Folylpolyglutamate synthetase mRNA G-quadruplexes regulate its cell protrusion localization and enhance a cancer cell invasive phenotype upon folate repletion.

BACKGROUND: Folates are crucial for the biosynthesis of nucleotides and amino acids, essential for cell proliferation and development. Folate deficiency induces DNA damage, developmental defects, and tumorigenicity. The obligatory enzyme folylpolyglutamate synthetase (FPGS) mediates intracellular folate retention via cytosolic and mitochondrial folate polyglutamylation. Our previous paper demonstrated the association of the cytosolic FPGS (cFPGS) with the cytoskeleton and various cell protrusion proteins. Based on these recent findings, the aim of the current study was to investigate the potential role of cFPGS at cell protrusions. RESULTS: Here we uncovered a central role for two G-quadruplex (GQ) motifs in the 3'UTR of FPGS mediating the localization of cFPGS mRNA and protein at cell protrusions. Using the MBSV6-loop reporter system and fluorescence microscopy, we demonstrate that following folate deprivation, cFPGS mRNA is retained in the endoplasmic reticulum, whereas upon 15 min of folate repletion, this mRNA is rapidly translocated to cell protrusions in a 3'UTR- and actin-dependent manner. The actin dependency of this folate-induced mRNA translocation is shown by treatment with Latrunculin B and inhibitors of the Ras homolog family member A (RhoA) pathway. Upon folate repletion, the FPGS 3'UTR GQs induce an amoeboid/mesenchymal hybrid cell phenotype during migration and invasion through a collagen gel matrix. Targeted disruption of the 3'UTR GQ motifs by introducing point mutations or masking them by antisense oligonucleotides abrogated cell protrusion targeting of cFPGS mRNA. CONCLUSIONS: Collectively, the GQ motifs within the 3'UTR of FPGS regulate its transcript and protein localization at cell protrusions in response to a folate cue, inducing cancer cell invasive phenotype. These novel findings suggest that the 3'UTR GQ motifs of FPGS constitute an attractive druggable target aimed at inhibition of cancer invasion and metastasis.

Doubly Stimulated Corrole for Organelle-Selective Antitumor Cytotoxicity.

Balancing between safety and efficacy of cancer chemotherapeutics is achievable by relying on internal and/or external stimuli for selective and on-demand antitumor cytotoxicity. We now introduce the difluorophosphorus(V) corrole PC-Im, a theranostic agent with a pH-sensitive N-methylimidazole moiety. Structure/activity relationships, via comparison with the permanently charged PC-ImM+ and the lipophilic PC, uncovered the exceptional features of PC-Im: nanoparticular and monomeric at neutral and low pH, respectively, 10-fold increased light-induced singlet oxygen production at acidic pH, internalization into malignant cells within minutes, and selective accumulation within lysosomes. Submillimolar PC-Im concentrations are tolerable in the dark, while illumination induces nanomolar cytotoxic effects due to a multiplicity of cellular deleterious events: endoplasmic reticulum fragmentation, lysosome fusion and exocytosis, calcium leakage, mitochondrial fission, and swelling. PC-Im emerges as an antitumor agent, whose potency is triggered by endogenous and exogenous stimuli, assuring its cytotoxicity will occur selectively upon lysosomal accumulation and solely upon light activation.

Folylpoly-É£-glutamate synthetase association to the cytoskeleton: Implications to folate metabolon compartmentalization.

