tension permitting epidermal tissue development and responds to retinoic acid committing cells to initiate development of surface epidermal layer. AJUBA regulates proliferation of skin stem cells through Hippo and Wnt signalling and encourages mitotic commitment through Aurora-A, Aurora-B and CDK1. In addition, AJUBA also induces epidermal differentiation to maintain appropriate epidermal thickness and barrier function by activating Notch signalling and stabilizing catenins and actin during cellular remodelling. AJUBA also plays an imperative context-dependent tumor-promoting and tumor-suppressive role within epithelial cancers. AJUBA's abundant roles within the epidermis signify its importance as a molecular switchboard, vetting multiple signalling pathways to control epidermal biology.
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http://dx.doi.org/10.1111/exd.14272DOI Listing
April 2021

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity.

J Vis Exp 2020 11 2(165). Epub 2020 Nov 2.

Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital; Department of Molecular Genetics, University of Toronto;

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.
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http://dx.doi.org/10.3791/61693DOI Listing
November 2020

The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

Science 2020 10;370(6514):351-356

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada.

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.
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http://dx.doi.org/10.1126/science.aaz0863DOI Listing
October 2020

CRISPR screens reveal potent driver mutations in head and neck cancers.

Mol Cell Oncol 2020 13;7(4):1758541. Epub 2020 May 13.

Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

We have recently tested the transforming potential of 484 'long-tail' genes, which are recurrently albeit infrequently mutated in head and neck cancers (HNSCC). We identified 15 novel tumor suppressors and our top hits converge on regulating the NOTCH signaling pathway. Therapeutic approaches activating NOTCH signaling could be a promising strategy to treat two-thirds of human HNSCC patients.
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http://dx.doi.org/10.1080/23723556.2020.1758541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469622PMC
May 2020

Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer.

Nat Commun 2020 07 24;11(1):3701. Epub 2020 Jul 24.

Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, 2950, Chemin de la Polytechnique, Montréal, QC, H3T 1J4, Canada.

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.
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http://dx.doi.org/10.1038/s41467-020-17549-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381612PMC
July 2020

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell 2020 07 25;182(2):345-356.e16. Epub 2020 Jun 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Molecular Architecture of Life Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada. Electronic address:

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
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http://dx.doi.org/10.1016/j.cell.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316060PMC
July 2020

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma.

Cell 2020 06 22;181(6):1329-1345.e24. Epub 2020 May 22.

The Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5G 1L7, Canada.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
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http://dx.doi.org/10.1016/j.cell.2020.04.047DOI Listing
June 2020

Toronto Workshop on Late Recurrence in Estrogen Receptor-Positive Breast Cancer: Part 1: Late Recurrence: Current Understanding, Clinical Considerations.

JNCI Cancer Spectr 2019 Dec 10;3(4):pkz050. Epub 2019 Aug 10.

Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Sinai Health System, Toronto, ON, Canada.

Disease recurrence (locoregional, distant) exerts a significant clinical impact on the survival of estrogen receptor-positive breast cancer patients. Many of these recurrences occur late, more than 5 years after original diagnosis, and represent a major obstacle to the effective treatment of this disease. Indeed, methods to identify patients at risk of late recurrence and therapeutic strategies designed to avert or treat these recurrences are lacking. Therefore, an international workshop was convened in Toronto, Canada, in February 2018 to review the current understanding of late recurrence and to identify critical issues that require future study. In this article, the major issues surrounding late recurrence are defined and current approaches that may be applicable to this challenge are discussed. Specifically, diagnostic tests with potential utility in late-recurrence prediction are described as well as a variety of patient-related factors that may influence recurrence risk. Clinical and therapeutic approaches are also reviewed, with a focus on patient surveillance and the implementation of extended endocrine therapy in the context of late-recurrence prevention. Understanding and treating late recurrence in estrogen receptor-positive breast cancer is a major unmet clinical need. A concerted effort of basic and clinical research is required to confront late recurrence and improve disease management and patient survival.
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http://dx.doi.org/10.1093/jncics/pkz050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049988PMC
December 2019

Toronto Workshop on Late Recurrence in Estrogen Receptor-Positive Breast Cancer: Part 2: Approaches to Predict and Identify Late Recurrence, Research Directions.

JNCI Cancer Spectr 2019 Dec 10;3(4):pkz049. Epub 2019 Aug 10.

Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Sinai Health System, Toronto, ON, Canada.

Late disease recurrence (more than 5 years after initial diagnosis) represents a clinical challenge in the treatment and management of estrogen receptor-positive breast cancer (BC). An international workshop was convened in Toronto, Canada, in February 2018 to review the current understanding of late recurrence and to identify critical issues that require future study. The underlying biological causes of late recurrence are complex, with the processes governing cancer cell dormancy, including immunosurveillance, cell proliferation, angiogenesis, and cellular stemness, being integral to disease progression. These critical processes are described herein as well as their role in influencing risk of recurrence. Moreover, observational and interventional clinical trials are proposed, with a focus on methods to identify patients at risk of recurrence and possible strategies to combat this in patients with estrogen receptor-positive BC. Because the problem of late BC recurrence of great importance, recent advances in disease detection and patient monitoring should be incorporated into novel clinical trials to evaluate approaches to enhance patient management. Indeed, future research on these issues is planned and will offer new options for effective late recurrence treatment and prevention strategies.
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http://dx.doi.org/10.1093/jncics/pkz049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050024PMC
December 2019

Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.

Science 2020 03;367(6483):1264-1269

Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, and suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. and mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.
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http://dx.doi.org/10.1126/science.aax0902DOI Listing
March 2020

Mutant ACVR1 Arrests Glial Cell Differentiation to Drive Tumorigenesis in Pediatric Gliomas.

Cancer Cell 2020 03 5;37(3):308-323.e12. Epub 2020 Mar 5.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address:

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1 arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3b and Pik3ca to generate high-grade diffuse gliomas. Mechanistically, Acvr1 upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.
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http://dx.doi.org/10.1016/j.ccell.2020.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105820PMC
March 2020

CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci.

Nat Commun 2019 11 29;10(1):5454. Epub 2019 Nov 29.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Dr. Bohr Gasse 3, 1030, Vienna, Austria.

CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.
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http://dx.doi.org/10.1038/s41467-019-13403-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884486PMC
November 2019

Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy.

EMBO Mol Med 2019 08 24;11(8):e9266. Epub 2019 Jun 24.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria.

Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.
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http://dx.doi.org/10.15252/emmm.201809266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685079PMC
August 2019

AIF-regulated oxidative phosphorylation supports lung cancer development.

Cell Res 2019 07 27;29(7):579-591. Epub 2019 May 27.

Department of Thoracic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.

Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a Kras-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of Kras-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout Kras mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.
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http://dx.doi.org/10.1038/s41422-019-0181-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796841PMC
July 2019

Author Correction: Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Nat Genet 2019 01;51(1):196

Department of Pathology, Montreal Children's Hospital, McGill University Health Centre, Montreal, Quebec, Canada.

In the version of this article originally published, the main-text sentence "In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor" misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.
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http://dx.doi.org/10.1038/s41588-018-0304-8DOI Listing
January 2019

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Nat Genet 2018 12 29;50(12):1650-1657. Epub 2018 Oct 29.

Department of Pathology, Montreal Children's Hospital, McGill University Health Centre, Montreal, Quebec, Canada.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.
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http://dx.doi.org/10.1038/s41588-018-0251-4DOI Listing
December 2018

Afatinib restrains K-RAS-driven lung tumorigenesis.

Sci Transl Med 2018 06;10(446)

Department of Physiology, Center of Physiology and Pharmacology and Comprehensive Cancer Center (CCC), Medical University of Vienna, AT-1090 Vienna, Austria.

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog () mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat -mutated NSCLC.
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http://dx.doi.org/10.1126/scitranslmed.aao2301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610658PMC
June 2018

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

Nat Methods 2017 Dec 16;14(12):1191-1197. Epub 2017 Oct 16.

Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), Vienna Biocenter (VBC), Vienna, Austria.

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.
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http://dx.doi.org/10.1038/nmeth.4466DOI Listing
December 2017

Translation from unconventional 5' start sites drives tumour initiation.

Nature 2017 01 11;541(7638):494-499. Epub 2017 Jan 11.

Robin Chemers Neustein Laboratory of Mammalian Development and Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.
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http://dx.doi.org/10.1038/nature21036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5287289PMC
January 2017

Aberrant regulation of RANKL/OPG in women at high risk of developing breast cancer.

