30 results on '"Brachmann SM"'
Search Results
2. Direct and selective pharmacological disruption of the YAP-TEAD interface by IAG933 inhibits Hippo-dependent and RAS-MAPK-altered cancers.
- Author
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Chapeau EA, Sansregret L, Galli GG, Chène P, Wartmann M, Mourikis TP, Jaaks P, Baltschukat S, Barbosa IAM, Bauer D, Brachmann SM, Delaunay C, Estadieu C, Faris JE, Furet P, Harlfinger S, Hueber A, Jiménez Núñez E, Kodack DP, Mandon E, Martin T, Mesrouze Y, Romanet V, Scheufler C, Sellner H, Stamm C, Sterker D, Tordella L, Hofmann F, Soldermann N, and Schmelzle T
- Subjects
- Humans, Animals, Mice, Cell Line, Tumor, Adaptor Proteins, Signal Transducing metabolism, YAP-Signaling Proteins metabolism, Neoplasms drug therapy, Neoplasms metabolism, DNA-Binding Proteins metabolism, Signal Transduction drug effects, TEA Domain Transcription Factors, ras Proteins metabolism, Female, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Transcription Factors metabolism, Protein Serine-Threonine Kinases metabolism, Hippo Signaling Pathway, Xenograft Model Antitumor Assays
- Abstract
The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
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3. CRISPR Screening Identifies Mechanisms of Resistance to KRASG12C and SHP2 Inhibitor Combinations in Non-Small Cell Lung Cancer.
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Prahallad A, Weiss A, Voshol H, Kerr G, Sprouffske K, Yuan T, Ruddy D, Meistertzheim M, Kazic-Legueux M, Kottarathil T, Piquet M, Cao Y, Martinuzzi-Duboc L, Buhles A, Adler F, Mannino S, Tordella L, Sansregret L, Maira SM, Graus Porta D, Fedele C, and Brachmann SM
- Subjects
- Humans, Phosphatidylinositol 3-Kinases genetics, Clustered Regularly Interspaced Short Palindromic Repeats, Proto-Oncogene Proteins p21(ras) genetics, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Early Detection of Cancer, Enzyme Inhibitors therapeutic use, Mutation, Cell Line, Tumor, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung pathology, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Lung Neoplasms pathology
- Abstract
Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non-small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments., Significance: Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005., (©2023 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2023
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4. JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRAS G12C for the Treatment of Solid Tumors.
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Lorthiois E, Gerspacher M, Beyer KS, Vaupel A, Leblanc C, Stringer R, Weiss A, Wilcken R, Guthy DA, Lingel A, Bomio-Confaglia C, Machauer R, Rigollier P, Ottl J, Arz D, Bernet P, Desjonqueres G, Dussauge S, Kazic-Legueux M, Lozac'h MA, Mura C, Sorge M, Todorov M, Warin N, Zink F, Voshol H, Zecri FJ, Sedrani RC, Ostermann N, Brachmann SM, and Cotesta S
- Subjects
- Animals, Humans, Mice, Disease Models, Animal, Drug Design, Mutation, Pyrazoles pharmacology, Pyrazoles therapeutic use, Neoplasms drug therapy, Neoplasms genetics, Proto-Oncogene Proteins p21(ras)
- Abstract
Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRAS
G12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).- Published
- 2022
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5. Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.
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Weiss A, Lorthiois E, Barys L, Beyer KS, Bomio-Confaglia C, Burks H, Chen X, Cui X, de Kanter R, Dharmarajan L, Fedele C, Gerspacher M, Guthy DA, Head V, Jaeger A, Núñez EJ, Kearns JD, Leblanc C, Maira SM, Murphy J, Oakman H, Ostermann N, Ottl J, Rigollier P, Roman D, Schnell C, Sedrani R, Shimizu T, Stringer R, Vaupel A, Voshol H, Wessels P, Widmer T, Wilcken R, Xu K, Zecri F, Farago AF, Cotesta S, and Brachmann SM
- Subjects
- Humans, Mutation, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Indazoles chemistry, Indazoles pharmacology, Neoplasms drug therapy, Neoplasms enzymology, Neoplasms genetics, Proto-Oncogene Proteins p21(ras) antagonists & inhibitors, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism
- Abstract
Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors., Significance: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397., (©2022 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2022
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6. A RAC-GEF network critical for early intestinal tumourigenesis.
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Pickering KA, Gilroy K, Cassidy JW, Fey SK, Najumudeen AK, Zeiger LB, Vincent DF, Gay DM, Johansson J, Fordham RP, Miller B, Clark W, Hedley A, Unal EB, Kiel C, McGhee E, Machesky LM, Nixon C, Johnsson AE, Bain M, Strathdee D, van Hoof SR, Medema JP, Anderson KI, Brachmann SM, Stucke VM, Malliri A, Drysdale M, Turner M, Serrano L, Myant K, Campbell AD, and Sansom OJ
- Subjects
- Adenomatous Polyposis Coli Protein metabolism, Animals, Carcinogenesis genetics, Homeostasis, Intestines ultrastructure, Mice, Knockout, Mutation genetics, Organ Specificity, Phenotype, Proto-Oncogene Proteins c-vav metabolism, Proto-Oncogene Proteins p21(ras) genetics, T-Lymphoma Invasion and Metastasis-inducing Protein 1 metabolism, Up-Regulation, Wnt Signaling Pathway, Mice, Carcinogenesis metabolism, Carcinogenesis pathology, Guanine Nucleotide Exchange Factors metabolism, Intestines pathology, Signal Transduction, rac1 GTP-Binding Protein metabolism
- Abstract
RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2
-/- Vav3-/- Tiam1-/- ), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.- Published
- 2021
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7. Genetic heterogeneity and clonal evolution during metastasis in breast cancer patient-derived tumor xenograft models.