Folates are essential for nucleotide biosynthesis, amino acid metabolism and cellular proliferation. Following carrier-mediated uptake, folates are polyglutamylated by folylpoly-É£-glutamate synthetase (FPGS), resulting in their intracellular retention. FPGS appears as a long isoform, directed to mitochondria via a leader sequence, and a short isoform reported as a soluble cytosolic protein (cFPGS). However, since folates are labile and folate metabolism is compartmentalized, we herein hypothesized that cFPGS is associated with the cytoskeleton, to couple folate uptake and polyglutamylation and channel folate polyglutamates to metabolon compartments. We show that cFPGS is a cytoskeleton-microtubule associated protein: Western blot analysis revealed that endogenous cFPGS is associated with the insoluble cellular fraction, i.e., cytoskeleton and membranes, but not with the cytosol. Mass spectrometry analysis identified the putative cFPGS interactome primarily consisting of microtubule subunits and cytoskeletal motor proteins. Consistently, immunofluorescence microscopy with cytosol-depleted cells demonstrated the association of cFPGS with the cytoskeleton and unconventional myosin-1c. Furthermore, since anti-microtubule, anti-actin cytoskeleton, and coatomer dissociation-inducing agents yielded perinuclear pausing of cFPGS, we propose an actin- and microtubule-dependent transport of cFPGS between the ER-Golgi and the plasma membrane. These novel findings support the coupling of folate transport with polyglutamylation and folate channeling to intracellular metabolon compartments. SIGNIFICANCE: FPGS, an essential enzyme catalyzing intracellular folate polyglutamylation and efficient retention, was described as a soluble cytosolic enzyme in the past 40 years. However, based on the lability of folates and the compartmentalization of folate metabolism and nucleotide biosynthesis, we herein hypothesized that cytoplasmic FPGS is associated with the cytoskeleton, to couple folate transport and polyglutamylation as well as channel folate polyglutamates to biosynthetic metabolon compartments. Indeed, using complementary techniques including Mass-spectrometry proteomics and fluorescence microscopy, we show that cytoplasmic FPGS is associated with the cytoskeleton and unconventional myosin-1c. This novel cytoskeletal localization of cytoplasmic FPGS supports the dynamic channeling of polyglutamylated folates to metabolon compartments to avoid oxidation and intracellular dilution of folates, while enhancing folate-dependent de novo biosynthesis of nucleotides and DNA/protein methylation.

Deciphering molecular mechanisms underlying chemoresistance in relapsed AML patients: towards precision medicine overcoming drug resistance.

BACKGROUND: Acute myeloid leukemia (AML) remains a devastating disease with a 5-year survival rate of less than 30%. AML treatment has undergone significant changes in recent years, incorporating novel targeted therapies along with improvements in allogeneic bone marrow transplantation techniques. However, the standard of care remains cytarabine and anthracyclines, and the primary hindrance towards curative treatment is the frequent emergence of intrinsic and acquired anticancer drug resistance. In this respect, patients presenting with chemoresistant AML face dismal prognosis even with most advanced therapies. Herein, we aimed to explore the potential implementation of the characterization of chemoresistance mechanisms in individual AML patients towards efficacious personalized medicine. METHODS: Towards the identification of tailored treatments for individual patients, we herein present the cases of relapsed AML patients, and compare them to patients displaying durable remissions following the same chemotherapeutic induction treatment. We quantified the expression levels of specific genes mediating drug transport and metabolism, nucleotide biosynthesis, and apoptosis, in order to decipher the molecular mechanisms underlying intrinsic and/or acquired chemoresistance modalities in relapsed patients. This was achieved by real-time PCR using patient cDNA, and could be readily implemented in the clinical setting. RESULTS: This analysis revealed pre-existing differences in gene expression levels between the relapsed patients and patients with lasting remissions, as well as drug-induced alterations at different relapse stages compared to diagnosis. Each of the relapsed patients displayed unique chemoresistance mechanisms following similar treatment protocols, which could have been missed in a large study aimed at identifying common drug resistance determinants. CONCLUSIONS: Our findings emphasize the need for standardized evaluation of key drug transport and metabolism genes as an integral component of routine AML management, thereby allowing for the selection of treatments of choice for individual patients. This approach could facilitate the design of efficacious personalized treatment regimens, thereby reducing relapse rates of therapy refractory disease.

The Lysosomotropic Activity of Hydrophobic Weak Base Drugs is Mediated via Their Intercalation into the Lysosomal Membrane.

Lipophilic weak base therapeutic agents, termed lysosomotropic drugs (LDs), undergo marked sequestration and concentration within lysosomes, hence altering lysosomal functions. This lysosomal drug entrapment has been described as luminal drug compartmentalization. Consistent with our recent finding that LDs inflict a pH-dependent membrane fluidization, we herein demonstrate that LDs undergo intercalation and concentration within lysosomal membranes. The latter was revealed experimentally and computationally by (a) confocal microscopy of fluorescent compounds and drugs within lysosomal membranes, and (b) molecular dynamics modeling of the pH-dependent membrane insertion and accumulation of an assortment of LDs, including anticancer drugs. Based on the multiple functions of the lysosome as a central nutrient sensory hub and a degradation center, we discuss the molecular mechanisms underlying the alteration of morphology and impairment of lysosomal functions as consequences of LDs' intercalation into lysosomes. Our findings bear important implications for drug design, drug induced lysosomal damage, diseases and pertaining therapeutics.

Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine.

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines' sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.

Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity.

