39 results on '"Johannessen CM"'
Search Results
2. Author Correction: Comprehensive mutational scanning of EGFR reveals TKI sensitivities of extracellular domain mutants.
- Author
-
Hayes TK, Aquilanti E, Persky NS, Yang X, Kim EE, Brenan L, Goodale AB, Alan D, Sharpe T, Shue RE, Westlake L, Golomb L, Silverman BR, Morris MD, Fisher TR, Beyene E, Li YY, Cherniack AD, Piccioni F, Hicks JK, Chi AS, Cahill DP, Dietrich J, Batchelor TT, Root DE, Johannessen CM, and Meyerson M
- Published
- 2024
- Full Text
- View/download PDF
3. Comprehensive mutational scanning of EGFR reveals TKI sensitivities of extracellular domain mutants.
- Author
-
Hayes TK, Aquilanti E, Persky NS, Yang X, Kim EE, Brenan L, Goodale AB, Alan D, Sharpe T, Shue RE, Westlake L, Golomb L, Silverman BR, Morris MD, Fisher TR, Beyene E, Li YY, Cherniack AD, Piccioni F, Hicks JK, Chi AS, Cahill DP, Dietrich J, Batchelor TT, Root DE, Johannessen CM, and Meyerson M
- Subjects
- Humans, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, ErbB Receptors metabolism, Mutation, Glioblastoma drug therapy, Lung Neoplasms drug therapy, Lung Neoplasms genetics
- Abstract
The epidermal growth factor receptor, EGFR, is frequently activated in lung cancer and glioblastoma by genomic alterations including missense mutations. The different mutation spectra in these diseases are reflected in divergent responses to EGFR inhibition: significant patient benefit in lung cancer, but limited in glioblastoma. Here, we report a comprehensive mutational analysis of EGFR function. We perform saturation mutagenesis of EGFR and assess function of ~22,500 variants in a human EGFR-dependent lung cancer cell line. This approach reveals enrichment of erlotinib-insensitive variants of known and unknown significance in the dimerization, transmembrane, and kinase domains. Multiple EGFR extracellular domain variants, not associated with approved targeted therapies, are sensitive to afatinib and dacomitinib in vitro. Two glioblastoma patients with somatic EGFR G598V dimerization domain mutations show responses to dacomitinib treatment followed by within-pathway resistance mutation in one case. In summary, this comprehensive screen expands the landscape of functional EGFR variants and suggests broader clinical investigation of EGFR inhibition for cancers harboring extracellular domain mutations., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
4. Systematic identification of biomarker-driven drug combinations to overcome resistance.
- Author
-
Rees MG, Brenan L, do Carmo M, Duggan P, Bajrami B, Arciprete M, Boghossian A, Vaimberg E, Ferrara SJ, Lewis TA, Rosenberg D, Sangpo T, Roth JA, Kaushik VK, Piccioni F, Doench JG, Root DE, and Johannessen CM
- Subjects
- Biomarkers, Cell Survival, Drug Combinations, Humans, Histone Demethylases metabolism, Monoacylglycerol Lipases
- Abstract
The ability to understand and predict variable responses to therapeutic agents may improve outcomes in patients with cancer. We hypothesized that the basal gene-transcription state of cancer cell lines, coupled with cell viability profiles of small molecules, might be leveraged to nominate specific mechanisms of intrinsic resistance and to predict drug combinations that overcome resistance. We analyzed 564,424 sensitivity profiles to identify candidate gene-compound pairs, and validated nine such relationships. We determined the mechanism of a novel relationship, in which expression of the serine hydrolase enzymes monoacylglycerol lipase (MGLL) or carboxylesterase 1 (CES1) confers resistance to the histone lysine demethylase inhibitor GSK-J4 by direct enzymatic modification. Insensitive cell lines could be sensitized to GSK-J4 by inhibition or gene knockout. These analytical and mechanistic studies highlight the potential of integrating gene-expression features with small-molecule response to identify patient populations that are likely to benefit from treatment, to nominate rational candidates for combinations and to provide insights into mechanisms of action., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
- Full Text
- View/download PDF
5. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers.
- Author
-
Ito T, Young MJ, Li R, Jain S, Wernitznig A, Krill-Burger JM, Lemke CT, Monducci D, Rodriguez DJ, Chang L, Dutta S, Pal D, Paolella BR, Rothberg MV, Root DE, Johannessen CM, Parida L, Getz G, Vazquez F, Doench JG, Zamanighomi M, and Sellers WR
- Subjects
- Cell Line, Tumor, Clustered Regularly Interspaced Short Palindromic Repeats, Enzyme Activation, GTP Phosphohydrolases genetics, Gene Knockout Techniques, Humans, Melanoma, Experimental genetics, Melanoma, Experimental therapy, Membrane Proteins genetics, Neoplasms enzymology, Neoplasms metabolism, Neoplasms therapy, Proto-Oncogene Proteins B-raf genetics, Dual Specificity Phosphatase 6 genetics, Dual-Specificity Phosphatases genetics, MAP Kinase Signaling System, Mitogen-Activated Protein Kinase Phosphatases genetics, Neoplasms genetics
- Abstract
Although single-gene perturbation screens have revealed a number of new targets, vulnerabilities specific to frequently altered drivers have not been uncovered. An important question is whether the compensatory relationship between functionally redundant genes masks potential therapeutic targets in single-gene perturbation studies. To identify digenic dependencies, we developed a CRISPR paralog targeting library to investigate the viability effects of disrupting 3,284 genes, 5,065 paralog pairs and 815 paralog families. We identified that dual inactivation of DUSP4 and DUSP6 selectively impairs growth in NRAS and BRAF mutant cells through the hyperactivation of MAPK signaling. Furthermore, cells resistant to MAPK pathway therapeutics become cross-sensitized to DUSP4 and DUSP6 perturbations such that the mechanisms of resistance to the inhibitors reinforce this mechanism of vulnerability. Together, multigene perturbation technologies unveil previously unrecognized digenic vulnerabilities that may be leveraged as new therapeutic targets in cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2021
- Full Text
- View/download PDF
6. Comprehensive Mutational Analysis of the BRCA1-Associated DNA Helicase and Tumor-Suppressor FANCJ/BACH1/BRIP1.
- Author
-
Calvo JA, Fritchman B, Hernandez D, Persky NS, Johannessen CM, Piccioni F, Kelch BA, and Cantor SB
- Subjects
- Cell Line, Tumor, Cisplatin pharmacology, Codon, Nonsense, Cross-Linking Reagents pharmacology, Gene Knockout Techniques, HeLa Cells, Humans, Loss of Function Mutation, Mitomycin pharmacology, Mutation drug effects, Mutation, Missense, Neoplasms pathology, BRCA1 Protein genetics, DNA Helicases genetics, DNA Mutational Analysis methods, Fanconi Anemia Complementation Group Proteins genetics, Mutation genetics, Neoplasms genetics, RNA Helicases genetics
- Abstract
FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient-derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. IMPLICATIONS: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients., (©2021 American Association for Cancer Research.)
- Published
- 2021
- Full Text
- View/download PDF
7. CloneSifter: enrichment of rare clones from heterogeneous cell populations.
- Author
-
Feldman D, Tsai F, Garrity AJ, O'Rourke R, Brenan L, Ho P, Gonzalez E, Konermann S, Johannessen CM, Beroukhim R, Bandopadhayay P, and Blainey PC
- Subjects
- Cells, Cultured, Clone Cells, Cloning, Organism methods, Clustered Regularly Interspaced Short Palindromic Repeats, RNA metabolism
- Abstract
Background: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones., Results: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population., Conclusions: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.