- Author
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Sprouffske K, Kerr G, Li C, Prahallad A, Rebmann R, Waehle V, Naumann U, Bitter H, Jensen MR, Hofmann F, Brachmann SM, Ferretti S, and Kauffmann A
- Abstract
Genetic heterogeneity within a tumor arises by clonal evolution, and patients with highly heterogeneous tumors are more likely to be resistant to therapy and have reduced survival. Clonal evolution also occurs when a subset of cells leave the primary tumor to form metastases, which leads to reduced genetic heterogeneity at the metastatic site. Although this process has been observed in human cancer, experimental models which recapitulate this process are lacking. Patient-derived tumor xenografts (PDX) have been shown to recapitulate the patient's original tumor's intra-tumor genetic heterogeneity, as well as its genomics and response to treatment, but whether they can be used to model clonal evolution in the metastatic process is currently unknown. Here, we address this question by following genetic changes in two breast cancer PDX models during metastasis. First, we discovered that mouse stroma can be a confounding factor in assessing intra-tumor heterogeneity by whole exome sequencing, thus we developed a new bioinformatic approach to correct for this. Finally, in a spontaneous, but not experimental (tail-vein) metastasis model we observed a loss of heterogeneity in PDX metastases compared to their orthotopic "primary" tumors, confirming that PDX models can faithfully mimic the clonal evolution process undergone in human patients during metastatic spreading., (© 2020 The Authors.)
- Published
- 2020
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8. SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.
- Author
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Lu H, Liu C, Velazquez R, Wang H, Dunkl LM, Kazic-Legueux M, Haberkorn A, Billy E, Manchado E, Brachmann SM, Moody SE, Engelman JA, Hammerman PS, Caponigro G, Mohseni M, and Hao HX
- Subjects
- Acrylonitrile pharmacology, Acrylonitrile therapeutic use, Aniline Compounds pharmacology, Animals, Cell Line, Tumor, Female, Humans, Mice, Mice, Nude, Neoplasms metabolism, Transfection, Xenograft Model Antitumor Assays, Acrylonitrile analogs & derivatives, Aniline Compounds therapeutic use, Neoplasms genetics, Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation in vitro and in vivo These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic., (©2019 American Association for Cancer Research.)
- Published
- 2019
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9. Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments.
- Author
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Martin KJ, McGhee EJ, Schwarz JP, Drysdale M, Brachmann SM, Stucke V, Sansom OJ, and Anderson KI
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- Biosensing Techniques, Fluorescence, Protein Binding, Fluorescence Resonance Energy Transfer methods, Luminescent Proteins metabolism
- Abstract
FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.
- Published
- 2018
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10. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.
- Author
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McDonald ER 3rd, de Weck A, Schlabach MR, Billy E, Mavrakis KJ, Hoffman GR, Belur D, Castelletti D, Frias E, Gampa K, Golji J, Kao I, Li L, Megel P, Perkins TA, Ramadan N, Ruddy DA, Silver SJ, Sovath S, Stump M, Weber O, Widmer R, Yu J, Yu K, Yue Y, Abramowski D, Ackley E, Barrett R, Berger J, Bernard JL, Billig R, Brachmann SM, Buxton F, Caothien R, Caushi JX, Chung FS, Cortés-Cros M, deBeaumont RS, Delaunay C, Desplat A, Duong W, Dwoske DA, Eldridge RS, Farsidjani A, Feng F, Feng J, Flemming D, Forrester W, Galli GG, Gao Z, Gauter F, Gibaja V, Haas K, Hattenberger M, Hood T, Hurov KE, Jagani Z, Jenal M, Johnson JA, Jones MD, Kapoor A, Korn J, Liu J, Liu Q, Liu S, Liu Y, Loo AT, Macchi KJ, Martin T, McAllister G, Meyer A, Mollé S, Pagliarini RA, Phadke T, Repko B, Schouwey T, Shanahan F, Shen Q, Stamm C, Stephan C, Stucke VM, Tiedt R, Varadarajan M, Venkatesan K, Vitari AC, Wallroth M, Weiler J, Zhang J, Mickanin C, Myer VE, Porter JA, Lai A, Bitter H, Lees E, Keen N, Kauffmann A, Stegmeier F, Hofmann F, Schmelzle T, and Sellers WR
- Subjects
- Cell Line, Tumor, Gene Library, Gene Regulatory Networks, Humans, Multiprotein Complexes metabolism, Neoplasms metabolism, Oncogenes, RNA, Small Interfering, Signal Transduction, Transcription Factors metabolism, Neoplasms genetics, Neoplasms pathology, RNA Interference
- Abstract
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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11. Maximizing the Efficacy of MAPK-Targeted Treatment in PTENLOF/BRAFMUT Melanoma through PI3K and IGF1R Inhibition.
