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Targeting phospholipid biosynthesis, specifically phosphatidylcholine (PC), which is enhanced in tumor cells, has been proven a suitable antitumor strategy. In fact, the overexpression of the choline kinase α1 (ChoKα1) isoform has been found in malignant cells and tumors, thus becoming an excellent antitumor target. ChoKα1 inhibitors are being synthesized at the present that show a large inhibitory activity. Two of them have been chosen in this study as representatives of different structural families: a biscationic biphenyl derivative of thieno[3,2-d]pyrimidinium substituted with a cyclic amine (here referred to as Fa22) and a biscationic biphenyl thioethano derivative of 7-chloro-quinolinium substituted with a pyrrolidinic moiety (here referred to as PL48). However, the potential use of these types of compounds in systemic treatments is hampered because of their low specificity. In fact, to enter the cell and reach their target, these inhibitors use choline transporters and inhibit choline uptake, being that one of the causes of their toxicity. One way to solve this problem could be allowing their entrance into the cells by alternative ways. With this goal, MamC-mediated magnetic nanoparticles (BMNPs), already proven effective drug nanocarriers, have been used to immobilize Fa22 and PL48. The idea is to let BMNPs enter the cell (they enter the cell by endocytosis) carrying these molecules, and, therefore, offering another way in for these compounds. In the present study, we demonstrate that the coupling of Fa22 and PL48 to BMNPs allows these molecules to enter the tumoral cell without completely inhibiting choline uptake, so, therefore, the use of Fa22 and PL48 in these nanoformulations reduces the toxicity compared to that of the soluble drugs. Moreover, the nanoassemblies Fa22-BMNPs and PL48-BMNPs allow the combination of chemotherapy and local hyperthermia therapies for a enhanced cytotoxic effect on the tumoral HepG2 cell line. The consistency of the results, independently of the drug structure, may indicate that this behavior could be extended to other ChoKα1 inhibitors, opening up a possibility for their potential use in clinics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB , encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7β1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P 2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P 2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB ., (© 2024 Tavasoli and McMaster.)

Purpose: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future., Methodology: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes., Results: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a V max of 1.9 ± 0.1 µmol/min/mg. Its K m for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn 2+ was used as the co-factor instead of Mg 2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting., Conclusions: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. K m and V max of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC 50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC 50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.

The synovial intimal lining is mainly governed by fibroblast-like synoviocytes (FLS), which portray a transformed tumor-like phenotype in rheumatoid arthritis (RA). Among the diverse cytokines that engender FLS, interleukin-21 (IL-21) was reported to stimulate hyperproliferation and perpetuate inflammation. Recently, choline kinase (ChoKα) has been reported to be an essential enzyme aiding RA-FLS hyperproliferation by altering phosphatidylcholine biosynthesis. The current study aimed to elucidate the therapeutic efficacy of myricetin, a flavonoid, in abating the IL-21-induced tumor-like phenotype of adjuvant-induced arthritis (AIA)-FLS via the ChoKα signaling cascade. Our results showed that myricetin suppressed IL-21 receptor expression and activation of the ChoKα signaling cascade (N-Ras, Ral-GDS, and PI3K) in IL-21-induced AIA-FLS. Consequently, myricetin treatment decreased ChoKα and PLD2 enzymatic activity and inhibited the proliferative, migratory, and invasive properties of AIA-FLSs. Our results demonstrated that myricetin could be a promising anti-arthritic compound by abating IL-21-induced hyperproliferation, migration, and invasive behavior of AIA-FLS by downregulating the ChoKα signaling cascade., (© 2023. The Society for In Vitro Biology.)

Choline kinase (CK) is reportedly overexpressed in various malignancies. Among its isoforms, CKα overexpression is presumably related to oncogenic change. Choline positron emission tomography (PET) is reportedly useful for detecting and evaluating therapy outcomes in malignancies. In this study, we investigated the correlation between CKα expression and 11 C-choline accumulation in breast cancer cells. We also compared the CKα expression level with other pathological findings for investigating tumour activity. Fifty-six patients with breast cancer (mean age: 51 years) who underwent their first medical examination between May 2007 and December 2008 were enrolled. All the patients underwent 11 C-choline PET/computed tomography imaging prior to surgery. The maximum standardised uptake value was recorded for evaluating 11 C-choline accumulation. The intensity of CKα expression was classified using immunostaining. A significant correlation was observed between CKα expression and 11 C-choline accumulation (P < 0.0001). A comparison of breast cancer mortality demonstrated that strong CKα expression was associated with a shorter survival time (P < 0.0001). 11 C-choline accumulation was also negatively correlated with survival time (P < 0.0001). Tumours with strong CKα expression are reportedly highly active in breast cancer. A correlation was observed between CKα expression and 11 C-choline accumulation, suggesting their role as prognostic indicators of breast cancer., (© 2023. Springer Nature Limited.)