- Published
- 2020
- Full Text
- View/download PDF
8. Rhabdoid Tumors Are Sensitive to the Protein-Translation Inhibitor Homoharringtonine.
- Author
-
Howard TP, Oberlick EM, Rees MG, Arnoff TE, Pham MT, Brenan L, DoCarmo M, Hong AL, Kugener G, Chou HC, Drosos Y, Mathias KM, Ramos P, Seashore-Ludlow B, Giacomelli AO, Wang X, Freeman BB 3rd, Blankenship K, Hoffmann L, Tiv HL, Gokhale PC, Johannessen CM, Stewart EA, Schreiber SL, Hahn WC, and Roberts CWM
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Tumor, Cell Proliferation drug effects, Drug Resistance, Neoplasm genetics, Female, Gene Expression Regulation, Neoplastic, Homoharringtonine therapeutic use, Humans, Mice, Rhabdoid Tumor pathology, Xenograft Model Antitumor Assays, bcl-X Protein genetics, Homoharringtonine pharmacology, Protein Biosynthesis drug effects, Rhabdoid Tumor drug therapy
- Abstract
Purpose: Rhabdoid tumors are devastating pediatric cancers in need of improved therapies. We sought to identify small molecules that exhibit in vitro and in vivo efficacy against preclinical models of rhabdoid tumor., Experimental Design: We screened eight rhabdoid tumor cell lines with 481 small molecules and compared their sensitivity with that of 879 other cancer cell lines. Genome-scale CRISPR-Cas9 inactivation screens in rhabdoid tumors were analyzed to confirm target vulnerabilities. Gene expression and CRISPR-Cas9 data were queried across cell lines and primary rhabdoid tumors to discover biomarkers of small-molecule sensitivity. Molecular correlates were validated by manipulating gene expression. Subcutaneous rhabdoid tumor xenografts were treated with the most effective drug to confirm in vitro results., Results: Small-molecule screening identified the protein-translation inhibitor homoharringtonine (HHT), an FDA-approved treatment for chronic myelogenous leukemia (CML), as the sole drug to which all rhabdoid tumor cell lines were selectively sensitive. Validation studies confirmed the sensitivity of rhabdoid tumor to HHT was comparable with that of CML cell lines. Low expression of the antiapoptotic gene BCL2L1 , which encodes Bcl-XL, was the strongest predictor of HHT sensitivity, and HHT treatment consistently depleted Mcl-1, the synthetic-lethal antiapoptotic partner of Bcl-XL. Rhabdoid tumor cell lines and primary-tumor samples expressed low BCL2L1 , and overexpression of BCL2L1 induced resistance to HHT in rhabdoid tumor cells. Furthermore, HHT treatment inhibited rhabdoid tumor cell line and patient-derived xenograft growth in vivo ., Conclusions: Rhabdoid tumor cell lines and xenografts are highly sensitive to HHT, at least partially due to their low expression of BCL2L1 . HHT may have therapeutic potential against rhabdoid tumors., (©2020 American Association for Cancer Research.)
- Published
- 2020
- Full Text
- View/download PDF
9. Genotype-Fitness Maps of EGFR-Mutant Lung Adenocarcinoma Chart the Evolutionary Landscape of Resistance for Combination Therapy Optimization.
- Author
-
Bolan PO, Zviran A, Brenan L, Schiffman JS, Dusaj N, Goodale A, Piccioni F, Johannessen CM, and Landau DA
- Subjects
- Acrylamides administration & dosage, Acrylamides pharmacology, Adenocarcinoma of Lung enzymology, Aniline Compounds administration & dosage, Aniline Compounds pharmacology, Antineoplastic Combined Chemotherapy Protocols pharmacology, Benzimidazoles administration & dosage, Benzimidazoles pharmacology, Drug Resistance, Neoplasm genetics, ErbB Receptors genetics, ErbB Receptors metabolism, Erlotinib Hydrochloride administration & dosage, Erlotinib Hydrochloride pharmacology, Genetic Fitness, Genotype, Humans, Lung Neoplasms enzymology, MAP Kinase Signaling System, Adenocarcinoma of Lung drug therapy, Adenocarcinoma of Lung genetics, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Mutation
- Abstract
Cancer evolution poses a central obstacle to cure, as resistant clones expand under therapeutic selection pressures. Genome sequencing of relapsed disease can nominate genomic alterations conferring resistance but sample collection lags behind, limiting therapeutic innovation. Genome-wide screens offer a complementary approach to chart the compendium of escape genotypes, anticipating clinical resistance. We report genome-wide open reading frame (ORF) resistance screens for first- and third-generation epidermal growth factor receptor (EGFR) inhibitors and a MEK inhibitor. Using serial sampling, dose gradients, and mathematical modeling, we generate genotype-fitness maps across therapeutic contexts and identify alterations that escape therapy. Our data expose varying dose-fitness relationship across genotypes, ranging from complete dose invariance to paradoxical dose dependency where fitness increases in higher doses. We predict fitness with combination therapy and compare these estimates to genome-wide fitness maps of drug combinations, identifying genotypes where combination therapy results in unexpected inferior effectiveness. These data are applied to nominate combination optimization strategies to forestall resistant disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
10. Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.
- Author
-
Persky NS, Hernandez D, Do Carmo M, Brenan L, Cohen O, Kitajima S, Nayar U, Walker A, Pantel S, Lee Y, Cordova J, Sathappa M, Zhu C, Hayes TK, Ram P, Pancholi P, Mikkelsen TS, Barbie DA, Yang X, Haq R, Piccioni F, Root DE, and Johannessen CM
- Subjects
- Adenosine Triphosphate metabolism, Amino Acid Sequence, Animals, Drug Discovery, Drug Resistance, Neoplasm, Humans, Models, Molecular, Neoplasms drug therapy, Neoplasms genetics, Neoplasms metabolism, Protein Kinases chemistry, Protein Kinases metabolism, Proteomics, Drug Resistance, Point Mutation, Protein Kinase Inhibitors pharmacology, Protein Kinases genetics
- Abstract
Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.
- Published
- 2020
- Full Text
- View/download PDF
11. Pooled Genomic Screens Identify Anti-apoptotic Genes as Targetable Mediators of Chemotherapy Resistance in Ovarian Cancer.
- Author
-
Stover EH, Baco MB, Cohen O, Li YY, Christie EL, Bagul M, Goodale A, Lee Y, Pantel S, Rees MG, Wei G, Presser AG, Gelbard MK, Zhang W, Zervantonakis IK, Bhola PD, Ryan J, Guerriero JL, Montero J, Liang FJ, Cherniack AD, Piccioni F, Matulonis UA, Bowtell DDL, Sarosiek KA, Letai A, Garraway LA, Johannessen CM, and Meyerson M
- Subjects
- Cell Line, Tumor, Cisplatin pharmacology, Female, Genomics, Humans, Myeloid Cell Leukemia Sequence 1 Protein genetics, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Ovarian Neoplasms drug therapy, Paclitaxel pharmacology, bcl-X Protein genetics, bcl-X Protein metabolism, Antineoplastic Agents pharmacology, Apoptosis genetics, Apoptosis Regulatory Proteins genetics, Drug Resistance, Neoplasm, Myeloid Cell Leukemia Sequence 1 Protein antagonists & inhibitors, Ovarian Neoplasms genetics, bcl-X Protein antagonists & inhibitors
- Abstract
High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. IMPLICATIONS: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy., (©2019 American Association for Cancer Research.)
