Publications by authors named "John Brognard"

38 Publications

TNIK is a therapeutic target in Lung Squamous Cell Carcinoma and regulates FAK activation through Merlin.

Cancer Discov 2021 Jan 25. Epub 2021 Jan 25.

Laboratory of Cell and Developmental Signaling, National Cancer Institute

Lung squamous cell carcinoma (LSCC) is the second most prevalent type of lung cancer. Despite extensive genomic characterization, no targeted therapies are approved for the treatment of LSCC. Distal amplification of the 3q chromosome is the most frequent genomic alteration in LSCC, and there is an urgent need to identify efficacious druggable targets within this amplicon. We identify the protein kinase TNIK as a therapeutic target in LSCC. TNIK is amplified in approximately 50% of LSCC cases. TNIK genetic depletion or pharmacological inhibition reduces the growth of LSCC cells in vitro and in vivo. In addition, TNIK inhibition showed antitumor activity and increased apoptosis in established LSCC patient-derived xenografts. Mechanistically, we identified the tumor suppressor Merlin/NF2 as a novel TNIK substrate and showed that TNIK and Merlin are required for the activation of focal adhesion kinase. In conclusion, our data identify targeting TNIK as a potential therapeutic strategy in LSCC.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0797DOI Listing
January 2021

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells.

J Biol Chem 2020 06 30;295(25):8470-8479. Epub 2020 Apr 30.

Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, USA

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.
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http://dx.doi.org/10.1074/jbc.RA119.012365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307187PMC
June 2020

Combing the Cancer Genome for Novel Kinase Drivers and New Therapeutic Targets.

Cancers (Basel) 2019 Dec 7;11(12). Epub 2019 Dec 7.

Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.

Protein kinases are critical regulators of signaling cascades that control cellular proliferation, growth, survival, metabolism, migration, and invasion. Deregulation of kinase activity can lead to aberrant regulation of biological processes and to the onset of diseases, including cancer. In this review, we focus on oncogenic kinases and the signaling pathways they regulate that underpin tumor development. We highlight genomic biomarker-based precision medicine intervention strategies that match kinase inhibitors alone or in combination to mutationally activated kinase drivers, as well as progress towards implementation of these treatment strategies in the clinic. We also discuss the challenges for identification of novel protein kinase cancer drivers in the genomic era.
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http://dx.doi.org/10.3390/cancers11121972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966563PMC
December 2019

Upregulation of MLK4 promotes migratory and invasive potential of breast cancer cells.

Oncogene 2019 04 14;38(15):2860-2875. Epub 2018 Dec 14.

Laboratory of Experimental Medicine, Centre of New Technologies, University of Warsaw, Warsaw, Poland.

Metastasis to distant organs is a major cause for solid cancer mortality, and the acquisition of migratory and invasive phenotype is a key factor in initiation of malignancy. In this study we investigated the contribution of Mixed-Lineage Kinase 4 (MLK4) to aggressive phenotype of breast cancer cells. Our TCGA cancer genomic data analysis revealed that amplification or mRNA upregulation of MLK4 occurred in 23% of invasive breast carcinoma cases. To find the association between MLK4 expression and the specific subtype of breast cancer, we performed a transcriptomic analysis of multiple datasets, which showed that MLK4 is highly expressed in triple-negative breast cancer compared to other molecular subtypes. Depletion of MLK4 in cell lines with high MLK4 expression impaired proliferation and anchorage-dependent colony formation. Moreover, silencing of MLK4 expression significantly reduced the migratory potential and invasiveness of breast cancer cells as well as the number of spheroids formed in 3D cultures. These in vitro findings translate into slower rate of tumor growth in mice upon MLK4 knock-down. Furthermore, we established that MLK4 activates NF-κB signaling and promotes a mesenchymal phenotype in breast cancer cells. Immunohistochemical staining of samples obtained from breast cancer patients revealed a strong positive correlation between high expression of MLK4 and metastatic potential of tumors, which was predominantly observed in TNBC subgroup. Taken together, our results show that high expression of MLK4 promotes migratory and invasive phenotype of breast cancer and may represent a novel target for anticancer treatment.
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http://dx.doi.org/10.1038/s41388-018-0618-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484767PMC
April 2019

Orange is the new black: Kinases are the new master regulators of tumor suppression.

IUBMB Life 2019 06 11;71(6):738-748. Epub 2018 Dec 11.

Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD.