The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn 2+ over the typical Mg 2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn 2+ , the activity of EhCK1 increased by approximately 108-fold compared to that in Mg 2+ . Specifically, in Mg 2+ , EhCK1 exhibited a V max and K 0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn 2+ , it displayed a V max of 149.1 ± 2.5 U/mg and a K 0.5 of 9.5 ± 0.1 mM. Moreover, when Mg 2+ was present at a constant concentration of 12 mM, the K 0.5 value for Mn 2+ was ~ 2.4-fold lower than that in Mn 2+ alone, without affecting its V max . Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn 2+ , it is worth noting that its K m for choline and ATP were higher than in equimolar of Mg 2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn 2+ , exhibiting Michaelis-Menten kinetic with ethanolamine (K m  = 312 ± 27 µM) and cooperativity with ATP (K 0.5  = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg 2+ , while choline kinase β differentially recognized choline and ethanolamine in Mg 2+ and Mn 2+ , respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn 2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Twist (TWIST1) is a gene required for cell fate specification in embryos and its expression in mammary epithelium can initiate tumorigenesis through the epithelial-mesenchymal transition. To identify downstream target genes of Twist in breast cancer, we performed microarray analysis on the transgenic breast cancer cell line, MCF-7/Twist. One of the targets identified was choline kinase whose upregulation resulted in increased cellular phosphocholine and total choline containing compounds-a characteristic observed in highly aggressive metastatic cancers. To study the interactions between Twist, choline kinase, and their effect on the microenvironment, we used 1 H magnetic resonance spectroscopy and found significantly higher phosphocholine and total choline, as well as increased phosphocholine/glycerophosphocholine ratio in MCF-7/Twist cells. We also observed significant increases in extracellular glucose, lactate, and [H +] ion concentrations in the MCF-7/Twist cells. Magnetic resonance imaging of MCF-7/Twist orthotopic breast tumors showed a significant increase in vascular volume and permeability surface area product compared to control tumors. In addition, by reverse transcription-quantitative polymerase chain reaction, we discovered that Twist upregulated choline kinase expression in estrogen receptor negative breast cancer cell lines through FOXA1 downregulation. Moreover, using The Cancer Genome Atlas database, we observed a significant inverse relationship between FOXA1 and choline kinase expression and propose that it could act as a modulator of the Twist/choline kinase axis. The data presented indicate that Twist is a driver of choline kinase expression in breast cancer cells via FOXA1 resulting in the generation of an aggressive breast cancer phenotype., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Choline kinase (ChoK) has been well documented as a major enzyme involved in the anomalous cellular lipid metabolic profile of chronic inflammatory disorders. However, new research has now been unveiled that helps us to better understand how changes in lipid metabolism influence the transformational phenotype, drug resistance, and antiapoptotic characteristics of invasive cells, leading to rheumatoid arthritis (RA) disease progression. It is still unknown how ChoK modulates the lipid metabolic aberrations that may promote altered cell phenotype and functionality in RA. Herein, we review the current understanding of ChoK's role in altered metabolism in diverse cell types involved in RA progression, and for the first time, we take a step forward to complete the puzzle and summarise striking facts that link choline metabolism to its transformed phenotype, in order to postulate ChoK as a robust therapeutic target in RA. This review forms a foundation on which ChoK can be tackled as a potential biomarker, opening doors for RA diagnosis and prognosis. It frameworks several ChoK inhibitors that rewire the lipid metabolic profile in the inflammatory disease landscape and envisages its being translated to clinics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The short-chain dehydrogenase/reductase (SDR) superfamily has essential roles in lipid metabolism and redox sensing. In recent years, accumulating evidence highlights the emerging association between SDR family enzymes and cancer. Dehydrogenase/reductase member 2(DHRS2) belongs to the NADH/NADPH-dependent SDR family, and extensively participates in the regulation of the proliferation, migration, and chemoresistance of cancer cells. However, the underlying mechanism has not been well defined. In the present study, we have demonstrated that DHRS2 inhibits the growth and metastasis of ovarian cancer (OC) cells in vitro and in vivo. Mechanistically, the combination of transcriptome and metabolome reveals an interruption of choline metabolism by DHRS2. DHRS2 post-transcriptionally downregulates choline kinase α (CHKα) to inhibit AKT signaling activation and reduce phosphorylcholine (PC)/glycerophosphorylcholine (GPC) ratio, impeding choline metabolism reprogramming in OC. These actions mainly account for the tumor-suppressive role of DHRS2 in OC. Overall, our findings establish the mechanistic connection among metabolic enzymes, metabolites, and the malignant phenotype of cancer cells. This could result in further development of novel pharmacological tools against OC by the induction of DHRS2 to disrupt the choline metabolic pathway., (© 2022. The Author(s).)

Background: Choline kinase beta (CHKB) catalyzes the first step in the de novo biosynthesis of phosphatidyl choline and phosphatidylethanolamine via the Kennedy pathway. Derangement of this pathway might also influence the homeostasis of mitochondrial membranes. Autosomal recessive CHKB mutations cause a rare form of congenital muscular dystrophy known as megaconial congenital muscular dystrophy (MCMD)., Case Presentation: We describe a novel proband presenting MCMD due to unpublished CHKB mutations. The patient is a 6-year-old boy who came to our attention for cognitive impairment and slowly progressive muscular weakness. He was the first son of non-consanguineous healthy parents from Sri Lanka. Neurological examination showed proximal weakness at four limbs, weak osteotendinous reflexes, Gowers' maneuver, and waddling gate. Creatine kinase levels were mildly increased. EMG and brain MRI were normal. Left quadriceps skeletal muscle biopsy showed a myopathic pattern with nuclear centralizations and connective tissue increase. Histological and histochemical staining suggested subsarcolemmal localization and dimensional increase of mitochondria. Ultrastructural analysis confirmed the presence of enlarged ("megaconial") mitochondria. Direct sequencing of CHKB identified two novel defects: the c.1060G > C (p.Gly354Arg) substitution and the c.448-56&#95;29del intronic deletion, segregating from father and mother, respectively. Subcloning of RT-PCR amplicons from patient's muscle RNA showed that c.448-56&#95;29del results in the partial retention (14 nucleotides) of intron 3, altering physiological splicing and transcript stability. Biochemical studies showed reduced levels of the mitochondrial fission factor DRP1 and the severe impairment of mitochondrial respiratory chain activity in patient's muscle compared to controls., Conclusions: This report expands the molecular findings associated with MCMD and confirms the importance of considering CHKB variants in the differential diagnosis of patients presenting with muscular dystrophy and mental retardation. The clinical outcome of MCMD patients seems to be influenced by CHKB molecular defects. Histological and ultrastructural examination of muscle biopsy directed molecular studies and allowed the identification and characterization of an intronic mutation, usually escaping standard molecular testing., (© 2022. The Author(s).)