- Published
- 2019
- Full Text
- View/download PDF
12. DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance.
- Author
-
Togami K, Pastika T, Stephansky J, Ghandi M, Christie AL, Jones KL, Johnson CA, Lindsay RW, Brooks CL, Letai A, Craig JW, Pozdnyakova O, Weinstock DM, Montero J, Aster JC, Johannessen CM, and Lane AA
- Subjects
- Animals, Azacitidine pharmacology, Cell Line, Tumor, DNA Methylation, Dendritic Cells pathology, Female, Humans, Male, Mice, Mice, Nude, Minor Histocompatibility Antigens metabolism, Recombinant Fusion Proteins pharmacology, Tumor Suppressor Proteins metabolism, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols pharmacology, Dendritic Cells metabolism, Drug Delivery Systems, Hematologic Neoplasms drug therapy, Hematologic Neoplasms metabolism, Hematologic Neoplasms pathology, Interleukin-3 Receptor alpha Subunit metabolism, Leukemia, Myeloid, Acute drug therapy, Leukemia, Myeloid, Acute metabolism, Leukemia, Myeloid, Acute pathology, Neoplasm Proteins metabolism
- Abstract
The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.
- Published
- 2019
- Full Text
- View/download PDF
13. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies.
- Author
-
Guièze R, Liu VM, Rosebrock D, Jourdain AA, Hernández-Sánchez M, Martinez Zurita A, Sun J, Ten Hacken E, Baranowski K, Thompson PA, Heo JM, Cartun Z, Aygün O, Iorgulescu JB, Zhang W, Notarangelo G, Livitz D, Li S, Davids MS, Biran A, Fernandes SM, Brown JR, Lako A, Ciantra ZB, Lawlor MA, Keskin DB, Udeshi ND, Wierda WG, Livak KJ, Letai AG, Neuberg D, Harper JW, Carr SA, Piccioni F, Ott CJ, Leshchiner I, Johannessen CM, Doench J, Mootha VK, Getz G, and Wu CJ
- Subjects
- Adult, Aged, Aged, 80 and over, Animals, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Apoptosis drug effects, Apoptosis genetics, Bridged Bicyclo Compounds, Heterocyclic therapeutic use, Cell Line, Tumor, Clonal Evolution drug effects, Disease Progression, Drug Resistance, Neoplasm drug effects, Drug Resistance, Neoplasm genetics, Energy Metabolism drug effects, Energy Metabolism genetics, Female, Gene Expression Regulation, Neoplastic, Humans, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Male, Mice, Middle Aged, Mitochondria drug effects, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Oxidative Phosphorylation drug effects, Proto-Oncogene Proteins c-bcl-2 metabolism, Sulfonamides therapeutic use, Treatment Outcome, Xenograft Model Antitumor Assays, Antineoplastic Combined Chemotherapy Protocols pharmacology, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Mitochondria pathology, Proto-Oncogene Proteins c-bcl-2 antagonists & inhibitors, Sulfonamides pharmacology
- Abstract
Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
14. Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma.
- Author
-
Bandopadhayay P, Piccioni F, O'Rourke R, Ho P, Gonzalez EM, Buchan G, Qian K, Gionet G, Girard E, Coxon M, Rees MG, Brenan L, Dubois F, Shapira O, Greenwald NF, Pages M, Balboni Iniguez A, Paolella BR, Meng A, Sinai C, Roti G, Dharia NV, Creech A, Tanenbaum B, Khadka P, Tracy A, Tiv HL, Hong AL, Coy S, Rashid R, Lin JR, Cowley GS, Lam FC, Goodale A, Lee Y, Schoolcraft K, Vazquez F, Hahn WC, Tsherniak A, Bradner JE, Yaffe MB, Milde T, Pfister SM, Qi J, Schenone M, Carr SA, Ligon KL, Kieran MW, Santagata S, Olson JM, Gokhale PC, Jaffe JD, Root DE, Stegmaier K, Johannessen CM, and Beroukhim R
- Subjects
- Animals, Basic Helix-Loop-Helix Transcription Factors drug effects, Basic Helix-Loop-Helix Transcription Factors metabolism, CRISPR-Cas Systems, Cell Cycle Proteins drug effects, Cell Cycle Proteins metabolism, Cell Line, Tumor, Cell Lineage, Cerebellar Neoplasms genetics, Cyclin D2 drug effects, Cyclin D2 metabolism, Cyclin-Dependent Kinase 4 antagonists & inhibitors, Cyclin-Dependent Kinase 6 antagonists & inhibitors, Drug Resistance, Neoplasm, Gene Expression Profiling, Humans, Medulloblastoma genetics, Mice, Neural Stem Cells drug effects, Neural Stem Cells metabolism, Proto-Oncogene Proteins c-myc genetics, S Phase drug effects, Azepines pharmacology, Cell Cycle drug effects, Cerebellar Neoplasms drug therapy, Medulloblastoma drug therapy, Neurogenesis drug effects, Proteins antagonists & inhibitors, Triazoles pharmacology
- Abstract
BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.
- Published
- 2019
- Full Text
- View/download PDF
15. Next-generation characterization of the Cancer Cell Line Encyclopedia.
- Author
-
Ghandi M, Huang FW, Jané-Valbuena J, Kryukov GV, Lo CC, McDonald ER 3rd, Barretina J, Gelfand ET, Bielski CM, Li H, Hu K, Andreev-Drakhlin AY, Kim J, Hess JM, Haas BJ, Aguet F, Weir BA, Rothberg MV, Paolella BR, Lawrence MS, Akbani R, Lu Y, Tiv HL, Gokhale PC, de Weck A, Mansour AA, Oh C, Shih J, Hadi K, Rosen Y, Bistline J, Venkatesan K, Reddy A, Sonkin D, Liu M, Lehar J, Korn JM, Porter DA, Jones MD, Golji J, Caponigro G, Taylor JE, Dunning CM, Creech AL, Warren AC, McFarland JM, Zamanighomi M, Kauffmann A, Stransky N, Imielinski M, Maruvka YE, Cherniack AD, Tsherniak A, Vazquez F, Jaffe JD, Lane AA, Weinstock DM, Johannessen CM, Morrissey MP, Stegmeier F, Schlegel R, Hahn WC, Getz G, Mills GB, Boehm JS, Golub TR, Garraway LA, and Sellers WR
- Subjects
- Antineoplastic Agents pharmacology, Biomarkers, Tumor, DNA Methylation, Drug Resistance, Neoplasm, Ethnicity genetics, Gene Editing, Histones metabolism, Humans, MicroRNAs genetics, Molecular Targeted Therapy, Neoplasms metabolism, Protein Array Analysis, RNA Splicing, Cell Line, Tumor, Neoplasms genetics, Neoplasms pathology