Transcription factor EB (TFEB) is a master transcriptional regulator playing a key role in lysosomal biogenesis, autophagy and lysosomal exocytosis. TFEB activity is inhibited following its phosphorylation by mammalian target of rapamycin complex 1 (mTORC1) on the surface of the lysosome. Phosphorylated TFEB is bound by 14-3-3 proteins, resulting in its cytoplasmic retention in an inactive state. It was suggested that the calcium-dependent phosphatase calcineurin is responsible for dephosphorylation and subsequent activation of TFEB under conditions of lysosomal stress. We have recently demonstrated that TFEB is activated following exposure of cancer cells to lysosomotropic anticancer drugs, resulting in lysosome-mediated cancer drug resistance via increased lysosomal biogenesis, lysosomal drug sequestration, and drug extrusion through lysosomal exocytosis. Herein, we studied the molecular mechanism underlying lysosomotropic-drug-induced activation of TFEB. We demonstrate that accumulation of lysosomotropic drugs results in membrane fluidization of lysosome-like liposomes, which is strictly dependent on the acidity of the liposomal lumen. Lysosomal accumulation of lysosomotropic drugs and the consequent fluidization of the lysosomal membrane, facilitated the dissociation of mTOR from the lysosomal membrane and inhibited the kinase activity of mTORC1, which is necessary and sufficient for the rapid translocation of TFEB to the nucleus. We further show that while lysosomotropic drug sequestration induces Ca2+ release into the cytoplasm, facilitating calcineurin activation, chelation of cytosolic Ca2+, or direct inhibition of calcineurin activity, do not interfere with drug-induced nuclear translocation of TFEB. We thus suggest that lysosomotropic drug-induced activation of TFEB is mediated by mTORC1 inhibition due to lysosomal membrane fluidization and not by calcineurin activation. We further postulate that apart from calcineurin, other constitutively active phosphatase(s) partake in TFEB dephosphorylation and consequent activation. Moreover, a rapid export of TFEB from the nucleus to the cytosol occurs upon relief of mTORC1 inhibition, suggesting that dephosphorylated TFEB constantly travels between the nucleus and the cytosol, acting as a rapidly responding sensor of mTORC1 activity.

The JmjN domain as a dimerization interface and a targeted inhibitor of KDM4 demethylase activity.

Histone methylation is regulated to shape the epigenome by modulating DNA compaction, thus playing central roles in fundamental chromatin-based processes including transcriptional regulation, DNA repair and cell proliferation. Histone methylation is erased by demethylases including the well-established KDM4 subfamily members, however, little is known about their dimerization capacity and its impact on their demethylase activity. Using the powerful bimolecular fluorescence complementation technique, we herein show the in situ formation of human KDM4A and KDM4C homodimers and heterodimers in nuclei of live transfectant cells and evaluate their H3K9me3 demethylation activity. Using size exclusion HPLC as well as Western blot analysis, we show that endogenous KDM4C undergoes dimerization under physiological conditions. Importantly, we identify the JmjN domain as the KDM4C dimerization interface and pin-point specific charged residues therein to be essential for this dimerization. We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides. In contrast, KDM4B does not dimerize and functions as a monomer, and hence was not affected by free JmjN expression. KDM4 proteins are overexpressed in numerous malignancies and their pharmacological inhibition or depletion in cancer cells was shown to impair tumor cell proliferation, invasion and metastasis. Thus, the KDM4 dimer-interactome emerging from the present study bears potential implications for cancer therapeutics via selective inhibition of KDM4A/C demethylase activity using JmjN-based peptidomimetics.

Structural recognition of tubulysin B derivatives by multidrug resistance efflux transporters in human cancer cells.

Multidrug resistance (MDR) is a major hindrance to curative chemotherapy of various human malignancies. Hence, novel chemotherapeutics must be evaluated for their recognition by MDR efflux transporters. Herein we explored the cytotoxic activity of synthetic tubulysin B (Tub-B, EC1009) derivatives (Tub-B-hydrazide/EC0347 and Tub-B bis-ether/EC1820), and their recognition by the MDR efflux transporters P-glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Originally isolated from Myxobacteria, tubulysins exhibited potent cytotoxic activity via microtubule depolymerization, and evaded recognition by these MDR efflux pumps. We show that subtle modifications in the natural Tub-B structure enhance its cytotoxicity and drug efflux efficiency. Whereas increasing the lipophilicity of Tub-B drugs enhanced their diffusion into the cell and consequently decreased the IC50 values (≥ 0.27 nM), increasing drug polarity enhanced their recognition by P-gp (>200-fold resistance in P-gp-overexpressing cells). Furthermore, restricting drug exposure time to the clinically relevant 4 h pulse, markedly enhanced efflux by P-gp, resulting in a 1000-fold increased resistance, which was further enhanced upon increased P-gp levels (i.e. an additional 3-fold increase in P-gp levels resulted in >6,000-fold resistance). The unique ability of EC1009 to evade recognition by MDR efflux pumps warrants drug development of tubulysin B derivatives as potent antitumor agents which overcome MDR in cancer.

