309 research outputs found
Targeting N-myristoylation for therapy of B-cell lymphomas
N-myristoyltransferases (NMTs) target many signaling proteins to membranes. Here the authors show an NMT inhibitor named PCLX-001 selectively kills lymphoma cells by shutting down their main survival signaling pathway and offers an additional treatment strategy for lymphoma patients
Future therapeutic strategies: Implications for Brk targeting
The official published article can be found at the link below - Copyright @ 2011 InTech. This Article has been provided by the Brunel Open Access Publishing Fund.Rajpal Burmi was supported by a Breast
Cancer Campaign project grant
Synthetic lethality driven by N-Myristoyltransferase inhibition in MYC deregulated cancers.
N-myristoylation is the irreversible attachment of myristate (a C14 fatty acid) to the N-terminal glycine of a given substrate. The enzyme responsible for this reaction is N-myristoyl transferase (NMT), a protein shown to be essential for many organisms, ranging from eukaryotes, plants, fungi, to infectious parasites. Initially, the NMTs of the fungi and infectious parasites sparked the interest of researchers in drug discovery to target these pathogens. However, the question remained whether one could target NMT in cancer. While early studies suggested potential upregulation of NMT1 in some early stage cancers, it remained unclear which cancer types to target and for which mechanistical reason.
In this study, data from pharmacogenomics screens across hundreds of cancer cell lines, treated with three different NMT inhibitors, were analysed. Haematological malignancies were amongst the most responsive cell lines; however, also cancers originating from other tissues were sensitive, indicating a more complicated picture. Detailed phenotypical and omics-based analysis of the effects of NMT inhibition in an example cancer cell line from the haematological malignancies, and an unbiased bioinformatics approach across the pharmacogenomics data hinted at the same protooncogene: MYC. Two different isogenic system with inducible MYC confirmed that MYC deregulated cells are highly dependent on myristoylation.
This newly uncovered synthetic lethality has potentially wide implications as MYC, a key transcription factor, is commonly deregulated in cancer and involved in most of the hallmarks of cancer. Targeting MYC or its downstream program attracted wide attention of the field; however, to date no drug has been approved to specifically target either. Novel approaches to target MYC, in the context of cancer, are urgently needed, and this study identified a potential new one.Open Acces
AMP-Activated Protein Kinase:Do We Need Activators or Inhibitors to Treat or Prevent Cancer?
AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance. In response to metabolic stress, it acts to redress energy imbalance through promotion of ATP-generating catabolic processes and inhibition of ATP-consuming processes, including cell growth and proliferation. While findings that AMPK was a downstream effector of the tumour suppressor LKB1 indicated that it might act to repress tumourigenesis, more recent evidence suggests that AMPK can either suppress or promote cancer, depending on the context. Prior to tumourigenesis AMPK may indeed restrain aberrant growth, but once a cancer has arisen, AMPK may instead support survival of the cancer cells by adjusting their rate of growth to match their energy supply, as well as promoting genome stability. The two isoforms of the AMPK catalytic subunit may have distinct functions in human cancers, with the AMPK-α1 gene often being amplified, while the AMPK-α2 gene is more often mutated. The prevalence of metabolic disorders, such as obesity and Type 2 diabetes, has led to the development of a wide range of AMPK-activating drugs. While these might be useful as preventative therapeutics in individuals predisposed to cancer, it seems more likely that AMPK inhibitors, whose development has lagged behind that of activators, would be efficacious for the treatment of pre-existing cancers
Expression of Bcl-2 family member Bid in normal and malignant tissues