- Abstract
Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
- Published
- 2019
- Full Text
- View/download PDF
16. A Functional Landscape of Resistance to MEK1/2 and CDK4/6 Inhibition in NRAS-Mutant Melanoma.
- Author
-
Hayes TK, Luo F, Cohen O, Goodale AB, Lee Y, Pantel S, Bagul M, Piccioni F, Root DE, Garraway LA, Meyerson M, and Johannessen CM
- Subjects
- Antineoplastic Agents pharmacology, Apoptosis, Cell Cycle Checkpoints, Cell Proliferation, Humans, Melanoma genetics, Melanoma pathology, Phosphorylation, Signal Transduction drug effects, Tumor Cells, Cultured, Cyclin-Dependent Kinase 4 antagonists & inhibitors, Cyclin-Dependent Kinase 6 antagonists & inhibitors, Drug Resistance, Neoplasm genetics, GTP Phosphohydrolases genetics, MAP Kinase Kinase 1 antagonists & inhibitors, MAP Kinase Kinase 2 antagonists & inhibitors, Melanoma drug therapy, Membrane Proteins genetics, Mutation
- Abstract
Combinatorial inhibition of MEK1/2 and CDK4/6 is currently undergoing clinical investigation in NRAS-mutant melanoma. To prospectively map the landscape of resistance to this investigational regimen, we utilized a series of gain- and loss-of-function forward genetic screens to identify modulators of resistance to clinical inhibitors of MEK1/2 and CDK4/6 alone and in combination. First, we identified NRAS-mutant melanoma cell lines that were dependent on NRAS for proliferation and sensitive to MEK1/2 and CDK4/6 combination treatment. We then used a genome-scale ORF overexpression screen and a CRISPR knockout screen to identify modulators of resistance to each inhibitor alone or in combination. These orthogonal screening approaches revealed concordant means of achieving resistance to this therapeutic modality, including tyrosine kinases, RAF, RAS, AKT, and PI3K signaling. Activated KRAS was sufficient to cause resistance to combined MEK/CDK inhibition and to replace genetic depletion of oncogenic NRAS. In summary, our comprehensive functional genetic screening approach revealed modulation of resistance to the inhibition of MEK1/2, CDK4/6, or their combination in NRAS-mutant melanoma. SIGNIFICANCE: These findings reveal that NRAS-mutant melanomas can acquire resistance to genetic ablation of NRAS or combination MEK1/2 and CDK4/6 inhibition by upregulating activity of the RTK-RAS-RAF and RTK-PI3K-AKT signaling cascade., (©2019 American Association for Cancer Research.)
- Published
- 2019
- Full Text
- View/download PDF
17. Mutational processes shape the landscape of TP53 mutations in human cancer.
- Author
-
Giacomelli AO, Yang X, Lintner RE, McFarland JM, Duby M, Kim J, Howard TP, Takeda DY, Ly SH, Kim E, Gannon HS, Hurhula B, Sharpe T, Goodale A, Fritchman B, Steelman S, Vazquez F, Tsherniak A, Aguirre AJ, Doench JG, Piccioni F, Roberts CWM, Meyerson M, Getz G, Johannessen CM, Root DE, and Hahn WC
- Subjects
- A549 Cells, Alleles, CRISPR-Cas Systems, Cells, Cultured, DNA Mutational Analysis, Databases, Genetic, High-Throughput Nucleotide Sequencing, Humans, Neoplasms pathology, Sequence Analysis, DNA, Mutagenesis physiology, Mutation, Neoplasms genetics, Tumor Suppressor Protein p53 genetics
- Abstract
Unlike most tumor suppressor genes, the most common genetic alterations in tumor protein p53 (TP53) are missense mutations
1,2 . Mutant p53 protein is often abundantly expressed in cancers and specific allelic variants exhibit dominant-negative or gain-of-function activities in experimental models3-8 . To gain a systematic view of p53 function, we interrogated loss-of-function screens conducted in hundreds of human cancer cell lines and performed TP53 saturation mutagenesis screens in an isogenic pair of TP53 wild-type and null cell lines. We found that loss or dominant-negative inhibition of wild-type p53 function reliably enhanced cellular fitness. By integrating these data with the Catalog of Somatic Mutations in Cancer (COSMIC) mutational signatures database9,10 , we developed a statistical model that describes the TP53 mutational spectrum as a function of the baseline probability of acquiring each mutation and the fitness advantage conferred by attenuation of p53 activity. Collectively, these observations show that widely-acting and tissue-specific mutational processes combine with phenotypic selection to dictate the frequencies of recurrent TP53 mutations.- Published
- 2018
- Full Text
- View/download PDF
18. A Convergence-Based Framework for Cancer Drug Resistance.
- Author
-
Konieczkowski DJ, Johannessen CM, and Garraway LA
- Subjects
- Humans, Immunotherapy, Molecular Targeted Therapy, Neoplasms drug therapy, Precision Medicine, Biomarkers, Tumor metabolism, Drug Resistance, Neoplasm, Neoplasms metabolism
- Abstract
Despite advances in cancer biology and therapeutics, drug resistance remains problematic. Resistance is often multifactorial, heterogeneous, and prone to undersampling. Nonetheless, many individual mechanisms of targeted therapy resistance may coalesce into a smaller number of convergences, including pathway reactivation (downstream re-engagement of original effectors), pathway bypass (recruitment of a parallel pathway converging on the same downstream output), and pathway indifference (development of a cellular state independent of the initial therapeutic target). Similar convergences may also underpin immunotherapy resistance. Such parsimonious, convergence-based frameworks may help explain resistance across tumor types and therapeutic categories and may also suggest strategies to overcome it., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
19. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.
- Author
-
Adalsteinsson VA, Ha G, Freeman SS, Choudhury AD, Stover DG, Parsons HA, Gydush G, Reed SC, Rotem D, Rhoades J, Loginov D, Livitz D, Rosebrock D, Leshchiner I, Kim J, Stewart C, Rosenberg M, Francis JM, Zhang CZ, Cohen O, Oh C, Ding H, Polak P, Lloyd M, Mahmud S, Helvie K, Merrill MS, Santiago RA, O'Connor EP, Jeong SH, Leeson R, Barry RM, Kramkowski JF, Zhang Z, Polacek L, Lohr JG, Schleicher M, Lipscomb E, Saltzman A, Oliver NM, Marini L, Waks AG, Harshman LC, Tolaney SM, Van Allen EM, Winer EP, Lin NU, Nakabayashi M, Taplin ME, Johannessen CM, Garraway LA, Golub TR, Boehm JS, Wagle N, Getz G, Love JC, and Meyerson M
- Subjects
- Antigens, Neoplasm genetics, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Breast Neoplasms secondary, Cell-Free Nucleic Acids blood, DNA Mutational Analysis, DNA, Neoplasm blood, Female, Gene Dosage, Humans, Male, Neoplasm Metastasis drug therapy, Prospective Studies, Prostatic Neoplasms drug therapy, Prostatic Neoplasms genetics, Prostatic Neoplasms secondary, Software, Exome Sequencing statistics & numerical data, Cell-Free Nucleic Acids genetics, DNA, Neoplasm genetics, Neoplasm Metastasis genetics, Exome Sequencing methods
- Abstract
Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.
- Published
- 2017
- Full Text
- View/download PDF
20. Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool.