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Herkert B, Kauffmann A, Mollé S, Schnell C, Ferrat T, Voshol H, Juengert J, Erasimus H, Marszalek G, Kazic-Legueux M, Billy E, Ruddy D, Stump M, Guthy D, Ristov M, Calkins K, Maira SM, Sellers WR, Hofmann F, Hall MN, and Brachmann SM
- Subjects
- Apoptosis, Cell Death, Cell Proliferation, Humans, Melanoma pathology, Proteomics, MAP Kinase Signaling System genetics, Melanoma genetics, PTEN Phosphohydrolase metabolism, Proto-Oncogene Proteins B-raf genetics, Receptor, IGF Type 1 metabolism
- Abstract
The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kβ inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kβ inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kβ, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kβ + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response., (©2015 American Association for Cancer Research.)
- Published
- 2016
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12. Characterization of the novel and specific PI3Kα inhibitor NVP-BYL719 and development of the patient stratification strategy for clinical trials.
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Fritsch C, Huang A, Chatenay-Rivauday C, Schnell C, Reddy A, Liu M, Kauffmann A, Guthy D, Erdmann D, De Pover A, Furet P, Gao H, Ferretti S, Wang Y, Trappe J, Brachmann SM, Maira SM, Wilson C, Boehm M, Garcia-Echeverria C, Chene P, Wiesmann M, Cozens R, Lehar J, Schlegel R, Caravatti G, Hofmann F, and Sellers WR
- Subjects
- Animals, Antineoplastic Agents pharmacokinetics, Cell Line, Tumor, Class I Phosphatidylinositol 3-Kinases, Disease Models, Animal, Drug Evaluation, Preclinical, Drug Resistance, Neoplasm genetics, Female, Humans, Inhibitory Concentration 50, Mice, Mutation, Neoplasms drug therapy, Neoplasms genetics, Neoplasms metabolism, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase metabolism, Phosphatidylinositol 3-Kinases genetics, Rats, Thiazoles pharmacokinetics, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Phosphoinositide-3 Kinase Inhibitors, Thiazoles pharmacology
- Abstract
Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.
- Published
- 2014
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13. Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations.
- Author
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Brachmann SM, Kleylein-Sohn J, Gaulis S, Kauffmann A, Blommers MJ, Kazic-Legueux M, Laborde L, Hattenberger M, Stauffer F, Vaxelaire J, Romanet V, Henry C, Murakami M, Guthy DA, Sterker D, Bergling S, Wilson C, Brümmendorf T, Fritsch C, Garcia-Echeverria C, Sellers WR, Hofmann F, and Maira SM
- Subjects
- Animals, Cell Cycle Checkpoints drug effects, Cell Line, Tumor, Gene Expression Profiling, Gene Expression Regulation, Neoplastic drug effects, Humans, Indazoles pharmacology, Mice, Mitosis drug effects, Protein Multimerization drug effects, Rats, Sulfonamides pharmacology, Tubulin metabolism, Aminopyridines pharmacology, Morpholines pharmacology, Phosphoinositide-3 Kinase Inhibitors
- Abstract
The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.
- Published
- 2012
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14. K-RAS mutant pancreatic tumors show higher sensitivity to MEK than to PI3K inhibition in vivo.
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Hofmann I, Weiss A, Elain G, Schwaederle M, Sterker D, Romanet V, Schmelzle T, Lai A, Brachmann SM, Bentires-Alj M, Roberts TM, Sellers WR, Hofmann F, and Maira SM
- Subjects
- Animals, Benzimidazoles pharmacology, Cell Line, Tumor, Cell Proliferation drug effects, Extracellular Signal-Regulated MAP Kinases metabolism, Female, Gene Knockdown Techniques, HEK293 Cells, Humans, Indazoles pharmacology, Mice, Mice, Nude, Mitogen-Activated Protein Kinase Kinases metabolism, Models, Biological, Pancreatic Neoplasms pathology, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation drug effects, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-akt metabolism, Proto-Oncogene Proteins p21(ras), Sulfonamides pharmacology, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mutation genetics, Pancreatic Neoplasms enzymology, Pancreatic Neoplasms genetics, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins genetics, Xenograft Model Antitumor Assays, ras Proteins genetics
- Abstract
Activating K-RAS mutations occur at a frequency of 90% in pancreatic cancer, and to date no therapies exist targeting this oncogene. K-RAS signals via downstream effector pathways such as the MAPK and the PI3K signaling pathways, and much effort has been focused on developing drugs targeting components of these pathways. To better understand the requirements for K-RAS and its downstream signaling pathways MAPK and PI3K in pancreatic tumor maintenance, we established an inducible K-RAS knock down system that allowed us to ablate K-RAS in established tumors. Knock down of K-RAS resulted in impaired tumor growth in all pancreatic xenograft models tested, demonstrating that K-RAS expression is indeed required for tumor maintenance of K-RAS mutant pancreatic tumors. We further examined signaling downstream of K-RAS, and detected a robust reduction of pERK levels upon K-RAS knock down. In contrast, no effect on pAKT levels could be observed due to almost undetectable basal expression levels. To investigate the requirement of the MAPK and the PI3K pathways on tumor maintenance, three selected pancreatic xenograft models were tested for their response to MEK or PI3K inhibition. Tumors of all three models regressed upon MEK inhibition, but showed less pronounced response to PI3K inhibition. The effect of MEK inhibition on pancreatic xenografts could be enhanced further by combined application of a PI3K inhibitor. These data provide further rationale for testing combinations of MEK and PI3K inhibitors in clinical trials comprising a patient population with pancreatic cancer harboring mutations in K-RAS.