Oncotarget 2017 Jan;8(3):3811-3825

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their lifetime in North America and Europe. Receptor Activator of NF-kB Ligand (RANKL), its receptor RANK and the natural antagonist osteoprotegerin (OPG) are essential regulators of bone resorption. We have initially shown that RANKL/RANK are essential for hormone-driven mammary epithelial proliferation in pregnancy and RANKL/RANK have been implicated in mammary stem cell biology. Using genetic mouse-models, we and others identified the RANKL/RANK system as a key regulator of sex hormone, BRCA1-mutation, and oncogene-driven breast cancer and we proposed that RANKL/RANK might be involved in the initiation of breast tumors. We now report that in postmenopausal women without known genetic predisposition, high RANKL and progesterone serum levels stratify a subpopulation of women at high risk of developing breast cancer 12-24 months before diagnosis (5.33-fold risk, 95%CI 1.5-25.4; P=0.02). In women with established breast cancer, we demonstrate that RANKL/OPG ratios change dependent on the presence of circulating tumor cells (CTCs). Finally, we show in a prospective human breast cancer cohort that alterations in RANKL/OPG ratios are significantly associated with breast cancer manifestation. These data indicate that the RANKL/RANK/OPG system is deregulated in post-menopausal women at high risk for breast cancer and in women with circulating tumor cells. Thus, serum levels of RANKL/OPG are potentially indicative of predisposition and progression of breast cancer in humans. Advancement of our findings towards clinical application awaits prior validation in independent patient cohorts.
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http://dx.doi.org/10.18632/oncotarget.14013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354797PMC
January 2017

Phase 1 study to evaluate the effect of the MEK inhibitor trametinib on cardiac repolarization in patients with solid tumours.

Cancer Chemother Pharmacol 2016 Sep 8;78(3):491-500. Epub 2016 Jul 8.

Huntsman Cancer Institute, 2000 Circle of Hope Drive, Salt Lake City, UT, 84112, USA.

Purpose: Trametinib is a reversible, selective inhibitor of the mitogen-activated extracellular signal-regulated kinase 1 (MEK1) and 2 (MEK2). Cardiotoxicity (congestive heart failure, decreased heart rate, left ventricular dysfunction, and hypertension) related to trametinib is an infrequent, but serious, adverse event (AE). Prolongation of the QT interval increases the risk of life-threatening cardiac arrhythmia. Thus, the risk of trametinib inducing QT prolongation at putative supratherapeutic exposure was evaluated.

Methods: Eligible patients with solid tumours received placebo on day 1, once-daily trametinib 2-mg doses on days 2-14, and a single trametinib 3-mg dose on day 15 to achieve supratherapeutic dosing for QTc measurement. Electrocardiogram was assessed by 12-lead ambulatory 24-h Holter monitoring pre-dose, and on day 1 and day 15. Pharmacokinetic (PK) and pharmacodynamics (PD) parameters were measured.

Results: Thirty-two of 35 patients completed the study. There was no effect of trametinib when compared with time-matched placebo on the change from baseline in QTcF, QTcB, or QTcI interval. Mean AUC0-24 and C max following trametinib 2-mg repeat doses were 364 ng.h/mL and 22.9 ng/mL, respectively; the corresponding values for the 3-mg dose were 454 ng.h/mL and 29.2 ng/mL. Median T max was approximately 2 h for both doses. Statistical analysis and PK/PD modelling showed no significant relationship between QTcF interval and trametinib plasma concentrations. AEs were consistent with those reported previously. No electrocardiogram abnormalities were reported as AEs.

Conclusions: The results of this study suggest trametinib has no significant effect on QT prolongation at supratherapeutic exposure.
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http://dx.doi.org/10.1007/s00280-016-3090-yDOI Listing
September 2016

RANKL/RANK control Brca1 mutation- .

Cell Res 2016 07 31;26(7):761-74. Epub 2016 May 31.

Department of Experimental Pathology and Pathology of Laboratory Animals, Medical University Vienna and University of Veterinary Medicine Vienna, Vienna 1090, Austria.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.
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http://dx.doi.org/10.1038/cr.2016.69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129883PMC
July 2016

ETS family transcriptional regulators drive chromatin dynamics and malignancy in squamous cell carcinomas.

Elife 2015 Nov 21;4:e10870. Epub 2015 Nov 21.