- Author
-
Donovan KF, Hegde M, Sullender M, Vaimberg EW, Johannessen CM, Root DE, and Doench JG
- Subjects
- Alleles, Cell Line, Tumor, Clustered Regularly Interspaced Short Palindromic Repeats, DNA End-Joining Repair, Gene Library, HEK293 Cells, Humans, INDEL Mutation, Indoles pharmacology, MAP Kinase Kinase 1 chemistry, Phenotype, Proto-Oncogene Proteins B-raf chemistry, RNA, Guide, CRISPR-Cas Systems genetics, Recombinant Fusion Proteins chemistry, Sequence Alignment, Sulfonamides pharmacology, Transduction, Genetic, Vemurafenib, CRISPR-Cas Systems, MAP Kinase Kinase 1 genetics, Mutagenesis, Protein Engineering methods, Proto-Oncogene Proteins B-raf genetics
- Abstract
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2017
- Full Text
- View/download PDF
21. Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants.
- Author
-
Brenan L, Andreev A, Cohen O, Pantel S, Kamburov A, Cacchiarelli D, Persky NS, Zhu C, Bagul M, Goetz EM, Burgin AB, Garraway LA, Getz G, Mikkelsen TS, Piccioni F, Root DE, and Johannessen CM
- Subjects
- Cell Line, Tumor, Drug Resistance, Neoplasm drug effects, Dual Specificity Phosphatase 6 metabolism, Humans, Models, Molecular, Phenotype, Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Reproducibility of Results, Mitogen-Activated Protein Kinase 1 genetics, Mutation, Missense genetics
- Abstract
Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
22. Characterizing genomic alterations in cancer by complementary functional associations.
- Author
-
Kim JW, Botvinnik OB, Abudayyeh O, Birger C, Rosenbluh J, Shrestha Y, Abazeed ME, Hammerman PS, DiCara D, Konieczkowski DJ, Johannessen CM, Liberzon A, Alizad-Rahvar AR, Alexe G, Aguirre A, Ghandi M, Greulich H, Vazquez F, Weir BA, Van Allen EM, Tsherniak A, Shao DD, Zack TI, Noble M, Getz G, Beroukhim R, Garraway LA, Ardakani M, Romualdi C, Sales G, Barbie DA, Boehm JS, Hahn WC, Mesirov JP, and Tamayo P
- Subjects
- Drug Resistance, Neoplasm genetics, Genes, Neoplasm genetics, Genetic Predisposition to Disease genetics, Genome, Human genetics, Humans, Mutation genetics, Neoplasms diagnosis, Signal Transduction genetics, Biomarkers, Tumor genetics, Chromosome Mapping methods, Genome-Wide Association Study methods, Neoplasm Proteins genetics, Neoplasms genetics, Polymorphism, Single Nucleotide genetics
- Abstract
Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.
- Published
- 2016
- Full Text
- View/download PDF
23. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq.
- Author
-
Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jané-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, and Garraway LA
- Subjects
- Base Sequence, Cell Communication, Cell Cycle, Drug Resistance, Neoplasm genetics, Endothelial Cells pathology, Genomics, Humans, Immunotherapy, Lymphocyte Activation, Melanoma therapy, Microphthalmia-Associated Transcription Factor metabolism, Neoplasm Metastasis, RNA genetics, Sequence Analysis, RNA, Single-Cell Analysis, Stromal Cells pathology, T-Lymphocytes immunology, T-Lymphocytes pathology, Transcriptome, Melanoma genetics, Melanoma secondary, Skin Neoplasms pathology, Tumor Microenvironment
- Abstract
To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies., (Copyright © 2016, American Association for the Advancement of Science.)
- Published
- 2016
- Full Text
- View/download PDF
24. A functional landscape of resistance to ALK inhibition in lung cancer.
- Author
-
Wilson FH, Johannessen CM, Piccioni F, Tamayo P, Kim JW, Van Allen EM, Corsello SM, Capelletti M, Calles A, Butaney M, Sharifnia T, Gabriel SB, Mesirov JP, Hahn WC, Engelman JA, Meyerson M, Root DE, Jänne PA, and Garraway LA
- Subjects
- Anaplastic Lymphoma Kinase, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung pathology, Cell Line, Tumor, Crizotinib, ErbB Receptors genetics, Humans, Lung Neoplasms drug therapy, Lung Neoplasms pathology, Pyrazoles therapeutic use, Pyridines therapeutic use, Receptor Protein-Tyrosine Kinases genetics, Receptor Protein-Tyrosine Kinases metabolism, Receptor, ErbB-2 genetics, Receptors, Purinergic P2Y genetics, Signal Transduction drug effects, Signal Transduction genetics, Carcinoma, Non-Small-Cell Lung genetics, Drug Resistance, Neoplasm genetics, Lung Neoplasms genetics, Pyrazoles pharmacology, Pyridines pharmacology, Receptor Protein-Tyrosine Kinases antagonists & inhibitors
- Abstract
We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein-coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein-kinase-C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors, whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naive controls, supporting a role for these identified mechanisms in clinical ALK inhibitor resistance., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
25. Integrating phenotypic small-molecule profiling and human genetics: the next phase in drug discovery.
- Author
-
Johannessen CM, Clemons PA, and Wagner BK
- Subjects
- Drug Resistance genetics, Genome, Human genetics, Humans, Phenotype, Drug Discovery, Genetics, Medical, Small Molecule Libraries pharmacology
- Abstract
Over the past decade, tremendous progress in high-throughput small molecule-screening methods has facilitated the rapid expansion of phenotype-based data. Parallel advances in genomic characterization methods have complemented these efforts by providing a growing list of annotated cell line features. Together, these developments have paved the way for feature-based identification of novel, exploitable cellular dependencies, subsequently expanding our therapeutic toolkit in cancer and other diseases. Here, we provide an overview of the evolution of phenotypic small-molecule profiling and discuss the most significant and recent profiling and analytical efforts, their impact on the field, and their clinical ramifications. We additionally provide a perspective for future developments in phenotypic profiling efforts guided by genomic science., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
26. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors.
- Author
-
Konieczkowski DJ, Johannessen CM, Abudayyeh O, Kim JW, Cooper ZA, Piris A, Frederick DT, Barzily-Rokni M, Straussman R, Haq R, Fisher DE, Mesirov JP, Hahn WC, Flaherty KT, Wargo JA, Tamayo P, and Garraway LA
- Subjects
- Anilides pharmacology, Benzimidazoles pharmacology, Benzocycloheptenes pharmacology, Cell Line, Tumor, Cells, Cultured, Gene Expression Regulation, Neoplastic drug effects, Hepatocyte Growth Factor metabolism, Humans, Indoles pharmacology, Melanocytes cytology, Melanocytes drug effects, Melanocytes metabolism, Melanoma drug therapy, Microphthalmia-Associated Transcription Factor genetics, Microphthalmia-Associated Transcription Factor metabolism, Proto-Oncogene Proteins c-met metabolism, Pyridines pharmacology, Quinolines pharmacology, Sulfonamides pharmacology, Triazoles pharmacology, Drug Resistance, Neoplasm drug effects, MAP Kinase Signaling System drug effects, Melanoma genetics, Melanoma pathology, NF-kappa B p50 Subunit metabolism, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins B-raf genetics
- Abstract
Unlabelled: Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma., Significance: Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors., (©2014 American Association for Cancer Research.)
- Published
- 2014
- Full Text
- View/download PDF
27. MAP kinase pathway alterations in BRAF-mutant melanoma patients with acquired resistance to combined RAF/MEK inhibition.