- Published
- 2012
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15. Identification of elongation factor G as the conserved cellular target of argyrin B.
- Author
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Nyfeler B, Hoepfner D, Palestrant D, Kirby CA, Whitehead L, Yu R, Deng G, Caughlan RE, Woods AL, Jones AK, Barnes SW, Walker JR, Gaulis S, Hauy E, Brachmann SM, Krastel P, Studer C, Riedl R, Estoppey D, Aust T, Movva NR, Wang Z, Salcius M, Michaud GA, McAllister G, Murphy LO, Tallarico JA, Wilson CJ, and Dean CR
- Subjects
- Allosteric Site, Amino Acid Sequence, Animals, Burkholderia drug effects, Cell Line, Tumor, Conserved Sequence, Crystallography, X-Ray, Humans, Mammals, Microbial Sensitivity Tests, Mitochondrial Proteins metabolism, Molecular Sequence Data, Mutant Proteins chemistry, Mutant Proteins metabolism, Oligopeptides chemistry, Oligopeptides pharmacology, Peptide Elongation Factor G antagonists & inhibitors, Peptide Elongation Factor G chemistry, Protein Binding drug effects, Pseudomonas aeruginosa drug effects, Saccharomyces cerevisiae metabolism, Sequence Homology, Amino Acid, Oligopeptides metabolism, Peptide Elongation Factor G metabolism
- Abstract
Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.
- Published
- 2012
- Full Text
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16. Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells.
- Author
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Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, Lane H, Wang S, Garcia-Echeverria C, and Maira SM
- Subjects
- Apoptosis genetics, Apoptosis physiology, Breast Neoplasms genetics, Breast Neoplasms metabolism, Breast Neoplasms pathology, Caspase 9 metabolism, Cell Line, Tumor, Class I Phosphatidylinositol 3-Kinases, Drug Resistance, Neoplasm genetics, Drug Resistance, Neoplasm physiology, Enzyme Inhibitors pharmacology, Female, Gene Amplification, Humans, Mutation, PTEN Phosphohydrolase genetics, Phosphatidylinositol 3-Kinases genetics, Poly(ADP-ribose) Polymerases metabolism, Signal Transduction drug effects, TOR Serine-Threonine Kinases, Apoptosis drug effects, Breast Neoplasms drug therapy, Genes, erbB-2 drug effects, Imidazoles pharmacology, Intracellular Signaling Peptides and Proteins drug effects, Phosphoinositide-3 Kinase Inhibitors, Protein Serine-Threonine Kinases drug effects, Quinolines pharmacology
- Abstract
NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.
- Published
- 2009
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17. p85 Associates with unphosphorylated PTEN and the PTEN-associated complex.
- Author
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Rabinovsky R, Pochanard P, McNear C, Brachmann SM, Duke-Cohan JS, Garraway LA, and Sellers WR
- Subjects
- Amino Acid Sequence, Antibodies immunology, Cell Line, Humans, Molecular Sequence Data, Molecular Weight, PTEN Phosphohydrolase chemistry, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase immunology, Phosphorylation, Protein Binding, Protein Subunits metabolism, Receptor, ErbB-2 immunology, PTEN Phosphohydrolase metabolism, Phosphatidylinositol 3-Kinases metabolism
- Abstract
The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110beta isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.
- Published
- 2009
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18. Dual regulatory roles of phosphatidylinositol 3-kinase in IFN signaling.
- Author
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Kaur S, Sassano A, Joseph AM, Majchrzak-Kita B, Eklund EA, Verma A, Brachmann SM, Fish EN, and Platanias LC
- Subjects
- Animals, Blotting, Western, Cardiovirus Infections immunology, Encephalomyocarditis virus immunology, Fibroblasts immunology, Fibroblasts virology, Gene Expression, Interferons immunology, Mice, Mice, Knockout, Phosphatidylinositol 3-Kinases immunology, Phosphorylation, Protein Biosynthesis, Proto-Oncogene Proteins c-akt, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Fibroblasts metabolism, Gene Expression Regulation, Interferons metabolism, Phosphatidylinositol 3-Kinases metabolism, Signal Transduction immunology
- Abstract
PI3K is activated by the type I and II IFN receptors, but its precise role in the generation of IFN responses is not well understood. In the present study we used embryonic fibroblasts from mice with targeted disruption of the genes encoding for both the p85alpha and p85beta regulatory subunits of PI3'-kinase (p85alpha(-/-)beta(-/-)) to precisely define the role of PI3K in the control of IFN-induced biological responses. Our data demonstrate that PI3K plays dual regulatory roles in the induction of IFN responses by controlling both IFN-alpha- and IFN-gamma-dependent transcriptional regulation of IFN-sensitive genes and simultaneously regulating the subsequent initiation of mRNA translation for such genes. These processes include the Isg15, Cxcl10, and/or Irf7 genes, whose functions are important in the generation of the biological effects of IFNs. Consistent with this, the induction of IFN antiviral responses is defective in double p85alpha/p85beta knockout cells. Thus, integration of signals via PI3K is a critical event during engagement of the IFN receptors that complements both the transcriptional activity of Jak-STAT pathways and controls initiation of mRNA translation.