For many decades, kinases have predominantly been characterized as oncogenes and drivers of tumorigenesis, because activating mutations in kinases occur in cancer with high frequency. The oncogenic functions of kinases relate to their roles as growth factor receptors and as critical mediators of mitogen-activated pathways. Indeed, some of the most promising cancer therapeutic agents are kinase inhibitors. However, cancer genomics studies, especially screens that utilize high-throughput identification of loss-of-function somatic mutations, are beginning to shed light on a widespread role for kinases as tumor suppressors. The initial characterization of tumor-suppressing kinases- in particular members of the protein kinase C (PKC) family, MKK4 of the mitogen-activated protein kinase kinase family, and DAPK3 of the death-associated protein kinase family- laid the foundation for bioinformatic approaches that enable the identification of other tumor-suppressing kinases. In this review, we discuss the important role that kinases play as tumor suppressors, using several examples to illustrate the history of their discovery and highlight the modern approaches that presently aid in the identification of tumor-suppressing kinases. © 2018 IUBMB Life, 71(6):738-748, 2019.
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http://dx.doi.org/10.1002/iub.1981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563145PMC
June 2019

Shipping Out MEK Inhibitor Resistance with SHP2 Inhibitors.

Cancer Discov 2018 10;8(10):1210-1212

Signaling Networks in Cancer Section, Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, Maryland.

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http://dx.doi.org/10.1158/2159-8290.CD-18-0915DOI Listing
October 2018

Signaling pathway screening platforms are an efficient approach to identify therapeutic targets in cancers that lack known driver mutations: a case report for a cancer of unknown primary origin.

NPJ Genom Med 2018 20;3:15. Epub 2018 Jun 20.

1Signalling Networks in Cancer Group, Cancer Research UK, Manchester Institute, University of Manchester, Manchester, M20 4BX UK.

Precision medicine aims to tailor cancer therapies to target specific tumor-promoting aberrations. For tumors that lack actionable drivers, which occurs frequently in the clinic, extensive molecular characterization and pre-clinical drug efficacy studies will be required. A cell line maintained at low passage and a patient- derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patient's DNA. However, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the and genes, respectively. We then conducted pathway analysis, which revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis will significantly aid in identifying actionable alterations in rare tumors and guide patient stratification into early-phase clinical trials.
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http://dx.doi.org/10.1038/s41525-018-0055-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010465PMC
June 2018

Protein kinase Cα gain-of-function variant in Alzheimer's disease displays enhanced catalysis by a mechanism that evades down-regulation.

Proc Natl Acad Sci U S A 2018 06 29;115(24):E5497-E5505. Epub 2018 May 29.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKCα in Alzheimer's disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ∼30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKCα-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKCα-M489V mutation. Our results unveil how an AD-associated mutation in PKCα permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKCα.
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http://dx.doi.org/10.1073/pnas.1805046115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004447PMC
June 2018

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

Sci Signal 2018 04 17;11(526). Epub 2018 Apr 17.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK.

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer.
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http://dx.doi.org/10.1126/scisignal.aan6776DOI Listing
April 2018

Survival of Head and Neck Cancer Cells Relies upon LZK Kinase-Mediated Stabilization of Mutant p53.

Cancer Res 2017 09 31;77(18):4961-4972. Epub 2017 Jul 31.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, United Kingdom.

Head and neck squamous cell carcinoma (HNSCC) includes epithelial cancers of the oral and nasal cavity, larynx, and pharynx and accounts for ∼350,000 deaths per year worldwide. Smoking-related HNSCC is associated with few targetable mutations but is defined by frequent copy-number alteration, the most common of which is gain at 3q. Critical 3q target genes have not been conclusively determined for HNSCC. Here, we present data indicating that (encoding LZK) is an amplified driver gene in HNSCC. Copy-number gain at 3q resulted in increased mRNA in HNSCC tumor samples and cell lines. Silencing LZK reduced cell viability and proliferation of HNSCC cells with 3q gain but not control cell lines. Inducible silencing of LZK caused near-complete loss of colony-forming ability in cells harboring 3q gain. These results were validated by evidence that LZK silencing was sufficient to reduce tumor growth in a xenograft model of HNSCC. Our results establish LZK as critical for maintaining expression of mutant stabilized p53. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0267DOI Listing
September 2017

Reversing the Paradigm: Protein Kinase C as a Tumor Suppressor.