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial β-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb -/- affected muscle. Treatment of Chkb -/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for β-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury., (© 2022. The Author(s).)

The CHKB gene encodes choline kinase β, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb -/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb +/- mice. Unlike wildtype mice, 60% of the Chkb +/- and all Chkb -/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb +/- and Chkb -/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb -/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb +/- and Chkb -/- mice., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A , a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA , respectively) altered the promoter usage of CERS2 and CHKA . Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

An evaluation of the impact of vitamin E deficiency on expression of the alpha-tocopherol transfer protein (α-TTP) and related CRAL&#95;TRIO genes was undertaken using livers from adult zebrafish based on the hypothesis that increased lipid peroxidation would modulate gene expression. Zebrafish were fed either a vitamin E sufficient (E+) or deficient (E-) diet for 9 months, then fish were euthanized, and livers were harvested. Livers from the E+ relative to E- fish contained 40-times more α-tocopherol (P <0.0001) and one fourth the malondialdehyde (P = 0.0153). RNA was extracted from E+ and E- livers, then subject to evaluation of gene expression of ttpa and other genes of the CRAL&#95;TRIO family, genes of antioxidant markers, and genes related to lipid metabolism. Ttpa expression was not altered by vitamin E status. However, one member of the CRAL&#95;TRIO family, tyrosine-protein phosphatase non-receptor type 9 gene (ptpn9a), showed a 2.4-fold increase (P = 0.029) in E- relative to E+ livers. Further, we identified that the gene for choline kinase alpha (chka) showed a 3.0-fold increase (P = 0.010) in E- livers. These outcomes are consistent with our previous findings that show vitamin E deficiency increased lipid peroxidation causing increases in phospholipid turnover., (Copyright © 2021. Published by Elsevier Inc.)

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lipid droplets are important for cancer cell growth and survival. However, the mechanism underlying the initiation of lipid droplet lipolysis is not well understood. We demonstrate here that glucose deprivation induces the binding of choline kinase (CHK) α2 to lipid droplets, which is sequentially mediated by AMPK-dependent CHKα2 S279 phosphorylation and KAT5-dependent CHKα2 K247 acetylation. Importantly, CHKα2 with altered catalytic domain conformation functions as a protein kinase and phosphorylates PLIN2 at Y232 and PLIN3 at Y251. The phosphorylated PLIN2/3 dissociate from lipid droplets and are degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, fatty acid oxidation, and brain tumor growth. In addition, levels of CHKα2 S279 phosphorylation, CHKα2 K247 acetylation, and PLIN2/3 phosphorylation are positively correlated with one another in human glioblastoma specimens and are associated with poor prognosis in glioblastoma patients. These findings underscore the role of CHKα2 as a protein kinase in lipolysis and glioblastoma development., Competing Interests: Declaration of interests Dr. Zhimin Lu owns shares in Signalway Biotechnology (Pearland, TX), which supplied rabbit antibodies that recognize CHKα2 pS279, CHKα2 AcK247, PLIN2 pY232, and PLIN3 pY251. Dr. Lu’s interest in this company had no bearing on its being chosen to supply these reagents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKβ, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases., (© 2020 Wiley Periodicals LLC.)

Seeking for new anticancer drugs with strong antiproliferative activity and simple molecular structure, we designed a novel series of compounds based on our previous reported pharmacophore model composed of five moieties. Antiproliferative assays on four tumoral cell lines and evaluation of Human Choline Kinase CKα1 enzymatic activity was performed for these compounds. Among tested molecules, those ones with biphenyl spacer showed betters enzymatic and antiproliferative activities (n-v). Docking and crystallization studies validate the hypothesis and confirm the results. The most active compound (t) induces a significant arrest of the cell cycle in G0/G1 phase that ultimately lead to apoptosis, following the mitochondrial pathway, as demonstrated for other choline kinase inhibitors. However additional assays reveal that the inhibition of choline uptake could also be involved in the antiproliferative outcome of this class of compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Next generation antiandrogens such as enzalutamide (Enz) are effective initially for the treatment of castration-resistant prostate cancer (CRPC). However, the disease often relapses and the underlying mechanisms remain elusive. By performing H3-lysine-27 acetylation (H3K27ac) ChIP-seq in Enz-resistant CRPC cells, we identified a group of super enhancers (SEs) that are abnormally activated in Enz-resistant CRPC cells and associated with enhanced transcription of a subset of tumor promoting genes such as CHPT1, which catalyzes phosphatidylcholine (PtdCho) synthesis and regulates choline metabolism. Increased CHPT1 conferred CRPC resistance to Enz in vitro and in mice. While androgen receptor (AR) primarily binds to a putative CHPT1 enhancer and mediates androgen-dependent expression of CHPT1 gene in Enz-sensitive prostate cancer cells, AR binds to a different enhancer within the CHPT1 SE locus and facilities androgen-independent expression of CHPT1 in Enz-resistant cells. We further identified a long-non coding RNA transcribed at CHPT1 enhancer (also known as enhancer RNA) that binds to the H3K27ac reader BRD4 and participates in regulating CHPT1 SE activity and CHPT1 gene expression. Our findings demonstrate that aberrantly activated SE upregulates CHPT1 expression and confers Enz resistance in CRPC, suggesting that SE-mediated expression of downstream effectors such as CHPT1 can be viable targets to overcome Enz resistance in PCa.

Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 μM, respectively, and were shown to be 2-4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 μM and 10 μM, respectively, and the minimum lethal concentration (MLC) was 1.6 μM and 20 μM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae.

Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB., Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies., Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth., Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB., Lay Summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer., Competing Interests: Conflict of interest Prof. Josep M. Llovet is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb and Ipsen, and consulting fees from Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Eli Lilly, Exelixis, Merck, Ipsen, Glycotest, Navigant, Leerink Swann LLC, Midatech Ltd, Fortress Biotech, Sprink Pharmaceuticals and Nucleix and CANFITE. CA has a research contract with CHIOME Biosciences Inc. The other authors report no conflicts of interest in this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S . pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Moad Khalifa et al.)

Purpose : The spatio-molecular distribution of choline and its metabolites in tumors is highly heterogeneous. Due to regulation of choline metabolism by hypoxic transcriptional signaling and other survival factors, we envisage that detection of such heterogeneity in patient tumors could provide the basis for advanced localized therapy. However, non-invasive methods to assess this phenomenon in patients are limited. We investigated such heterogeneity in Non-Small Cell Lung Cancer (NSCLC) with [ 18 F]fluoromethyl-(1,2- 2 H 4 ) choline ([ 18 F]D4-FCH) and positron emission tomography/computed tomography (PET/CT). Experimental design : [ 18 F]D4-FCH (300.5±72.9MBq [147.60-363.6MBq]) was administered intravenously to 17 newly diagnosed NSCLC patients. PET/CT scans were acquired concurrently with radioactive blood sampling to permit mathematical modelling of blood-tissue transcellular rate constants. Comparisons were made with biopsy-derived choline kinase-α (CHKα) expression and diagnostic [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) scans. Results : Oxidation of [ 18 F]D4-FCH to [ 18 F]D4-fluorobetaine was suppressed (48.58±0.31% parent at 60 min) likely due to the deuterium isotope effect embodied within the design of the radiotracer. Early (5 min) and late (60 min) images showed specific uptake of tracer in all 51 lesions (tumors, lymph nodes and metastases) from 17 patients analyzed. [ 18 F]D4-FCH-derived uptake (SUV 60max ) in index primary lesions (n=17) ranged between 2.87-10.13; lower than that of [ 18 F]FDG PET [6.89-22.64]. Mathematical modelling demonstrated net irreversible uptake of [ 18 F]D4-FCH at steady-state, and parametric mapping of the entire tumor showed large intratumorally heterogeneity in radiotracer retention, which is likely to have influenced correlations with biopsy-derived CHKα expression. Conclusions : [ 18 F]D4-FCH is detectable in NSCLC with large intratumorally heterogeneity, which could be exploited in the future for targeting localized therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Specific deletion of the tumor suppressor TRAF3 from B lymphocytes in mice leads to the prolonged survival of mature B cells and expanded B cell compartments in secondary lymphoid organs. In the current study, we investigated the metabolic basis of TRAF3-mediated regulation of B cell survival by employing metabolomic, lipidomic, and transcriptomic analyses. We compared the polar metabolites, lipids, and metabolic enzymes of resting splenic B cells purified from young adult B cell-specific Traf3 -/- and littermate control mice. We found that multiple metabolites, lipids, and enzymes regulated by TRAF3 in B cells are clustered in the choline metabolic pathway. Using stable isotope labeling, we demonstrated that phosphocholine and phosphatidylcholine biosynthesis was markedly elevated in Traf3 -/- mouse B cells and decreased in TRAF3-reconstituted human multiple myeloma cells. Furthermore, pharmacological inhibition of choline kinase α, an enzyme that catalyzes phosphocholine synthesis and was strikingly increased in Traf3 -/- B cells, substantially reversed the survival phenotype of Traf3 -/- B cells both in vitro and in vivo. Taken together, our results indicate that enhanced phosphocholine and phosphatidylcholine synthesis supports the prolonged survival of Traf3 -/- B lymphocytes. Our findings suggest that TRAF3-regulated choline metabolism has diagnostic and therapeutic value for B cell malignancies with TRAF3 deletions or relevant mutations., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Choline kinase alpha is a 457-residue protein that catalyzes the reaction between ATP and choline to yield ADP and phosphocholine. This metabolic action has been well studied because of choline kinase's link to cancer malignancy and poor patient prognosis. As the myriad of x-ray crystal structures available for this enzyme show, chemotherapeutic drug design has centered on stopping the catalytic activity of choline kinase and reducing the downstream metabolites it produces. Furthermore, these crystal structures only reveal the catalytic domain of the protein, residues 80-457. However, recent studies provide evidence for a non-catalytic protein-binding role for choline kinase alpha. Here, we show that choline kinase alpha interacts with the SH3 domain of c-Src. Co-precipitation assays, surface plasmon resonance, and crystallographic analysis of a 1.5 Å structure demonstrate that this interaction is specific and is mediated by the poly-proline region found N-terminal to the catalytic domain of choline kinase. Taken together, these data offer strong evidence that choline kinase alpha has a heretofore underappreciated role in protein-protein interactions, which offers an exciting new way to approach drug development against this cancer-enhancing protein.