- Published
- 2008
- Full Text
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19. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells.
- Author
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Isakoff SJ, Engelman JA, Irie HY, Luo J, Brachmann SM, Pearline RV, Cantley LC, and Brugge JS
- Subjects
- Breast Neoplasms pathology, Cell Adhesion genetics, Cell Growth Processes genetics, Cell Line, Tumor, Cell Survival genetics, Cell Transformation, Neoplastic metabolism, Class I Phosphatidylinositol 3-Kinases, Epithelial Cells enzymology, Epithelial Cells pathology, Humans, Mammary Glands, Human enzymology, Mammary Glands, Human pathology, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Breast Neoplasms enzymology, Breast Neoplasms genetics, Cell Transformation, Neoplastic genetics, Mutation, Phosphatidylinositol 3-Kinases genetics
- Abstract
Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110alpha yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110alpha mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110alpha isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition.
- Published
- 2005
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20. Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement.
- Author
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Brachmann SM, Yballe CM, Innocenti M, Deane JA, Fruman DA, Thomas SM, and Cantley LC
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- Animals, Cell Membrane drug effects, Cell Membrane genetics, Cell Membrane metabolism, Cell Surface Extensions drug effects, Embryo Loss enzymology, Embryo Loss genetics, Embryo Loss metabolism, Embryo, Mammalian embryology, Embryo, Mammalian enzymology, Embryo, Mammalian metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Isoenzymes chemistry, Isoenzymes deficiency, Isoenzymes genetics, Isoenzymes metabolism, Mice, Mice, Knockout, Molecular Weight, Phosphatidylinositol 3-Kinases chemistry, Phosphatidylinositol 3-Kinases deficiency, Phosphatidylinositol 3-Kinases genetics, Phosphorylation, Platelet-Derived Growth Factor pharmacology, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, Signal Transduction, Actins metabolism, Phosphatidylinositol 3-Kinases metabolism
- Abstract
Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.
- Published
- 2005
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21. Phosphoinositide 3-kinase catalytic subunit deletion and regulatory subunit deletion have opposite effects on insulin sensitivity in mice.
- Author
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Brachmann SM, Ueki K, Engelman JA, Kahn RC, and Cantley LC
- Subjects
- Animals, Blood Glucose genetics, Catalytic Domain, Fasting, Insulin blood, Insulin metabolism, Insulin-Like Growth Factor I physiology, Isoenzymes genetics, Isoenzymes physiology, Liver enzymology, Mice, Mice, Knockout, Muscle, Skeletal enzymology, Phosphatidylinositol 3-Kinases chemistry, Phosphatidylinositol 3-Kinases genetics, Protein Serine-Threonine Kinases metabolism, Protein Subunits genetics, Protein Subunits physiology, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, RNA Interference, Sequence Deletion genetics, Glucose Intolerance etiology, Hyperinsulinism etiology, Insulin Resistance, Phosphatidylinositol 3-Kinases physiology
- Abstract
Studies ex vivo have shown that phosphoinositide 3-kinase (PI3K) activity is necessary but not sufficient for insulin-stimulated glucose uptake. Unexpectedly, mice lacking either of the PI3K regulatory subunits p85alpha or p85beta exhibit increased insulin sensitivity. The insulin hypersensitivity is particularly unexpected in p85alpha-/- p55alpha-/- p50alpha-/- mice, where a decrease in p110alpha and p110beta catalytic subunits was observed in insulin-sensitive tissues. These results raised the possibility that decreasing total PI3K available for stimulation by insulin might circumvent negative feedback loops that ultimately shut off insulin-dependent glucose uptake in vivo. Here we present results arguing against this explanation. We show that p110alpha+/- p110beta+/- mice exhibit mild glucose intolerance and hyperinsulinemia in the fasted state. Unexpectedly, p110alpha+/- p110beta+/- mice showed a approximately 50% decrease in p85 expression in liver and muscle. Consistent with this in vivo observation, knockdown of p110 by RNA interference in mammalian cells resulted in loss of p85 proteins due to decreased protein stability. We propose that insulin sensitivity is regulated by a delicate balance between p85 and p110 subunits and that p85 subunits mediate a negative role in insulin signaling independent of their role as mediators of PI3K activation.
- Published
- 2005
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22. Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein.