- Author
-
Wagle N, Van Allen EM, Treacy DJ, Frederick DT, Cooper ZA, Taylor-Weiner A, Rosenberg M, Goetz EM, Sullivan RJ, Farlow DN, Friedrich DC, Anderka K, Perrin D, Johannessen CM, McKenna A, Cibulskis K, Kryukov G, Hodis E, Lawrence DP, Fisher S, Getz G, Gabriel SB, Carter SL, Flaherty KT, Wargo JA, and Garraway LA
- Subjects
- Aged, Antineoplastic Agents therapeutic use, Humans, Imidazoles therapeutic use, Male, Melanoma drug therapy, Melanoma metabolism, Middle Aged, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases genetics, Mitogen-Activated Protein Kinases genetics, Mitogen-Activated Protein Kinases metabolism, Mutation, Oximes therapeutic use, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf antagonists & inhibitors, Pyridones therapeutic use, Pyrimidinones therapeutic use, Signal Transduction, Skin Neoplasms drug therapy, Skin Neoplasms metabolism, raf Kinases antagonists & inhibitors, raf Kinases genetics, Drug Resistance, Neoplasm physiology, Melanoma genetics, Proto-Oncogene Proteins B-raf genetics, Skin Neoplasms genetics
- Abstract
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.
- Published
- 2014
- Full Text
- View/download PDF
28. The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma.
- Author
-
Van Allen EM, Wagle N, Sucker A, Treacy DJ, Johannessen CM, Goetz EM, Place CS, Taylor-Weiner A, Whittaker S, Kryukov GV, Hodis E, Rosenberg M, McKenna A, Cibulskis K, Farlow D, Zimmer L, Hillen U, Gutzmer R, Goldinger SM, Ugurel S, Gogas HJ, Egberts F, Berking C, Trefzer U, Loquai C, Weide B, Hassel JC, Gabriel SB, Carter SL, Getz G, Garraway LA, and Schadendorf D
- Subjects
- Cell Line, Tumor, Exome, Female, HEK293 Cells, Humans, Imidazoles therapeutic use, Indoles therapeutic use, MAP Kinase Kinase 1 genetics, MAP Kinase Kinase 2 genetics, Male, Melanoma drug therapy, Middle Aged, Mutation, Neoplasm Metastasis, Oximes therapeutic use, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins B-raf genetics, Sequence Analysis, DNA, Skin Neoplasms drug therapy, Sulfonamides therapeutic use, Vemurafenib, Antineoplastic Agents therapeutic use, Drug Resistance, Neoplasm genetics, Melanoma genetics, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf antagonists & inhibitors, Skin Neoplasms genetics
- Abstract
Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.
- Published
- 2014
- Full Text
- View/download PDF
29. A melanocyte lineage program confers resistance to MAP kinase pathway inhibition.
- Author
-
Johannessen CM, Johnson LA, Piccioni F, Townes A, Frederick DT, Donahue MK, Narayan R, Flaherty KT, Wargo JA, Root DE, and Garraway LA
- Subjects
- CREB-Binding Protein metabolism, Cell Line, Tumor, Cell Lineage, Cyclic AMP metabolism, Gene Expression Regulation, Neoplastic, HEK293 Cells, Humans, Melanocytes cytology, Melanocytes enzymology, Melanoma enzymology, Melanoma physiopathology, Signal Transduction, Transcription Factors genetics, Transcription Factors metabolism, Antineoplastic Agents pharmacology, Drug Resistance, Neoplasm genetics, Melanocytes drug effects, Mitogen-Activated Protein Kinases metabolism, Protein Kinase Inhibitors pharmacology
- Abstract
Malignant melanomas harbouring point mutations (Val600Glu) in the serine/threonine-protein kinase BRAF (BRAF(V600E)) depend on RAF-MEK-ERK signalling for tumour cell growth. RAF and MEK inhibitors show remarkable clinical efficacy in BRAF(V600E) melanoma; however, resistance to these agents remains a formidable challenge. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here we carried out systematic gain-of-function resistance studies by expressing more than 15,500 genes individually in a BRAF(V600E) melanoma cell line treated with RAF, MEK, ERK or combined RAF-MEK inhibitors. These studies revealed a cyclic-AMP-dependent melanocytic signalling network not previously associated with drug resistance, including G-protein-coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAF(V600E) melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF-MEK inhibition but restored in relapsing tumours. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAPK-pathway and histone-deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF-MEK-ERK inhibition, which may be overcome by combining signalling- and chromatin-directed therapeutics.
- Published
- 2013
- Full Text
- View/download PDF
30. MicroSCALE screening reveals genetic modifiers of therapeutic response in melanoma.
- Author
-
Wood KC, Konieczkowski DJ, Johannessen CM, Boehm JS, Tamayo P, Botvinnik OB, Mesirov JP, Hahn WC, Root DE, Garraway LA, and Sabatini DM
- Subjects
- Apoptosis, Blotting, Western, Cell Cycle, Cell Line, Tumor, Fluorescent Antibody Technique, Humans, MAP Kinase Signaling System drug effects, Melanoma genetics, Mutation, Pharmacogenetics, Proto-Oncogene Proteins B-raf genetics, Melanoma drug therapy
- Abstract
Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but owing to technical limitations they have been restricted for use with a narrow range of cell lines and short-term assays. Here, we describe MicroSCALE (Microarrays of Spatially Confined Adhesive Lentiviral Features), a cell microarray-based platform that enables application of this technology to a wide range of cell types and longer-term assays. We used MicroSCALE to uncover kinases that when overexpressed partially desensitized B-RAFV600E-mutant melanoma cells to inhibitors of the mitogen-activated protein kinase kinase kinase (MAPKKK) RAF, the MAPKKs MEK1 and 2 (MEK1/2, mitogen-activated protein kinase kinase 1 and 2), mTOR (mammalian target of rapamycin), or PI3K (phosphatidylinositol 3-kinase). These screens indicated that cells treated with inhibitors acting through common mechanisms were affected by a similar profile of overexpressed proteins. In contrast, screens involving inhibitors acting through distinct mechanisms yielded unique profiles, a finding that has potential relevance for small-molecule target identification and combination drugging studies. Further, by integrating large-scale functional screening results with cancer cell line gene expression and pharmacological sensitivity data, we validated the nuclear factor κB pathway as a potential mediator of resistance to MAPK pathway inhibitors. The MicroSCALE platform described here may enable new classes of large-scale, resource-efficient screens that were not previously feasible, including those involving combinations of cell lines, perturbations, and assay outputs or those involving limited numbers of cells and limited or expensive reagents.
- Published
- 2012
- Full Text
- View/download PDF
31. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling.