Trends Pharmacol Sci 2017 05 8;38(5):438-447. Epub 2017 Mar 8.

Laboratory of Cell and Developmental Signaling, National Cancer Institute at Frederick, Frederick, MD 21702, USA; Cancer Research UK Manchester Institute, Manchester, UK. Electronic address:

The discovery in the 1980s that protein kinase C (PKC) is a receptor for the tumor-promoting phorbol esters fueled the dogma that PKC is an oncoprotein. Yet 30+ years of clinical trials for cancer using PKC inhibitors not only failed, but in some instances worsened patient outcome. The recent analysis of cancer-associated mutations, from diverse cancers and throughout the PKC family, revealed that PKC isozymes are generally inactivated in cancer, supporting a tumor suppressive function. In keeping with a bona fide tumor suppressive role, germline causal loss-of-function (LOF) mutations in one isozyme have recently been identified in lymphoproliferative disorders. Thus, strategies in cancer treatment should focus on restoring rather than inhibiting PKC.
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http://dx.doi.org/10.1016/j.tips.2017.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403564PMC
May 2017

Somatically mutated ABL1 is an actionable and essential NSCLC survival gene.

EMBO Mol Med 2016 Feb;8(2):105-16

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute The University of Manchester, Manchester, UK

The lack of actionable mutations in patients with non-small cell lung cancer (NSCLC) presents a significant hurdle in the design of targeted therapies for this disease. Here, we identify somatically mutated ABL1 as a genetic dependency that is required to maintain NSCLC cell survival. We demonstrate that NSCLC cells with ABL1 mutations are sensitive to ABL inhibitors and we verify that the drug-induced effects on cell viability are specific to pharmacological inhibition of the ABL1 kinase. Furthermore, we confirm that imatinib suppresses lung tumor growth in vivo, specifically in lung cancer cells harboring a gain-of-function (GOF) mutation in ABL1. Consistent with structural modeling, we demonstrate that mutations in ABL1 identified in primary NSCLC tumors and a lung cancer cell line increase downstream pathway activation compared to wild-type ABL1. Finally, we observe that the ABL1 cancer mutants display an increased cytosolic localization, which is associated with the oncogenic properties of the ABL1 kinase. In summary, our results suggest that NSCLC patients with ABL1 mutations could be stratified for treatment with imatinib in combination with other therapies.
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http://dx.doi.org/10.15252/emmm.201505456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734836PMC
February 2016

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

Cancer Res 2016 Feb 4;76(3):724-35. Epub 2015 Dec 4.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, United Kingdom.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0701-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740929PMC
February 2016

Using large-scale genomics data to identify driver mutations in lung cancer: methods and challenges.

Pharmacogenomics 2015 31;16(10):1149-60. Epub 2015 Jul 31.

RNA Biology Group & Computational Biology Support Team, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, UK.

Lung cancer is the commonest cause of cancer death in the world and carries a poor prognosis for most patients. While precision targeting of mutated proteins has given some successes for never- and light-smoking patients, there are no proven targeted therapies for the majority of smokers with the disease. Despite sequencing hundreds of lung cancers, known driver mutations are lacking for a majority of tumors. Distinguishing driver mutations from inconsequential passenger mutations in a given lung tumor is extremely challenging due to the high mutational burden of smoking-related cancers. Here we discuss the methods employed to identify driver mutations from these large datasets. We examine different approaches based on bioinformatics, in silico structural modeling and biological dependency screens and discuss the limitations of these approaches.
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http://dx.doi.org/10.2217/pgs.15.60DOI Listing
June 2016

Transcriptional Repressor DAXX Promotes Prostate Cancer Tumorigenicity via Suppression of Autophagy.

J Biol Chem 2015 Jun 22;290(25):15406-20. Epub 2015 Apr 22.