- Author
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Lekmine F, Sassano A, Uddin S, Smith J, Majchrzak B, Brachmann SM, Hay N, Fish EN, and Platanias LC
- Subjects
- Bone Neoplasms, Cell Line, Tumor, Chromones pharmacology, Enzyme Activation, Enzyme Inhibitors pharmacology, Humans, Kinetics, Morpholines pharmacology, Osteosarcoma, Phosphorylation, Protein Biosynthesis, RNA, Messenger genetics, Interferon-gamma pharmacology, Ribosomal Protein S6 metabolism, Ribosomal Protein S6 Kinases, 70-kDa metabolism
- Abstract
The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.
- Published
- 2004
- Full Text
- View/download PDF
23. Transformation of mouse fibroblasts by Jaagsiekte sheep retrovirus envelope does not require phosphatidylinositol 3-kinase.
- Author
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Maeda N, Inoshima Y, Fruman DA, Brachmann SM, and Fan H
- Subjects
- 3T3 Cells, Amino Acid Motifs, Animals, Chromones pharmacology, Mice, Morpholines pharmacology, Platelet-Derived Growth Factor pharmacology, Signal Transduction, Viral Envelope Proteins physiology, Cell Transformation, Viral, Jaagsiekte sheep retrovirus physiology, Phosphatidylinositol 3-Kinases physiology
- Abstract
Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer of sheep. The envelope of JSRV may have oncogenic properties, since it can morphologically transform mouse NIH 3T3 cells and other fibroblast lines. Recently, we found that the cytoplasmic tail of the envelope transmembrane (TM) protein is necessary for transformation, and in particular a consensus binding motif (YXXM) for phosphatidylinositol 3-kinase (PI3K) is important. Moreover, JSRV-transformed cells show phosphorylation (activation) of Akt/protein kinase B, a downstream target of PI3K. In these studies, we directly tested for the involvement of PI3K in transformation by JSRV. Contrary to expectations, four different experiments indicated that PI3K is not necessary for JSRV-induced transformation: (i) cotransfection with a dominant negative truncated form of the PI3K regulatory subunit (Deltap85) did not affect transformation frequency, (ii) cells stably expressing Deltap85 showed the same frequencies of transformation as parental NIH 3T3 cells, (iii) fibroblasts established from double-knockout mice lacking PI3K p85alpha and p85beta could be transformed with JSRV envelope, and (iv) incubation of cells with the PI3K inhibitor LY294002 did not specifically inhibit transformation, nor did the drug reverse transformation of JSRV-transformed cells. One alternate explanation for the lack of transformation by YXXM mutants could be that they were defective in intracellular trafficking. However, confocal microscopy of epitope-tagged envelope proteins of both wild-type and nontransforming YXXM mutants showed a cell surface or plasma membrane localization. While PI3K is not required for JSRV-induced transformation of NIH 3T3 cells, the downstream target Akt kinase was found to be activated (phosphorylated) in JSRV-transformed PI3K-negative cells. Therefore, JSRV envelope can induce PI3K-independent phosphorylation of Akt.
- Published
- 2003
- Full Text
- View/download PDF
24. Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation.
- Author
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Woolsey AM, Sunwoo L, Petersen CA, Brachmann SM, Cantley LC, and Burleigh BA
- Subjects
- Actins metabolism, Animals, Antigens, CD metabolism, CHO Cells, Calcium metabolism, Cells, Cultured, Cricetinae, Cricetulus, Exocytosis physiology, Host-Parasite Interactions physiology, Lysosomal Membrane Proteins, Lysosomes metabolism, Membrane Proteins metabolism, Mice, Microscopy, Fluorescence, Rats, Vacuoles metabolism, Vesicular Transport Proteins, Cell Membrane metabolism, Phosphatidylinositol 3-Kinases metabolism, Phosphatidylinositol Phosphates metabolism, Trypanosoma cruzi physiology
- Abstract
Mammalian cell invasion by the protozoan parasite, Trypanosoma cruzi, is facilitated by the activation of host cell phosphatidylinositol 3 (PI 3)-kinases. We demonstrate that the well-characterized Ca2+-regulated lysosome-mediated parasite entry pathway is abolished by wortmannin pretreatment. In addition, we have characterized a novel route of T. cruzi invasion unexpectedly revealed in the course of this study. For over a decade, targeted exocytosis of lysosomes at the host cell plasma membrane was considered as the primary mechanism for T. cruzi entry into non-professional phagocytic cells. We now provide evidence that a significant fraction (50% or greater) of invading T. cruzi trypomastigotes exploit an alternate actin-independent entry pathway that involves formation of a tightly associated host cell plasma membrane-derived vacuole enriched in the lipid products of class I PI 3-kinases, PtdInsP3/PtdIns(3,4)P2. Initially devoid of lysosomal markers, the resultant parasite-containing vacuoles gradually acquire lysosome associated membrane protein 1 (lamp-1) and fluid phase endocytic tracer from the lysosomal compartment. In striking contrast to latex bead phagosomes, few T. cruzi vacuoles associate with the early endosomal marker, EEA1 and the 'maturation' process becomes refractory to PI 3-kinase inhibition immediately following parasite internalization. Jointly, these data provide a new paradigm for T. cruzi invasion of non-professional phagocytic cells and reveal a novel vacuole maturation process that appears to bypass the requirement for EEA1.