Lipids represent a diverse array of molecules essential to the cell's structure, defense, energy, and communication. Lipid metabolism can often become dysregulated during tumor development. During cancer therapy, targeted inhibition of cell proliferation can likewise cause widespread and drastic changes in lipid composition. Molecular imaging techniques have been developed to monitor altered lipid profiles as a biomarker for cancer diagnosis and treatment response. For decades, MRS has been the dominant non-invasive technique for studying lipid metabolite levels. Recent insights into the oncogenic transformations driving changes in lipid metabolism have revealed new mechanisms and signaling molecules that can be exploited using optical imaging, mass spectrometry imaging, and positron emission tomography. These novel imaging modalities have provided researchers with a diverse toolbox to examine changes in lipids in response to a wide array of anticancer strategies including chemotherapy, radiation therapy, signal transduction inhibitors, gene therapy, immunotherapy, or a combination of these strategies. The understanding of lipid metabolism in response to cancer therapy continues to evolve as each therapeutic method emerges, and this review seeks to summarize the current field and areas of unmet needs., (© 2019 John Wiley & Sons, Ltd.)

Congenital muscular dystrophy with megaconial myopathy (MDCMC) is an autosomal recessive disorder characterized by progressive muscle weakness and wasting. The observation of megamitochondria in skeletal muscle biopsies is exclusive to this type of MD. The disease is caused by loss of function mutations in the choline kinase beta (CHKB) gene which results in dysfunction of the Kennedy pathway for the synthesis of phosphatidylcholine. We have previously reported a rostrocaudal MD (rmd) mouse with a deletion in the Chkb gene resulting in an MDCMC-like phenotype, and we used this mouse to test gene therapy strategies for the rescue and alleviation of the dystrophic phenotype. Introduction of a muscle-specific Chkb transgene completely rescues motor and behavioral function in the rmd mouse model, confirming the cell-autonomous nature of the disease. Intramuscular gene therapy post-disease onset using an adeno-associated viral 6 (AAV6) vector carrying a functional copy of Chkb is also capable of rescuing the dystrophy phenotype. In addition, we examined the ability of choline kinase alpha (Chka), a gene paralog of Chkb, to improve dystrophic phenotypes when upregulated in skeletal muscles of rmd mutant mice using a similar AAV6 vector. The sum of our results in a preclinical model of disease suggest that replacement of the Chkb gene or upregulation of endogenous Chka could serve as potential lines of therapy for MDCMC patients., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Congenital Muscular Dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders presenting at birth with psychomotor delay, cognitive impairment, muscle weakness and hypotonia. Here we described an alteration of mitochondrial inner membrane potential and mitochondrial network in cells derived from Italian patients carrying three novel mutations in CHKB gene, recently associated with "megaconial CMD". On the bases of our findings, we hypothesize that the mitochondrial membrane potential alteration, presumably as a consequence of the altered biosynthesis of phosphatidylcholine, could be responsible for the peculiar morphological aspect of mitochondria in this disease and might be involved in the disease pathogenesis., (Copyright © 2019. Published by Elsevier B.V.)

Copy number variation (CNV) influences the mRNA transcription levels and phenotypic traits through gene dosage, position effects, alteration of downstream pathways, and modulation of the structure and position of chromosomes. A previous study using the read depth approach to genome resequencing analysis revealed CNVs of the choline kinase beta (CHKB) gene in the copy number variable regions (CNVRs) of yak breeds may influence muscle development and therefore the phenotypic traits of yak breeds. Further work is required to attain a more complete understanding and validate the importance of the detected CNVR of the CHKB gene found in yak breeds, because there is no association studies of the CHKB gene with yak growth traits that have been reported. The goal of this study was to determine the distribution of CHKB copy numbers in five Chinese domestic yak breeds and evaluate their impact on gene expression and growth traits. The data were analyzed using real-time quantitative PCR. In this study, the normal CNV of the CHKB gene was found to be significantly (p < 0.05) associated with greater chest girth and body weight for three age groups of Datong yaks. Our results indicated that the copy number of the CHKB gene is negatively correlated with the mRNA expression level. From this result, we conclude that CNVs of the CHKB gene could be novel markers for growth traits of Chinese domestic yak breeds and might therefore provide a novel opportunity to utilize data on CNVs in designing molecular markers for the selection of animal breeding programs for larger populations of various yaks.

Background: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase β (Chk β) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKβ are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria., Methods: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase β (Chk β) were analyzed for Pak1/2 phosphorylation., Results: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkβ and are a model for human diseases associated with CHKβ deficiency. Pak1/2 and Chkβ activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis., Conclusions: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKβ deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline), which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However, salt accumulates with the production and inhibits enzyme activity, and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore, when the mutant was used in fed-batch production, the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM, the productivity was 5.8 ± 0.1 mM·L -1  h -1 , and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry, and sheds light on alleviating effect of salt accumulation during industrial manufacturing process., (Copyright © 2018. Published by Elsevier B.V.)

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells., Competing Interests: The authors have declared no competing interests exist.