Folylpoly-γ-glutamate synthetase: A key determinant of folate homeostasis and antifolate resistance in cancer.

Mammalians are devoid of autonomous biosynthesis of folates and hence must obtain them from the diet. Reduced folate cofactors are B9-vitamins which play a key role as donors of one-carbon units in the biosynthesis of purine nucleotides, thymidylate and amino acids as well as in a multitude of methylation reactions including DNA, RNA, histone and non-histone proteins, phospholipids, as well as intermediate metabolites. The products of these S-adenosylmethionine (SAM)-dependent methylations are involved in the regulation of key biological processes including transcription, translation and intracellular signaling. Folate-dependent one-carbon metabolism occurs in several subcellular compartments including the cytoplasm, mitochondria, and nucleus. Since folates are essential for DNA replication, intracellular folate cofactors play a central role in cancer biology and inflammatory autoimmune disorders. In this respect, various folate-dependent enzymes catalyzing nucleotide biosynthesis have been targeted by specific folate antagonists known as antifolates. Currently, antifolates are used in drug treatment of multiple human cancers, non-malignant chronic inflammatory disorders as well as bacterial and parasitic infections. An obligatory key component of intracellular folate retention and intracellular homeostasis is (anti)folate polyglutamylation, mediated by the unique enzyme folylpoly-γ-glutamate synthetase (FPGS), which resides in both the cytoplasm and mitochondria. Consistently, knockout of the FPGS gene in mice results in embryonic lethality. FPGS catalyzes the addition of a long polyglutamate chain to folates and antifolates, hence rendering them polyanions which are efficiently retained in the cell and are now bound with enhanced affinity by various folate-dependent enzymes. The current review highlights the crucial role that FPGS plays in maintenance of folate homeostasis under physiological conditions and delineates the plethora of the molecular mechanisms underlying loss of FPGS function and consequent antifolate resistance in cancer.

Folylpolyglutamate synthetase splicing alterations in acute lymphoblastic leukemia are provoked by methotrexate and other chemotherapeutics and mediate chemoresistance.

Methotrexate (MTX), a folate antagonist which blocks de novo nucleotide biosynthesis and DNA replication, is an anchor drug in acute lymphoblastic leukemia (ALL) treatment. However, drug resistance is a primary hindrance to curative chemotherapy in leukemia and its molecular mechanisms remain poorly understood. We have recently shown that impaired folylpolyglutamate synthetase (FPGS) splicing possibly contributes to the loss of FPGS activity in MTX-resistant leukemia cell line models and adult leukemia patients. However, no information is available on the possible splicing alterations in FPGS in pediatric ALL. Here, using a comprehensive PCR-based screen we discovered and characterized a spectrum of FPGS splicing alterations including exon skipping and intron retention, all of which proved to frequently emerge in both pediatric and adult leukemia patient specimens. Furthermore, an FPGS activity assay revealed that these splicing alterations resulted in loss of FPGS function. Strikingly, pulse-exposure of leukemia cells to antifolates and other chemotherapeutics markedly enhanced the prevalence of several FPGS splicing alterations in antifolate-resistant cells, but not in their parental antifolate-sensitive counterparts. These novel findings suggest that an assortment of deleterious FPGS splicing alterations may constitute a mechanism of antifolate resistance in childhood ALL. Our findings have important implications for the rational overcoming of drug resistance in individual leukemia patients.

Binding of a Smad4/Ets-1 complex to a novel intragenic regulatory element in exon12 of FPGS underlies decreased gene expression and antifolate resistance in leukemia.

Polyglutamylation of antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-ß/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.

The roles of the catalytic and noncatalytic activities of Rpd3L and Rpd3S in the regulation of gene transcription in yeast.

In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.

Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.

Heterogeneous nuclear ribonucleoprotein H1/H2-dependent unsplicing of thymidine phosphorylase results in anticancer drug resistance.