From the Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 and

The DAXX transcriptional repressor was originally associated with apoptotic cell death. However, recent evidence that DAXX represses several tumor suppressor genes, including the DAPK1 and DAPK3 protein kinases, and is up-regulated in many cancers argues that a pro-survival role may predominate in a cancer context. Here, we report that DAXX has potent growth-enhancing effects on primary prostatic malignancy through inhibition of autophagy. Through stable gene knockdown and mouse subcutaneous xenograft studies, we demonstrate that DAXX promotes tumorigenicity of human ALVA-31 and PC3 prostate cancer (PCa) cells in vivo. Importantly, DAXX represses expression of essential autophagy modulators DAPK3 and ULK1 in vivo, revealing autophagy suppression as a mechanism through which DAXX promotes PCa tumorigenicity. Furthermore, DAXX knockdown increases autophagic flux in cultured PCa cells. Finally, interrogation of the Oncomine(TM) database suggests that DAXX overexpression is associated with malignant transformation in several human cancers, including prostate and pancreatic cancers. Thus, DAXX may represent a new cancer biomarker for the detection of aggressive disease, whose tissue-specific down-regulation can serve as an improved therapeutic modality. Our results establish DAXX as a pro-survival protein in PCa and reveal that, in the early stages of tumorigenesis, autophagy suppresses prostate tumor formation.
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http://dx.doi.org/10.1074/jbc.M115.658765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505457PMC
June 2015

Cancer-associated protein kinase C mutations reveal kinase's role as tumor suppressor.

Cell 2015 Jan 22;160(3):489-502. Epub 2015 Jan 22.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.
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http://dx.doi.org/10.1016/j.cell.2015.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313737PMC
January 2015

Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma.

Cancer Cell 2015 Jan 11;27(1):85-96. Epub 2014 Dec 11.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK. Electronic address:

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.
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http://dx.doi.org/10.1016/j.ccell.2014.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297292PMC
January 2015

Discrepancies in cancer genomic sequencing highlight opportunities for driver mutation discovery.

Cancer Res 2014 Nov 25;74(22):6390-6396. Epub 2014 Sep 25.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, M20 4BX, UK.

Cancer genome sequencing is being used at an increasing rate to identify actionable driver mutations that can inform therapeutic intervention strategies. A comparison of two of the most prominent cancer genome sequencing databases from different institutes (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer) revealed marked discrepancies in the detection of missense mutations in identical cell lines (57.38% conformity). The main reason for this discrepancy is inadequate sequencing of GC-rich areas of the exome. We have therefore mapped over 400 regions of consistent inadequate sequencing (cold-spots) in known cancer-causing genes and kinases, in 368 of which neither institute finds mutations. We demonstrate, using a newly identified PAK4 mutation as proof of principle, that specific targeting and sequencing of these GC-rich cold-spot regions can lead to the identification of novel driver mutations in known tumor suppressors and oncogenes. We highlight that cross-referencing between genomic databases is required to comprehensively assess genomic alterations in commonly used cell lines and that there are still significant opportunities to identify novel drivers of tumorigenesis in poorly sequenced areas of the exome. Finally, we assess other reasons for the observed discrepancy, such as variations in dbSNP filtering and the acquisition/loss of mutations, to give explanations as to why there is a discrepancy in pharmacogenomic studies, given recent concerns with poor reproducibility of data.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247168PMC
November 2014

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output.

Proc Natl Acad Sci U S A 2014 Sep 8;111(38):E3957-65. Epub 2014 Sep 8.

Departments of Pharmacology and

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.
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http://dx.doi.org/10.1073/pnas.1404221111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183331PMC
September 2014

Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors.

Nat Commun 2014 May 22;5:3901. Epub 2014 May 22.

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK.

RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2-18 months. Here we demonstrate that the mixed lineage kinases (MLK1-4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1-4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors.
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http://dx.doi.org/10.1038/ncomms4901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046110PMC
May 2014

Reply: To PMID 23666743.

Arthritis Rheumatol 2014 Jan;66(1):229-30

Hôpital Femme Mère Enfant, Hospices Civils de Lyon and Université de Lyon, Lyon, France.

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http://dx.doi.org/10.1002/art.38234DOI Listing
January 2014

Druggable drivers of lung cancer.

Oncotarget 2013 Sep;4(9):1334-5

Signalling Networks in Cancer Group, Cancer Research UK Manchester Institute and The University of Manchester, Manchester, UK.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824536PMC
http://dx.doi.org/10.18632/oncotarget.1223DOI Listing
September 2013

Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer.

Proc Natl Acad Sci U S A 2013 Jul 8;110(30):12426-31. Epub 2013 Jul 8.

Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

Approximately 70% of patients with non-small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ~50% of non-small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability.
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http://dx.doi.org/10.1073/pnas.1305207110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725071PMC
July 2013

Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

Arthritis Rheum 2013 Aug;65(8):2161-71

Centre de Référence des Maladies Rénales Rares, Hospices Civils de Lyon, INSERM U1111, UMS3444/US8, Université Claude Bernard Lyon 1, and Université de Lyon, Lyon, France.