In this article we present a series of non-cytotoxic potent human choline kinase (CK) inhibitors that exhibit nanomolar antiplasmodial activity in vitro. The most active antiplasmodial compounds, 10a-b, bearing a pyridinium cationic head were inactive against CK, while compounds 10g and 10j with a quinolinium moiety exhibit moderate inhibition of both the parasite and the enzyme. The results point towards an additional mechanism of action unrelated to CK inhibition that remains to be established., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: Choline kinase α inhibitors represent one of the newest classes of cytotoxic drugs for cancer treatment, since aberrant choline metabolism is a characteristic shared by many human cancers., Results: Here, we present a new class of asymmetrical pyridinium/quinolinium derivatives developed and designed based on drug optimization., Conclusion: Among all compounds described here, compound 8, bearing a 7-chloro-4N-methyl-p-chloroaniline quinolinium moiety, exhibited the greatest inhibitory activity at the enzyme (IC 50  = 0.29 μM) and antiproliferative activity in cellular assays (GI 50  = 0.29-0.92 μM). Specifically, compound 8 strongly induces a cell-cycle arrest in G1 phase, but it does not significantly induce apoptosis while causing senescence in the MDA-MB-231 cell line.

Esophageal squamous cell carcinoma (ESCC) is a major health threat worldwide. Research focused on molecular events associated with ESCC carcinogenesis for diagnosis, treatment and prevention is needed. Our goal is to discover novel biomarkers and investigate the underlying molecular mechanisms of ESCC progression by employing a global metabolomic approach. Sera from 34 ESCC patients and 32 age and sex matched healthy controls were profiled using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS). We identified 120 differential metabolites in ESCC patient serums compared to healthy controls. Several amino acids, serine, arginine, lysine and histidine were significantly changed in ESCC patients. Most importantly, we found dysregulated lipid metabolism as an important characteristic in ESCC patients. Several free fat acids (FFA) and carnitines were found down-regulated in ESCC patients. Choline was significantly increased and phosphatidylcholines (PC) were significantly decreased in ESCC serum. The high expression of choline and low expression of total PC in patient serum were associated with the high expression of choline kinase (Chok) and activated Kennedy pathway in ESCC cells. Chok expression can serve as a significant biomarker for ESCC prognosis. In conclusion, metabolite profiles in the ESCC patient serum were significantly different from those in the healthy controls. Phosphatidylcholines and Chok, the key enzyme in the PC metabolism pathway, may serve as novel biomarkers for ESCC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although gemcitabine plus cisplatin is the gold standard chemotherapy regimen for advanced cholangiocarcinoma, the response rate has been disappointing. This study aims to investigate a novel therapeutic regimen [gemcitabine plus everolimus (rad001), an mTOR inhibitor] for cholangiocarcinoma. Gemcitabine, oxaliplatin, cetuximab and rad001 in various combinations were first evaluated in vitro using six cholangiocarcinoma cell lines. In vivo therapeutic efficacies of gemcitabine and rad001 alone and their combination were further evaluated using a xenograft mouse model and a chemically induced orthotopic cholangiocarcinoma rat model. In the in vitro study, gemcitabine plus rad001 exerted a synergistic therapeutic effect on the cholangiocarcinoma cells, irrespective of the KRAS mutation status. In the xenograft study, gemcitabine plus rad001 showed the best therapeutic effect on tumor volume change, and was associated with increased caspase-3 expression, decreased eIF4E expression, as well as overexpression of both death receptor- and mitochondrial apoptotic pathway-related genes. In a chemically induced cholangiocarcinoma-afflicted rat model, the gemcitabine plus rad001 treatment suppressed tumor glycolysis as measured by 18 F-fludeoxyglucose micro-positron emission tomography. Also, increased intratumoral free choline, decreased glycerophosphocholine and nearly undetectable phosphocholine levels were demonstrated by proton nuclear magnetic resonance, supported by results of decreased choline kinase expression in western blotting. We concluded that gemcitabine plus rad001 has a synergistic antiproliferative effect on cholangiocarcinoma, irrespective of the KRAS mutation status. The antitumor effect is associated with activation of both death receptor and mitochondrial pathways, as well as the downregulation of choline kinase activity, resulting in a characteristic change in choline metabolism., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The major phospholipid present in most eukaryotic membranes is phosphatidylcholine (PC), comprising ~ 50% of phospholipid content. PC metabolic pathways are highly conserved from yeast to humans. The main pathway for the synthesis of PC is the Kennedy (CDP-choline) pathway. In this pathway, choline is converted to phosphocholine by choline kinase, phosphocholine is metabolized to CDP-choline by the rate-determining enzyme for this pathway, CTP:phosphocholine cytidylyltransferase, and cholinephosphotransferase condenses CDP-choline with diacylglycerol to produce PC. This Review discusses how PC synthesis via the Kennedy pathway is regulated, its role in cellular and biological processes, as well as diseases known to be associated with defects in PC synthesis. Finally, we present the first model for the making of a membrane via PC synthesis., (© 2017 Federation of European Biochemical Societies.)

Choline kinase α (ChoKα) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic. As such, it makes a promising target for inhibiting tumor growth. Though there have been several inhibitors synthesized for ChoKα, not all of them demonstrate the same efficacy in vivo, though the reasons behind this difference in potency are not clear. One particular inhibitor, designated TCD-717, has recently completed phase I clinical trials. Cell culture and in vitro studies support the powerful inhibitory effect TCD-717 has on ChoKα, but an examination of the inhibitor's interaction with the ChoKα enzyme has been missing prior to this work. Here we detail the 2.35 Å structure of ChoKα in complex with TCD-717. Examination of this structure in conjunction with kinetic assays reveals that TCD-717 does not bind directly in the choline pocket as do previously characterized ChoKα inhibitors, but rather in a proximal but novel location near the surface of the enzyme. The unique binding site identified for TCD-717 lends insight for the future design of more potent in vivo inhibitors for ChoKα.