- Author
-
Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, Kehoe SM, Johannessen CM, Macconaill LE, Hahn WC, Meyerson M, and Garraway LA
- Subjects
- Adult, Antineoplastic Agents pharmacology, Disease Progression, Fatal Outcome, Gene Expression Profiling, Humans, Indoles pharmacology, Male, Melanoma secondary, Phosphatidylethanolamine Binding Protein genetics, Precision Medicine methods, Protein Kinase Inhibitors pharmacology, Skin Neoplasms pathology, Sulfonamides pharmacology, Vemurafenib, Antineoplastic Agents therapeutic use, DNA Mutational Analysis, Drug Resistance, Neoplasm genetics, Indoles therapeutic use, MAP Kinase Kinase 1 genetics, Melanoma drug therapy, Melanoma genetics, Mutation, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins B-raf genetics, Skin Neoplasms drug therapy, Skin Neoplasms genetics, Sulfonamides therapeutic use
- Abstract
A detailed understanding of the mechanisms by which tumors acquire resistance to targeted anticancer agents should speed the development of treatment strategies with lasting clinical efficacy. RAF inhibition in BRAF-mutant melanoma exemplifies the promise and challenge of many targeted drugs; although response rates are high, resistance invariably develops. Here, we articulate overarching principles of resistance to kinase inhibitors, as well as a translational approach to characterize resistance in the clinical setting through tumor mutation profiling. As a proof of principle, we performed targeted, massively parallel sequencing of 138 cancer genes in a tumor obtained from a patient with melanoma who developed resistance to PLX4032 after an initial dramatic response. The resulting profile identified an activating mutation at codon 121 in the downstream kinase MEK1 that was absent in the corresponding pretreatment tumor. The MEK1(C121S) mutation was shown to increase kinase activity and confer robust resistance to both RAF and MEK inhibition in vitro. Thus, MEK1(C121S) or functionally similar mutations are predicted to confer resistance to combined MEK/RAF inhibition. These results provide an instructive framework for assessing mechanisms of acquired resistance to kinase inhibition and illustrate the use of emerging technologies in a manner that may accelerate personalized cancer medicine.
- Published
- 2011
- Full Text
- View/download PDF
32. A public genome-scale lentiviral expression library of human ORFs.
- Author
-
Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, Lubonja R, Thomas SR, Alkan O, Bhimdi T, Green TM, Johannessen CM, Silver SJ, Nguyen C, Murray RR, Hieronymus H, Balcha D, Fan C, Lin C, Ghamsari L, Vidal M, Hahn WC, Hill DE, and Root DE
- Subjects
- Humans, Open Reading Frames, Cloning, Molecular methods, Genetic Vectors genetics, Genomic Library, Lentivirus genetics
- Abstract
Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.
- Published
- 2011
- Full Text
- View/download PDF
33. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.
- Author
-
Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP, Barretina J, Caponigro G, Hieronymus H, Murray RR, Salehi-Ashtiani K, Hill DE, Vidal M, Zhao JJ, Yang X, Alkan O, Kim S, Harris JL, Wilson CJ, Myer VE, Finan PM, Root DE, Roberts TM, Golub T, Flaherty KT, Dummer R, Weber BL, Sellers WR, Schlegel R, Wargo JA, Hahn WC, and Garraway LA
- Subjects
- Allosteric Regulation, Cell Line, Tumor, Clinical Trials as Topic, Enzyme Activation drug effects, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Gene Library, Humans, Indoles pharmacology, Indoles therapeutic use, MAP Kinase Kinase Kinases genetics, Melanoma drug therapy, Melanoma enzymology, Melanoma genetics, Melanoma metabolism, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases metabolism, Open Reading Frames genetics, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins B-raf chemistry, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism, Proto-Oncogene Proteins c-raf genetics, Proto-Oncogene Proteins c-raf metabolism, Sulfonamides pharmacology, Sulfonamides therapeutic use, Vemurafenib, Drug Resistance, Neoplasm drug effects, Drug Resistance, Neoplasm genetics, MAP Kinase Kinase Kinases metabolism, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins B-raf antagonists & inhibitors
- Abstract
Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
- Published
- 2010
- Full Text
- View/download PDF
34. TORC1 is essential for NF1-associated malignancies.
- Author
-
Johannessen CM, Johnson BW, Williams SM, Chan AW, Reczek EE, Lynch RC, Rioth MJ, McClatchey A, Ryeom S, and Cichowski K
- Subjects
- Animals, Cell Line, Cyclin D, Cyclins genetics, Cyclins metabolism, Disease Models, Animal, Gene Expression Regulation drug effects, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neoplasms metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, Sirolimus pharmacology, TOR Serine-Threonine Kinases, Transcription Factors metabolism, Genes, Neurofibromatosis 1, Neoplasms genetics, Transcription Factors physiology
- Abstract
Inactivating mutations in NF1 underlie the prevalent familial cancer syndrome neurofibromatosis type 1 [1]. The NF1-encoded protein is a Ras GTPase-activating protein (RasGAP) [2]. Accordingly, Ras is aberrantly activated in NF1-deficient tumors; however, it is unknown which effector pathways critically function in tumor development. Here we provide in vivo evidence that TORC1/mTOR activity is essential for tumorigenesis. Specifically, we show that the mTOR inhibitor rapamycin potently suppresses the growth of aggressive NF1-associated malignancies in a genetically engineered murine model. However, in these tumors rapamycin does not function via mechanisms generally assumed to mediate tumor suppression, including inhibition of HIF-1alpha and indirect suppression of AKT, but does suppress the mTOR target Cyclin D1 [3]. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for this commonly untreatable malignancy. Moreover, they indicate that mTOR inhibitors do not suppress all tumor types via the same mechanism, suggesting that current biomarkers that rely on HIF-1alpha suppression may not be informative for all cancers. Finally, our results reveal important differences between the effects of mTOR inhibition on the microvasculature in genetically engineered versus xenograft models and indicate that the former may be required for effective preclinical screening with this class of inhibitors.
- Published
- 2008
- Full Text
- View/download PDF
35. A negative feedback signaling network underlies oncogene-induced senescence.
- Author
-
Courtois-Cox S, Genther Williams SM, Reczek EE, Johnson BW, McGillicuddy LT, Johannessen CM, Hollstein PE, MacCollin M, and Cichowski K
- Subjects
- Animals, Cells, Cultured, Feedback, Genes, Neurofibromatosis 1 physiology, Genes, Retinoblastoma physiology, Genes, p53 physiology, Humans, Mice, Neoplasms genetics, Neoplasms pathology, Phosphatidylinositol 3-Kinases physiology, Stem Cells pathology, raf Kinases physiology, Cellular Senescence, Genes, ras physiology, Signal Transduction physiology
- Abstract
Oncogene-induced senescence functions to limit tumor development. However, a complete understanding of the signals that trigger this type of senescence is currently lacking. We found that mutations affecting NF1, Raf, and Ras induce a global negative feedback response that potently suppresses Ras and/or its effectors. Moreover, these signals promote senescence by inhibiting the Ras/PI3K pathway, which can impact the senescence machinery through HDM2 and FOXO. This negative feedback program is regulated in part by RasGEFs, Sprouty proteins, RasGAPs, and MKPs. Moreover, these signals function in vivo in benign human tumors. Thus, the ultimate response to the aberrant activation of the Ras pathway is a multifaceted negative feedback signaling network that terminates the oncogenic signal and participates in the senescence response.