Objective: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE.

Methods: We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology.

Results: We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype.

Conclusion: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.
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http://dx.doi.org/10.1002/art.38008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066615PMC
August 2013

Cancer-associated loss-of-function mutations implicate DAPK3 as a tumor-suppressing kinase.

Cancer Res 2011 Apr 12;71(8):3152-61. Epub 2011 Apr 12.

Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, The University of Manchester, Manchester, United Kingdom.

Cancer kinome sequencing studies have identified several protein kinases predicted to possess driver (i.e., causal) mutations. Using bioinformatic applications, we have pinpointed DAPK3 (ZIPK) as a novel cancer-associated kinase with functional mutations. Evaluation of nonsynonymous point mutations, discovered in DAPK3 in various tumors (T112M, D161N, and P216S), reveals that all three mutations decrease or abolish kinase activity. Furthermore, phenotypic assays indicate that the three mutations observed in cancer abrogate the function of the kinase to regulate both the cell cycle and cell survival. Coexpression of wild-type (WT) and cancer mutant kinases shows that the cancer mutants dominantly inhibit the function of the WT kinase. Reconstitution of a non-small cell lung cancer cell line that harbors an endogenous mutation in DAPK3 (P216S) with WT DAPK3 resulted in decreased cellular aggregation and increased sensitivity to chemotherapy. Our results suggest that DAPK3 is a tumor suppressor in which loss-of-function mutations promote increased cell survival, proliferation, cellular aggregation, and increased resistance to chemotherapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-3543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078168PMC
April 2011

Protein kinase signaling networks in cancer.

Curr Opin Genet Dev 2011 Feb 29;21(1):4-11. Epub 2010 Nov 29.

Signalling Networks in Cancer Group, Cancer Research UK, Paterson Institute for Cancer Research, The University of Manchester, Manchester, UK.

Protein kinases orchestrate the activation of signaling cascades in response to extracellular and intracellular stimuli to control cell growth, proliferation, and survival. The complexity of numerous intracellular signaling pathways is highlighted by the number of kinases encoded by the human genome (539) and the plethora of phosphorylation sites identified in phosphoproteomic studies. Perturbation of these signaling networks by mutations or abnormal protein expression underlies the cause of many diseases including cancer. Recent RNAi screens and cancer genomic sequencing studies have revealed that many more kinases than anticipated contribute to tumorigenesis and are potential targets for inhibitor drug development intervention. This review will highlight recent insights into known pathways essential for tumorigenesis and discuss exciting new pathways for therapeutic intervention.
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http://dx.doi.org/10.1016/j.gde.2010.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038181PMC
February 2011

The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

Mol Cell 2009 Aug;35(4):442-53

Department of Pharmacology, Case Comprehensive Caner Center, School of Medicine, Case Western Reserve University, 2109 Adelbert Road, Wood Building W343A, Cleveland, OH 44106, USA.

ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.
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http://dx.doi.org/10.1016/j.molcel.2009.06.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2736145PMC
August 2009

Common polymorphism in the phosphatase PHLPP2 results in reduced regulation of Akt and protein kinase C.

J Biol Chem 2009 May 26;284(22):15215-23. Epub 2009 Mar 26.

Department of Pharmacology and Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92093-0721, USA.

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.
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http://dx.doi.org/10.1074/jbc.M901468200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685702PMC
May 2009

PHLiPPing the switch on Akt and protein kinase C signaling.

Trends Endocrinol Metab 2008 Aug 27;19(6):223-30. Epub 2008 May 27.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0721, USA.

The Ser/Thr-specific phosphatase PHLPP [pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase] provides 'the brakes' for Akt and protein kinase C (PKC) signaling. The two isoforms of this recently discovered family, PHLPP1 and PHLPP2, control the amplitude and duration of signaling of Akt and PKC by catalyzing the dephosphorylation of the hydrophobic phosphorylation motif, a C-terminal phosphorylation switch that controls these kinases. Aberrant regulation of either kinase accompanies many diseases, notably diabetes and cancer. By specifically dephosphorylating the hydrophobic motif, PHLPP controls the degree of agonist-evoked signaling by Akt and the cellular levels of PKC. This review focuses on the function of PHLPP1 and PHLPP2 in modulating signaling by Akt and PKC.
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http://dx.doi.org/10.1016/j.tem.2008.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963565PMC
August 2008