- Published
- 2003
- Full Text
- View/download PDF
25. Activation of the p70 S6 kinase and phosphorylation of the 4E-BP1 repressor of mRNA translation by type I interferons.
- Author
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Lekmine F, Uddin S, Sassano A, Parmar S, Brachmann SM, Majchrzak B, Sonenberg N, Hay N, Fish EN, and Platanias LC
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Cell Cycle Proteins, Cell Line, Cell Nucleus metabolism, Cells, Cultured, Chromatography, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Eukaryotic Initiation Factors, Fibroblasts metabolism, Gene Expression Regulation, Genes, Reporter, Guanosine Diphosphate metabolism, Humans, Immunoblotting, Interferons metabolism, Luciferases metabolism, Mice, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation, Protein Binding, Protein Biosynthesis, Protein Kinases metabolism, Protein Structure, Tertiary, Recombinant Proteins metabolism, Sepharose metabolism, Signal Transduction, TOR Serine-Threonine Kinases, Tumor Cells, Cultured, Carrier Proteins metabolism, Interferon Type I metabolism, Phosphoproteins metabolism, RNA, Messenger metabolism, Ribosomal Protein S6 Kinases, 70-kDa metabolism
- Abstract
The Type I IFN receptor-generated signals required for initiation of mRNA translation and, ultimately, induction of protein products that mediate IFN responses, remain unknown. We have previously shown that IFNalpha and IFNbeta induce phosphorylation of insulin receptor substrate proteins and downstream engagement of the phosphatidylinositol (PI) 3'-kinase pathway. In the present study we provide evidence for the existence of a Type I IFN-dependent signaling cascade activated downstream of PI 3'-kinase, involving p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated on threonine 421 and serine 424 and is activated during treatment of cells with IFNalpha or IFNbeta. Such activation of p70 S6K is blocked by pharmacological inhibitors of the PI 3'-kinase or the FKBP 12-rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR). Consistent with this, the Type I IFN-dependent phosphorylation/activation of p70 S6K is defective in embryonic fibroblasts from mice with targeted disruption of the p85alpha and p85beta subunits of the PI 3'-kinase (p85alpha-/-beta-/-). Treatment of sensitive cell lines with IFNalpha or IFNbeta also results in phosphorylation/inactivation of the 4E-BP-1 repressor of mRNA translation. Such 4E-BP1 phosphorylation is also PI3'-kinase-dependent and rapamycin-sensitive, indicating that the Type I IFN-inducible activation of PI3'-kinase and FRAP/mTOR results in dissociation of 4E-BP1 from the eukaryotic initiation factor-4E (eIF4E) complex. Altogether, our data establish that the Type I IFN receptor-activated PI 3'-kinase pathway mediates activation of the p70 S6 kinase and inactivation of 4E-BP1, to regulate mRNA translation and induction of Type I IFN responses.
- Published
- 2003
- Full Text
- View/download PDF
26. Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1.
- Author
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Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, Di Fiore PP, and Scita G
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, COS Cells, Chromatography, High Pressure Liquid, Cytoskeletal Proteins, Enzyme Activation, Fibroblasts metabolism, Genetic Vectors, Glutathione Transferase metabolism, Mice, Microscopy, Fluorescence, Models, Biological, Molecular Sequence Data, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Transfection, Arabidopsis Proteins, Phosphatidylinositol 3-Kinases metabolism, Phosphoprotein Phosphatases metabolism, Proteins metabolism, SOS1 Protein metabolism, rac GTP-Binding Proteins metabolism
- Abstract
Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.
- Published
- 2003
- Full Text
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27. Inhibition of autophagy in mitotic animal cells.
- Author
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Eskelinen EL, Prescott AR, Cooper J, Brachmann SM, Wang L, Tang X, Backer JM, and Lucocq JM
- Subjects
- Adenine pharmacology, Anaphase, Androstadienes pharmacology, Animals, Blotting, Western, Cell Line, Chromones pharmacology, Endoplasmic Reticulum metabolism, Enzyme Inhibitors pharmacology, Golgi Apparatus metabolism, HeLa Cells, Humans, Immunohistochemistry, Metaphase, Microscopy, Immunoelectron, Microscopy, Phase-Contrast, Morpholines pharmacology, Nocodazole pharmacology, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Protein Kinases metabolism, Rats, Ribosomal Protein S6 Kinases, 70-kDa metabolism, Sirolimus pharmacology, TOR Serine-Threonine Kinases, Telophase, Time Factors, Wortmannin, Adenine analogs & derivatives, Autophagy, Mitosis
- Abstract
In nutrient-deprived cells autophagy recycles cytoplasmic constituents by engulfing and degrading them in membrane-bound autophagic vacuoles. The regulation of autophagic vacuole formation is poorly understood, but here we show this process is under strict cell-cycle control in cultured animal cells. We found strong inhibition of autophagic vacuole accumulation in nocodazole-arrested pseudo-prometaphase cells, and also in metaphase and anaphase cells generated on release from the nocodazole arrest. Autophagic vacuoles reappeared after closure of the nuclear envelope in telophase/G1. Treatment with phosphoinositide 3(PI3)-kinase inhibitors wortmannin, LY294002 and 3-methyladenine (known to inhibit the autophagic response in interphase cells) rescued autophagy in mitotic cells without inducing reassembly of vesiculated ER and Golgi compartments. The autophagy induced in mitotic cells was inhibited by amino acids, and the resulting autophagosomes contained proteins LC3 and Lamp1, known to be associated with autophagosomes in interphase cells. The mitotic inhibition of autophagy was not relieved by rapamycin treatment or in PDK1-/- embryonic stem cells, by microinjection of inhibitory antibodies against the class III PI3 kinase VPS34, or in cell lines lacking the p85 regulatory subunits of class IA PI3 kinases. Our results show that autophagy is under strict mitotic control and indicate a novel role for phosphoinositide 3-kinases or other wortmannin/LY294002-sensitive kinases in mitotic membrane traffic regulation.
