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Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition.

ABSTRACT: Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane association and depletes Notch1 mRNA. GGDPS inhibition increases phosphorylation of histone H2A.X, and inhibitors of ataxia telangiectasia-mutated kinase (ATM) mitigate GGDPS inhibitor-induced apoptosis. GGDPS inhibition also influences c-abl activity downstream of caspases, and inhibitors of these enzymes prevent GGDPS inhibitor-induced apoptosis. Surprisingly, induction of apoptosis by GGDPS inhibition is reduced by co-treatment with ?-secretase inhibitors. While inhibitors of ?-secretase deplete one specific form of the Notch1 intracellular domain (NICD), they also increase Notch1 mRNA expression and increase alternate forms of Notch1 protein expression in cells treated with a GGDPS inhibitor. Furthermore, inhibitors of ?-secretase and ATM increase Notch1 mRNA stability independent of GGDPS inhibition. These results provide a model by which T-ALL cells use Notch1 to avoid DNA-damage-induced apoptosis, and can be overcome by inhibition of GGDPS through effects on Notch1 expression and its subsequent response.

SUBMITTER: Agabiti SS

PROVIDER: S-EPMC6768865 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition.

Agabiti Sherry S SS Li Jin J Dong Willie W Poe Michael M MM Wiemer Andrew J AJ

Cell death & disease 20190930 10

Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane asso ...[more]

PMID: 31570763

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Project description:BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

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Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition.

Publications

Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition.

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