Despite the proven clinical antineoplastic activity of histone deacetylase inhibitors (HDACI), their effect has been reported to be lower than expected in B-cell lymphomas. Traditionally considered as "epigenetic drugs", HDACI modify the acetylation status of an extensive proteome, acting as general lysine deacetylase inhibitors (KDACI), and thus potentially impacting various branches of cellular metabolism. Here, we demonstrate through metabolomic profiling of patient plasma and cell lines that the KDACI panobinostat alters lipid metabolism and downstream survival signaling in diffuse large B-cell lymphomas (DLBCL). Specifically, panobinostat induces metabolic adaptations resulting in newly acquired dependency on the choline pathway and activation of PI3K signaling. This metabolic reprogramming decreased the antineoplastic effect of panobinostat. Conversely, inhibition of these metabolic adaptations resulted in superior anti-lymphoma effect as demonstrated by the combination of panobinostat with a choline pathway inhibitor. In conclusion, our study demonstrates the power of metabolomics in identifying unknown effects of KDACI, and emphasizes the need for a better understanding of these drugs in order to achieve successful clinical implementation., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a V max of 3.52μmol/min/mg and an apparent K m value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent K m for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the V max /K m decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glycerophosphodiesterase EDI3 (GPCPD1; GDE5; GDPD6) has been suggested to promote cell migration, adhesion, and spreading, but its mechanisms of action remain uncertain. In this study, we targeted the glycerol-3-phosphate acyltransferase GPAM along with choline kinase-α (CHKA), the enzymes that catabolize the products of EDI3 to determine which downstream pathway is relevant for migration. Our results clearly showed that GPAM influenced cell migration via the signaling lipid lysophosphatidic acid (LPA), linking it with GPAM to cell migration. Analysis of GPAM expression in different cancer types revealed a significant association between high GPAM expression and reduced overall survival in ovarian cancer. Silencing GPAM in ovarian cancer cells decreased cell migration and reduced the growth of tumor xenografts. In contrast to these observations, manipulating CHKA did not influence cell migration in the same set of cell lines. Overall, our findings show how GPAM influences intracellular LPA levels to promote cell migration and tumor growth. Cancer Res; 77(17); 4589-601. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose of Review: Prostate cancer (PCa) is the most commonly diagnosed noncutaneous cancer and second leading cause of death in men. Imaging evaluation of PCa is challenging because of the prostate's deep pelvic location, its complex zonal anatomy and its multifocal nature. Novel imaging modalities are needed to improve detection, reassessment in biochemical relapse, and disease progression in advanced metastatic stages., Recent Findings: Current imaging modalities have distinct strengths. However, all lack the ability to diagnose micrometastases, differentiate high from low-grade disease and diagnose advanced disease, especially at low prostate specific antigen values. There is a need to combine the existing imaging methods with concepts utilizing tumor biology to differentiate biologically aggressive from indolent tumors. PET imaging with novel tracers facilitate improved imaging of PCa, but also usher in new compounds that could be useful for directing treatment as well. Most tracers have limited sensitivity, with the exception of prostate-specific membrane antigen (PSMA)-targeting tracers, that offer relatively higher sensitivity and specificity., Summary: PSMA-PET appears promising in improving the imaging yield particularly in recurrent and advanced disease states. Incorporating PSMA-PET in these settings could open or prolong windows along the trajectory of the disease that could allow new treatments or more effective use of currently existing treatments. Prospective studies focusing on novel imaging enhancement and integration with therapeutic applications are needed.

Background: Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure in many countries. The aim of the study was to describe the profiling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver of Acetaminophen -induced liver injured mice., Methods: A time course study was carried out using C57BL/6 mice after intraperitoneal administration of 300 mg/kg Acetaminophen 1 h, 3 h, 6 h, 12 h and 24 h. A high-throughput liquid chromatography mass spectrometry (LC-MS) lipidomic method was utilized to detect phosphatidylcholine and phosphatidylethanolamine species in the plasma and liver. The expressions of phosphatidylcholine and phosphatidylethanolamine metabolism related genes in liver were detected by quantitative Reverse transcription polymerase chain reaction (qRT-PCR) and Western-blot., Results: Following Acetaminophen treatment, the content of many PC and PE species in plasma increased from 1 h time point, peaked at 3 h or 6 h, and tended to return to baseline at 24 h time point. The relative contents of almost all PC species in liver decreased from 1 h, appeared to be lowest at 6 h, and then return to normality at 24 h, which might be partly explained by the suppression of phospholipases mRNA expressions and the induction of choline kinase (Chka) expression. Inconsistent with PC profile, the relative contents of many PE species in liver increased upon Acetaminophen treatment, which might be caused by the down-regulation of phosphatidylethanolamine N-methyltransferase (Pemt)., Conclusions: Acetaminophen overdose induced dramatic change of many PC and PE species in plasma and liver, which might be caused by damaging hepatocytes and interfering the phospholipid metabolism in Acetaminophen -injured liver.