Bid is the only known Bcl-2 family member that can function as an agonist of proapoptotic Bcl-2-related proteins such as Bax and Bak. Expression of the proapoptotic Bcl-2 family protein Bid was assessed by immunoblotting and immunohistochemical methods in normal murine and human-tissues and in several types of human cancers and tumor cell lines. Bid expression in normal tissues varied widely, with prominent Bid immunostaining occurring in several types of short-lived cells (e.g., germinal center B cells, peripheral blood granulocytes, differentiated keratinocytes) and in apoptosissensitive cells (e.g., adult neurons). Analysis of Bid expression by immunostaining of 100 colon, 95 ovarian, and 254 prostate cancers, as well as 59 brain tumors and 50 lymphomas, revealed evidence of altered Bid regulation in sometypes of cancers. Correlations with clinical outcome data revealed association of higher levels of Bid with longer recurrence-free survival in men with locally advanced (T3 stage) prostate cancer (P=0.04). Immunoblot analysis of Bid protein levels in the NCI's panel of 60 human tumor cell lines revealed a correlation between higher levels of Bid and sensitivity to ribonucleotide reductase (RR)-inhibiting drugs (P<0.0005). Overexpression of Bid in a model tumor cell line by gene transfection resulted in increased sensitivity to apoptosis induction by a RR inhibitor. Taken together, these observations suggest a potential role for Bid in tumor responses to specific chemotherapeutic drugs, and lay a foundation for future investigations of this member of the Bcl-2 family in healthy and diseased tissues.ope
Genetic screens identify novel liabilities of senescent cells
Drugs that selectively kill senescent cells, senolytics, can improve the outcomes of cancer, fibrosis and age-related diseases. Despite their potential, our knowledge of the molecular pathways that affect the survival of senescent cells is limited. To identify novel senolytic targets, we performed RNAi and CRISPR screens and identified COPI (Coatomer Complex I)vesicle formation as a liability of senescent cells. Genetic or pharmacological inhibition ofCOPI results in Golgi dispersal, intracellular accumulation of secreted factors, and unfolded protein response-dependent cell death of senescent cells. Knockdown of COPI subunits improves the outcomes of cancer and fibrosis in mouse models. Drugs targeting COPI have poor pharmacological properties, but N-myristoyltransferase inhibitors (NMTi) phenocopy COPI inhibition and are potent senolytics. NMTi eliminate senescent cells, ameliorating lung fibrosis and liver steatosis in aged mice. Our results suggest that senescent cells rely on a hyperactive secretory apparatus, and that inhibiting trafficking kills senescent cells in various senescence-associated diseases and during ageing.Open Acces
Syk Inhibitors in Clinical Development for Hematological Malignancies
Spleen tyrosine kinase (Syk) is a cytosolic non-receptor protein tyrosine kinase (PTK) and is mainly expressed in hematopoietic cells. Syk was recognized as a critical element in the B-cell receptor signaling pathway. Syk is also a key component in signal transduction from other immune receptors like Fc receptors and adhesion receptors. Several oral Syk inhibitors including fostamatinib (R788), entospletinib (GS-9973), cerdulatinib (PRT062070), and TAK-659 are being assessed in clinical trials. The second generation compound, entospletinib, showed promising results in clinical trials against B-cell malignancies, mainly chronic lymphoid leukemia. Syk inhibitors are being evaluated in combination regimens in multiple malignancies
Functional characterization of FMNL1 as potential target for novel anti-tumor therapies
Formine spielen eine wichtige Bedeutung bei der Regulierung polarisierter
Aktin-gesteuerter Prozesse. Dies betrifft beispielsweise die Zellmigration, den
Vesikeltransport, die Morphogenese und die Zytokinese (Faix und Grosse
2006). In früheren Arbeiten konnte nachgewiesen werden, dass das Protein
Formin-like 1 (FMNL1) in Zellen von Patienten mit chronischer lymphatischer
Leukämie (CLL), anderen Leukämien und Lymphomen und in Zelllinien, die
von soliden Tumoren stammen, überexprimiert wird. Im gesunden Gewebe
wird es fast ausschließlich in hämatopoetischen Zellen exprimiert. Diese
selektive Expression macht FMNL1 zu einem attraktiven Ziel für neuartige
Immuntherapien bei malignen und entzündlichen Erkrankungen (Krackhardt,
Witzens et al. 2002; Schuster, Busch et al. 2007). In Vorarbeiten der Gruppe
wurde ein allorestringierter T-Zellklon mit einem definierten T-Zellrezeptor
identifiziert, der ein Peptid von FMNL1 erkennt und eine starke
Antitumor-Aktivität gegen Lymphom- und Nierenzellkarzinom-Zelllinien,
Epstein-Barr-Virus (EBV)-transformierte B-Zellen und von CLL-Zellen zeigt
(Schuster, Busch et al. 2007). Allerdings sind die Funktion und Regulation
von FMNL1 – beide wichtig für die Validierung dieses Proteins als Antigen –
noch nicht gut untersucht. Frühere Arbeiten haben eine Beteiligung von
FMNL1 bei der Neuausrichtung des MTOC (Mikrotubulin-organisierendes
Zentrum) in Richtung der immunologischen Synapse und zusätzlich bei der
Zytotoxizität von T-Zellen beschrieben (Gomez, Kumar et al gezeigt. 2007).
Darüber hinaus wurde gezeigt, dass das murine FRL, das zu 85% homolog
zum menschlichen FMNL1 ist, an der Zelladhäsion und Motilität von
Makrophagen sowie an der Fc-Rezeptor-vermittelten Phagozytose beteiligt
ist (Yayoshi-Yamamoto, et al Taniuchi hat. 2000; Seth, Otomo et al. 2006).