Howard Hughes Medical Institute, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, United States.

Tumor-initiating stem cells (SCs) exhibit distinct patterns of transcription factors and gene expression compared to healthy counterparts. Here, we show that dramatic shifts in large open-chromatin domain (super-enhancer) landscapes underlie these differences and reflect tumor microenvironment. By in vivo super-enhancer and transcriptional profiling, we uncover a dynamic cancer-specific epigenetic network selectively enriched for binding motifs of a transcription factor cohort expressed in squamous cell carcinoma SCs (SCC-SCs). Many of their genes, including Ets2 and Elk3, are themselves regulated by SCC-SC super-enhancers suggesting a cooperative feed-forward loop. Malignant progression requires these genes, whose knockdown severely impairs tumor growth and prohibits progression from benign papillomas to SCCs. ETS2-deficiency disrupts the SCC-SC super-enhancer landscape and downstream cancer genes while ETS2-overactivation in epidermal-SCs induces hyperproliferation and SCC super-enhancer-associated genes Fos, Junb and Klf5. Together, our findings unearth an essential regulatory network required for the SCC-SC chromatin landscape and unveil its importance in malignant progression.
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http://dx.doi.org/10.7554/eLife.10870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739765PMC
November 2015

Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis.

Nat Commun 2015 Mar 3;6:6285. Epub 2015 Mar 3.

1] Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090, Austria [2] Department of Physiology, Center of Physiology and Pharmacology, Comprehensive Cancer Center, Medical University of Vienna, Vienna 1090, Austria.

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased Kras(G12D)-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3-NF-κB-IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.
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http://dx.doi.org/10.1038/ncomms7285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366489PMC
March 2015

MKK7 and ARF: new players in the DNA damage response scenery.

Cell Cycle 2014 26;13(8):1227-36. Epub 2014 Mar 26.

Molecular Carcinogenesis Group; Department of Histology and Embryology; School of Medicine; University of Athens; Athens, Greece; Faculty Institute of Cancer Sciences; University of Manchester; Manchester Academic Health Science Centre; Manchester, UK; Manchester Centre for Cellular Metabolism; University of Manchester; Manchester Academic Health Science Centre; Manchester, UK; Biomedical Research Foundation; Academy of Athens; Athens, Greece.

Sensing, integrating, and processing of stressogenic signals must be followed by accurate differential response(s) for a cell to survive and avoid malignant transformation. The DNA damage response (DDR) pathway is vital in this process, as it deals with genotoxic/oncogenic insults, having p53 as a nodal effector that performs most of the above tasks. Accumulating data reveal that other pathways are also involved in the same or similar processes, conveying also to p53. Emerging questions are if, how, and when these additional pathways communicate with the DDR axis. Two such stress response pathways, involving the MKK7 stress-activated protein kinase (SAPK) and ARF, have been shown to be interlocked with the ATM/ATR-regulated DDR axis in a highly ordered manner. This creates a new landscape in the DDR orchestrated response to genotoxic/oncogenic insults that is currently discussed.
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http://dx.doi.org/10.4161/cc.28654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049959PMC
August 2015

A dual role for autophagy in a murine model of lung cancer.

Nat Commun 2014 ;5:3056

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna 1030, Austria.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.
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http://dx.doi.org/10.1038/ncomms4056DOI Listing
November 2015

Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas.

Science 2014 Jan;343(6168):309-13

Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10065, USA.

Mining modern genomics for cancer therapies is predicated on weeding out "bystander" alterations (nonconsequential mutations) and identifying "driver" mutations responsible for tumorigenesis and/or metastasis. We used a direct in vivo RNA interference (RNAi) strategy to screen for genes that upon repression predispose mice to squamous cell carcinomas (SCCs). Seven of our top hits-including Myh9, which encodes nonmuscle myosin IIa-have not been linked to tumor development, yet tissue-specific Myh9 RNAi and Myh9 knockout trigger invasive SCC formation on tumor-susceptible backgrounds. In human and mouse keratinocytes, myosin IIa's function is manifested not only in conventional actin-related processes but also in regulating posttranscriptional p53 stabilization. Myosin IIa is diminished in human SCCs with poor survival, which suggests that in vivo RNAi technology might be useful for identifying potent but low-penetrance tumor suppressors.
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http://dx.doi.org/10.1126/science.1248627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159249PMC
January 2014
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