Abnormal choline phospholipid metabolism is a hallmark of cancer. The magnetic resonance spectroscopy (MRS) detected total choline (tCho) signal can serve as an early noninvasive imaging biomarker of chemotherapy response in breast cancer. We have quantified the individual components of the tCho signal, glycerophosphocholine (GPC), phosphocholine (PC) and free choline (Cho), before and after treatment with the commonly used chemotherapeutic drug doxorubicin in weakly metastatic human MCF7 and triple-negative human MDA-MB-231 breast cancer cells. While the tCho concentration did not change following doxorubicin treatment, GPC significantly increased and PC decreased. Of the two phosphatidylcholine-specific PLD enzymes, only PLD1, but not PLD2, mRNA was down-regulated by doxorubicin treatment. For the two reported genes encoding GPC phosphodiesterase, the mRNA of GDPD6, but not GDPD5, decreased following doxorubicin treatment. mRNA levels of choline kinase α (ChKα), which converts Cho to PC, were reduced following doxorubicin treatment. PLD1 and ChKα protein levels decreased following doxorubicin treatment in a concentration dependent manner. Treatment with the PLD1 specific inhibitor VU0155069 sensitized MCF7 and MDA-MB-231 breast cancer cells to doxorubicin-induced cytotoxicity. Low concentrations of 100 nM of doxorubicin increased MDA-MB-231 cell migration. GDPD6, but not PLD1 or ChKα, silencing by siRNA abolished doxorubicin-induced breast cancer cell migration. Doxorubicin induced GPC increase and PC decrease are caused by reductions in PLD1, GDPD6, and ChKα mRNA and protein expression. We have shown that silencing or inhibiting these genes/proteins can promote drug effectiveness and reduce adverse drug effects. Our findings emphasize the importance of detecting PC and GPC individually., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication. IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the "membranous web" structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation., (Copyright © 2017 Wong and Chen.)

Background & Aims: Choline kinase α (CHKA) catalyzes conversion of choline to phosphocholine and can contribute to carcinogenesis. Little is known about the role of CHKA in the pathogenesis of hepatocellular carcinoma (HCC)., Methods: We performed whole-exome and transcriptome sequence analyses of 9 paired HCC and non-tumor-adjacent tissues. We performed tissue chip analyses of 120 primary HCC and non-tumor-adjacent tissues from patients who received surgery in Shanghai, China from January 2006 through December 2009; 48 sets of specimens (HCC and non-tumor-adjacent tissues) were also analyzed. CHKA gene copy number was quantified and findings were validated by quantitative reverse transcription polymerase chain reaction analysis. CHKA messenger RNA and protein levels were determined by polymerase chain reaction, immunohistochemical, and immunoblot analyses. CHKA was examined in 2 hepatocyte cell lines and 7 HCC-derived cell lines, and knocked down with small interfering RNAs in 3 HCC cell lines. Cells were analyzed in proliferation, wound healing, migration, and invasion assays. Cells were injected into tail veins of mice and tumor growth and metastasis were quantified. Immunoprecipitation and immunofluorescence assays were conducted to determine interactions between CHKA and the epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin complex 2., Results: Levels of CHKA messenger RNA were frequently increased in HCC tissues compared with nontumor tissues; increased expression was associated with amplification at the CHKA loci. Tumors that expressed high levels of CHKA had more aggressive phenotypes, and patients with these tumors had shorter survival times after surgery compared to patients whose tumors expressed low levels of CHKA. HCC cell lines that stably overexpressed CHKA had higher levels of migration and invasion than control HCC cells, and formed larger xenograft tumors with more metastases in mice compared to HCC cells that did not overexpress CHKA. CHKA was required for physical interaction between EGFR and mechanistic target of rapamycin complex 2. This complex was required for HCC cells to form metastatic xenograft tumors in mice and to become resistant to EGFR inhibitors., Conclusions: We found levels of CHKA to be increased in human HCCs compared to nontumor tissues, and increased expression to be associated with tumor aggressiveness and reduced survival times of patients. Overexpression of CHKA in HCC cell lines increased their invasiveness, resistance to EGFR inhibitors, and ability to form metastatic tumors in mice by promoting interaction of EGFR with mechanistic target of rapamycin complex 2., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Choline, a hydrophilic cation, has versatile physiological roles throughout the body, including cholinergic neurotransmission, memory consolidation and membrane biosynthesis and metabolism. Choline kinases possess enzyme activity that catalyses the conversion of choline to phosphocholine, which is further converted to cytidine diphosphate-coline (CDP-choline) in the biosynthesis of phosphatidylcholine (PC). PC is a major constituent of the phospholipid bilayer which constitutes the eukaryotic cell membrane, and regulates cell signal transduction. Choline Kinase consists of three isoforms, CHKα1, CHKα2 and CHKβ, encoded by two separate genes (CHKA(Human)/Chka(Mouse) and CHKB(Human)/Chkb(Mouse)). Both isoforms have similar structures and enzyme activity, but display some distinct molecular structural domains and differential tissue expression patterns. Whilst Choline Kinase was discovered in early 1950, its pivotal role in the development of muscular dystrophy, bone deformities, and cancer has only recently been identified. CHKα has been proposed as a cancer biomarker and its inhibition as an anti-cancer therapy. In contrast, restoration of CHKβ deficiency through CDP-choline supplements like citicoline may be beneficial for the treatment of muscular dystrophy, bone metabolic diseases, and cognitive conditions. The molecular structure and expression pattern of Choline Kinase, the differential roles of Choline Kinase isoforms and their potential as novel therapeutic targets for muscular dystrophy, bone deformities, cognitive conditions and cancer are discussed., (Copyright © 2016. Published by Elsevier Ltd.)