- Published
- 2006
- Full Text
- View/download PDF
36. Tumor-specific p73 up-regulation mediates p63 dependence in squamous cell carcinoma.
- Author
-
DeYoung MP, Johannessen CM, Leong CO, Faquin W, Rocco JW, and Ellisen LW
- Subjects
- Animals, Carcinoma, Squamous Cell pathology, Cell Cycle genetics, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21 biosynthesis, Cyclin-Dependent Kinase Inhibitor p21 genetics, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins genetics, Humans, Keratinocytes pathology, Mice, Mice, Knockout, Nuclear Proteins biosynthesis, Nuclear Proteins genetics, Phosphoproteins deficiency, Phosphoproteins genetics, RNA, Small Interfering pharmacology, Skin Neoplasms pathology, Trans-Activators deficiency, Trans-Activators genetics, Transcription Factors, Transduction, Genetic, Tumor Suppressor Protein p53 antagonists & inhibitors, Tumor Suppressor Protein p53 physiology, Tumor Suppressor Proteins biosynthesis, Tumor Suppressor Proteins genetics, Carcinoma, Squamous Cell genetics, DNA-Binding Proteins physiology, Keratinocytes metabolism, Neoplasm Proteins physiology, Nuclear Proteins physiology, Phosphoproteins physiology, Skin Neoplasms genetics, Trans-Activators physiology, Tumor Suppressor Proteins physiology
- Abstract
p63 is essential for normal epithelial development and is overexpressed in the vast majority of squamous cell carcinomas (SCC). Recent work had shown that DeltaNp63alpha is essential for survival of SCC cells, raising the possibility that the p63 pathway may be an attractive therapeutic target in these tumors. Nevertheless, it is unknown whether a therapeutic window exists for inhibiting p63 in tumor cells versus normal epithelia. Here, we show that SCC cells are uniquely dependent on DeltaNp63alpha for survival, unlike normal p63-expressing epithelial cells, and that dependence is mediated through tumor-specific up-regulation of the related protein p73. In normal primary human keratinocytes, we find that inhibition of endogenous p63 by RNA interference (RNAi) induces p21(CIP1) expression, inhibits cell cycle progression, and ultimately promotes cellular senescence. In contrast, p63 inhibition in SCC cells induces proapoptotic bcl-2 family members and rapidly triggers apoptosis. Expression of p73 is low in uncultured basal keratinocytes but is markedly up-regulated in both SCC cell lines and primary tumors in vivo. Whereas p21(CIP1) induction following loss of p63 in normal cells is independent of p53 and p73, both proapoptotic gene induction and cell death following p63 RNAi in tumor cells are p73 dependent. Finally, ectopic p73 expression in primary keratinocytes does not affect baseline cell proliferation but is sufficient to trigger cell death following loss of p63. Together, these findings define a specific molecular mechanism of p63 dependence through p73 up-regulation, and they provide a rationale for targeting the p63 pathway as a therapeutic strategy in SCCs.
- Published
- 2006
- Full Text
- View/download PDF
37. Regulation of mTOR and cell growth in response to energy stress by REDD1.
- Author
-
Sofer A, Lei K, Johannessen CM, and Ellisen LW
- Subjects
- AMP-Activated Protein Kinase Kinases, Animals, Cell Size, Energy Metabolism genetics, Enzyme Activation, Humans, Mice, Monomeric GTP-Binding Proteins genetics, Monomeric GTP-Binding Proteins metabolism, Neuropeptides genetics, Neuropeptides metabolism, Phosphorylation, Protein Kinases physiology, RNA Interference, Ras Homolog Enriched in Brain Protein, TOR Serine-Threonine Kinases, Transcription Factors genetics, Tuberous Sclerosis Complex 2 Protein, Energy Metabolism physiology, Protein Kinases metabolism, Repressor Proteins metabolism, Transcription Factors physiology, Tumor Suppressor Proteins metabolism
- Abstract
The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. We have recently identified the stress response REDD1 gene as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. Here, we demonstrate that REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, as regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway.
- Published
- 2005
- Full Text
- View/download PDF
38. The NF1 tumor suppressor critically regulates TSC2 and mTOR.
- Author
-
Johannessen CM, Reczek EE, James MF, Brems H, Legius E, and Cichowski K
- Subjects
- Animals, Genetic Vectors, Immunoprecipitation, Mice, NIH 3T3 Cells, Phosphatidylinositol 3-Kinases metabolism, TOR Serine-Threonine Kinases, Transfection, Tuberous Sclerosis Complex 2 Protein, ras Proteins metabolism, Neurofibromatosis 1 metabolism, Neurofibromin 1 metabolism, Protein Kinases metabolism, Repressor Proteins metabolism, Signal Transduction physiology, Tumor Suppressor Proteins metabolism
- Abstract
Loss-of-function mutations in the NF1 tumor suppressor gene underlie the familial cancer syndrome neurofibromatosis type I (NF1). The NF1-encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (RasGAP). Accordingly, deregulation of Ras is thought to contribute to NF1 development. However, the critical effector pathways involved in disease pathogenesis are still unknown. We show here that the mTOR pathway is tightly regulated by neurofibromin. mTOR is constitutively activated in both NF1-deficient primary cells and human tumors in the absence of growth factors. This aberrant activation depends on Ras and PI3 kinase, and is mediated by the phosphorylation and inactivation of the TSC2-encoded protein tuberin by AKT. Importantly, tumor cell lines derived from NF1 patients, and a genetically engineered cell system that requires Nf1-deficiency for transformation, are highly sensitive to the mTOR inhibitor rapamycin. Furthermore, while we show that the activation of endogenous Ras leads to constitutive mTOR signaling in this disease state, we also demonstrate that in normal cells Ras is differentially required for mTOR signaling in response to various growth factors. Thus, these findings identify the NF1 tumor suppressor as an indispensable regulator of TSC2 and mTOR. Furthermore, our results also demonstrate that Ras plays a critical role in the activation of mTOR in both normal and tumorigenic settings. Finally, these data suggest that rapamycin, or its derivatives, may represent a viable therapy for NF1.
- Published
- 2005
- Full Text
- View/download PDF
39. REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species.
- Author
-
Ellisen LW, Ramsayer KD, Johannessen CM, Yang A, Beppu H, Minda K, Oliner JD, McKeon F, and Haber DA
- Subjects
- Adenoviridae genetics, Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Blotting, Western, Cell Differentiation, Cloning, Molecular, Conserved Sequence, DNA-Binding Proteins, Down-Regulation, Epithelial Cells cytology, Evolution, Molecular, Fibroblasts metabolism, Genes, Tumor Suppressor, Humans, In Situ Hybridization, Keratinocytes metabolism, Mice, Microscopy, Fluorescence, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Oxidative Stress, Precipitin Tests, Promoter Regions, Genetic, Protein Isoforms, RNA, Messenger metabolism, Sequence Homology, Amino Acid, Transcription Factors, Transcriptional Activation, Tumor Suppressor Proteins, Ultraviolet Rays, Xenopus, beta-Galactosidase metabolism, Gene Expression Regulation, Membrane Proteins, Phosphoproteins metabolism, Reactive Oxygen Species, Trans-Activators metabolism, Transcription, Genetic, Tumor Suppressor Protein p53 metabolism, Up-Regulation, Xenopus Proteins
- Abstract
We identified REDD1 as a novel transcriptional target of p53 induced following DNA damage. During embryogenesis, REDD1 expression mirrors the tissue-specific pattern of the p53 family member p63, and TP63 null embryos show virtually no expression of REDD1, which is restored in mouse embryo fibroblasts following p63 expression. In differentiating primary keratinocytes, TP63 and REDD1 expression are coordinately downregulated, and ectopic expression of either gene inhibits in vitro differentiation. REDD1 appears to function in the regulation of reactive oxygen species (ROS); we show that TP63 null fibroblasts have decreased ROS levels and reduced sensitivity to oxidative stress, which are both increased following ectopic expression of either TP63 or REDD1. Thus, REDD1 encodes a shared transcriptional target that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation.
- Published
- 2002
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.