- Published
- 2002
- Full Text
- View/download PDF
28. Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signaling and survival.
- Author
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Ueki K, Fruman DA, Brachmann SM, Tseng YH, Cantley LC, and Kahn CR
- Subjects
- Animals, Apoptosis physiology, Cell Line, Cell Survival physiology, Dimerization, Gene Targeting, Insulin metabolism, Insulin Receptor Substrate Proteins, Insulin-Like Growth Factor I metabolism, Mice, Mice, Knockout, Phosphatidylinositol 3-Kinases classification, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol Phosphates metabolism, Phosphoproteins metabolism, Protein Subunits, Signal Transduction physiology, Phosphatidylinositol 3-Kinases chemistry, Phosphatidylinositol 3-Kinases metabolism
- Abstract
Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85alpha gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85alpha, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85alpha represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.
- Published
- 2002
- Full Text
- View/download PDF
29. Increased insulin sensitivity in mice lacking p85beta subunit of phosphoinositide 3-kinase.
- Author
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Ueki K, Yballe CM, Brachmann SM, Vicent D, Watt JM, Kahn CR, and Cantley LC
- Subjects
- Animals, Catalytic Domain, Mice, Mice, Transgenic, Models, Biological, Muscles metabolism, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation, Phosphotyrosine metabolism, Protein Binding, Signal Transduction, Time Factors, Up-Regulation, Insulin metabolism, Phosphatidylinositol 3-Kinases chemistry, Phosphatidylinositol 3-Kinases physiology
- Abstract
On the basis of ex vivo studies using insulin-responsive cells, activation of a Class IA phosphoinositide 3-kinase (PI3K) seems to be required for a wide variety of cellular responses downstream of insulin. The Class IA PI3K enzymes are heterodimers of catalytic and regulatory subunits. In mammals, insulin-responsive tissues express both the p85alpha and p85beta isoforms of the regulatory subunit. Surprisingly, recent studies have revealed that disruption of the p85alpha gene in the mouse (p85alpha(-/-) mice) results in hypoglycemia with decreased plasma insulin, and the p85alpha(+/-) mice exhibit significantly increased insulin sensitivity. These results suggest either that p85alpha negatively regulates insulin signaling, or that p85beta, which mediates the major fraction of Class IA PI3K signaling in the absence of p85alpha, is more efficient than p85alpha in mediating insulin responses. To address this question, we have generated mice in which the p85beta gene is deleted (p85beta(-/-) mice). As with the p85alpha(-/-) mice, the p85beta(-/-) mice showed hypoinsulinemia, hypoglycemia, and improved insulin sensitivity. At the molecular level, PI3K activity associated with phosphotyrosine complexes was preserved despite a 20-30% reduction in the total protein level of the regulatory subunits. Moreover, insulin-induced activation of AKT was significantly up-regulated in muscle from the p85beta(-/-) mice. In addition, insulin-dependent tyrosine phosphorylation of insulin receptor substrate-2 was enhanced in the p85beta(-/-) mice, a phenotype not observed in the p85alpha(-/-) mice. These results indicate that in addition to their roles in recruiting the catalytic subunit of PI3K to the insulin receptor substrate proteins, both p85alpha and p85beta play negative roles in insulin signaling.
- Published
- 2002
- Full Text
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30. Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation.
- Author
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Vieira OV, Botelho RJ, Rameh L, Brachmann SM, Matsuo T, Davidson HW, Schreiber A, Backer JM, Cantley LC, and Grinstein S
- Subjects
- Androstadienes pharmacology, Animals, Cells, Cultured, Enzyme Inhibitors metabolism, Fibroblasts metabolism, Genes, Reporter, Humans, Immunoglobulin G metabolism, Isoenzymes genetics, Isoenzymes metabolism, Lysosomes metabolism, Macrophages drug effects, Macrophages metabolism, Mice, Mice, Knockout, Microinjections, Phagosomes ultrastructure, Phosphatidylinositol 3-Kinases genetics, Phosphoinositide-3 Kinase Inhibitors, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Wortmannin, Phagocytosis physiology, Phagosomes metabolism, Phosphatidylinositol 3-Kinases metabolism
- Abstract
Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.
- Published
- 2001
- Full Text
- View/download PDF
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