Das Ziel dieses Projekts war es, die Funktion von FMNL1 für die weitere
Validierung dieses Proteins als mögliche Zielscheibe für neue
Anti-Tumor-Therapien zu untersuchen. Wir haben eine neue Spleißvariante
(FMNL1) identifiziert, die am C-terminalen Ende ein residuelles Intron
aufweist, welches einen Einfluss auf die
Diaphanous-autoinhibierende-Domäne (DAD) hat. Im Gegensatz zu anderen
FMNL1-Spleißvarianten, die eine zytoplasmatischen Lokalisierung aufweisen,
zeigt diese Speißvariante eine kortikale und membranständige Lokalisation in
verschiedenen Zelllinien. Eine FMNL1 Mutante, bei der die DAD-Domäne
fehlt (FMNL1ΔDAD), weist eine ähnliche Lokalisierung auf. Das weist darauf
hin, dass es bei FMNL1 zu einer Deregulierung der Autoinhibition kommt,
die zu einer konstitutiv aktiven Form von FMNL1 führt, die möglicherweise bei
der zellulären Transformation eine Rolle spielen könnte. FMNL1 und
FMNL1ΔDAD können eine polarisierte, nicht mit einer Apoptose-assoziierten
Blasenbildung an der Membran hervorrufen, die von Myosin, Aktin undTubulin abhängig ist, aber unabhängig von Src und ROCK zu sein scheint.
Wir haben außerdem nachgewiesen, dass FMNL1 als myristoyliertes Protein
vorliegt und konnten zeigen, dass die N-terminale Myristoylierung wichtig für
die Regulierung der Funktion von FMNL1 ist, indem sie eine schnelle und
reversible Membran-Lokalisierung ermöglicht.
Des Weiteren haben wir gezeigt, dass FMNL1, das auch am kontraktilen
Ring und Kortex von FMNL1-transfizierten mitotischen Zellen lokalsiert ist,
die Zellproliferation moderat verstärkt. Eine gemeinsame Lokalisierung von
menschlichem endogenem FMNL1 und -Tubulin am Kortex und den
mitotischenden Spindeln von sich teilenden T-Zellen weist ebenfalls auf eine
Rolle von FMNL1 in der Mitose und dem Zellwachstum hin. Überexpression
von FMNL1 konnte eine höhere Konzentration von intrazellulärem freiem
Calcium nach Zell-Stimulation induzieren, was auf eine Beteiligung von
FMNL1 am Calcium-Signalweg deutet.
Unsere Ergebnisse eröffnen neue Einblicke in die Regulation und Funktion
von FMNL1 und zeigen dessen Beteiligung an unterschiedlichen
Polarisierungsprozessen. Die Identifizierung von Interaktionspartnern von
FMNL1 in verschiedenen hämatopoetischen Zellen sowie die weitere
funktionelle Charakterisierung der Spleißvarianten wird von besonderer
Bedeutung sein, und möglicherweise zur Entwicklung einer spezifischen
therapeutischen Beinflussung maligner und entzündlicher Erkrankungen
beitragen
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A yeast-based assay for protein tyrosine kinase substrate specificity and inhibitor resistance
Phosphorylation of tyrosines by protein kinases is a fundamental mode of signal transduction in all eukaryotic cells, leading to a wide variety of cellular outcomes, including proliferation, differentiation, transcriptional activation, and programmed cell death. Perturbations to tyrosine kinase signaling networks by activation, overexpression, or mutation is the driving factor in many diseases, most notably cancers. The development of tyrosine kinase inhibitors, 37 of which are currently FDA-approved, has led to a revolution in cancer treatment. Imatinib, the first FDA-approved kinase inhibitor, has drastically improved prognosis for patients with Bcr-abl-positive leukemias. Despite this unprecedented success, however, up to one-third of patients lose response to imatinib due to mutations within the tyrosine kinase domain of Bcr-abl. Subsequent generations of Bcr-abl inhibitors, including dasatinib and ponatinib, have been developed to overcome these resistance mutations, but in each case, novel resistance mutations have arisen. We present a high-throughput yeast-based assay for the prediction of dasatinib- and ponatinib-resistant mutations in the ABL1 kinase domain. Our results not only recapitulate all known dasatinib-resistant mutations, but confirm recent patient data emphasizing the importance of compound mutations in ponatinib resistance. Furthermore, with hundreds of kinase inhibitors in development for the treatment of a wide range of diseases, understanding the cellular pathway of each kinase is critically important to the selection of ideal drug targets and avoiding potentially toxic side effects. Discovery of novel tyrosine kinase substrates is hindered by the presence of 90 human tyrosine kinases, which are often active in the same pathways. Phosphoproteomics, chemical genetics, and in vitro assays have been used to great success, yet only 30% of phosphorylated tyrosines in the human proteome have been assigned to a specific kinase. Recent advances in predicting tyrosine kinase substrates have been made by combining large data sets on kinase domain specificity, cellular localization, and protein-protein interactions in probabilistic algorithms. However, the high-quality data sets required for accurate predictions are often lacking. In chapter 2, we present a high-throughput yeastbased assay for screening millions of putative kinase substrates, which we then use to build a probabilistic model to accurately predict the in vitro phosphorylation of candidate substratesBiochemistr
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