Thymidine phosphorylase (TP) catalyzes the conversion of thymidine to thymine and 2-deoxyribose-1-phosphate. The latter plays an important role in induction of angiogenesis. As such, many human malignancies exhibit TP overexpression that correlates with increased microvessel density, formation of aggressive tumors, and dismal prognosis. Because TP is frequently overexpressed in cancer, pro-drugs were developed that utilize TP activity for their bioactivation to cytotoxic drugs. In this respect, TP is indispensable for the pharmacologic activity of the chemotherapeutic drug capecitabine, as it converts its intermediary metabolite 5'-deoxyfluorouridine to 5-fluorouracil. Thus, loss of TP function confers resistance to the prodrug capecitabine, currently used for the treatment of metastatic colorectal cancer and breast cancer. However, drug resistance phenomena may frequently emerge that compromise the pharmacologic activity of capecitabine. Deciphering the molecular mechanisms underlying resistance to TP-activated prodrugs is an important goal toward the overcoming of such drug resistance phenomena. Here, we discovered that lack of TP protein in drug-resistant tumor cells is due to unsplicing of its pre-mRNA. Advanced bioinformatics identified the family of heterogeneous nuclear ribonucleoproteins (hnRNP) H/F as candidate splicing factors potentially responsible for impaired TP splicing. Indeed, whereas parental cells lacked nuclear localization of hnRNPs H1/H2 and F, drug-resistant cells harbored marked levels of these splicing factors. Nuclear RNA immunoprecipitation experiments established a strong binding of hnRNP H1/H2 to TP pre-mRNA, hence implicating them in TP splicing. Moreover, introduction of hnRNP H2 into drug-sensitive parental cells recapitulated aberrant TP splicing and 5'-deoxyfluorouridine resistance. Thus, this is the first study identifying altered function of hnRNP H1/H2 in tumor cells as a novel determinant of aberrant TP splicing thereby resulting in acquired chemoresistance to TP-activated fluoropyrimidine anticancer drugs.

Arabinogalactan-folic acid-drug conjugate for targeted delivery and target-activated release of anticancer drugs to folate receptor-overexpressing cells.

Folic acid (FA) is a high affinity ligand (K(d) = 0.1-1 nM) of folate receptors (FRs) responsible for cellular uptake of folates via receptor-mediated endocytosis. FRs are frequently overexpressed in malignant epithelial cells including ovary, brain, kidney, breast, colon, and lung. FR has emerged as a target for the differential-delivery of anticancer chemotherapeutics with several FA-linked therapeutic agents currently undergoing clinical trials. Here we show that by tethering both FA and the anticancer drug methotrexate (MTX) to arabinogalactan (AG), a highly branched natural polysaccharide with unusual water solubility, a targeted biomacromolecular nanovehicle is formed, which can differentially deliver a cytotoxic cargo into FR-overexpressing cells. Moreover, by linking MTX via an endosomally cleavable peptide (GFLG), we demonstrate a target-activated release mechanism. This FA-AG-GFLG-MTX drug conjugate displayed 6.3-fold increased cytotoxic activity to FR-overexpressing cells compared to their FR-lacking counterparts. These findings establish a novel FA-tethered polymeric nanoconjugate for the targeted delivery of antitumor agents into cancer cells overexpressing FR.

Chemotherapeutic drug-induced ABCG2 promoter demethylation as a novel mechanism of acquired multidrug resistance.

ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

Functional elements in the minimal promoter of the human proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay. This analysis mapped the minimal promoter to 157bp upstream to the first ATG. Crucial GC-box sites were identified within the minimal promoter and in its close vicinity which substantially contribute to promoter activity, as their disruption resulted in 94% loss of luciferase activity. We also identified upstream enhancer elements including YY1 and AP1 which, although distantly located, prominently transactivated the minimal promoter, as their inactivation resulted in 50% decrease in reporter activity. This is the first functional identification of the minimal PCFT promoter harboring crucial GC-box elements that markedly contribute to its transcriptional activation via putative interaction with distal YY1 and AP1 enhancer elements.

Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of antifolate resistance in leukemia.

Folylpoly-gamma-gluatamate synthetase (FPGS) catalyzes the polyglutamylation and thus intracellular retention of folates and antifolates (eg, methotrexate; MTX) through the addition of multiple glutamate equivalents to their gamma-carboxyl residue. Since polyglutamylation of antifolates is crucial for their pharmacological activity in leukemia, loss of FPGS function results in decreased cellular levels of polyglutamylation-dependent antifolates and consequent drug resistance. Whereas resistance to pulse exposure to antifolates is frequently associated with loss of FPGS activity, the underlying molecular mechanism remains elusive. Here we explored the molecular basis of antifolate resistance in human MTX-resistant leukemia cell lines displaying marked loss of FPGS activity. We demonstrate that these MTX-resistant cells exhibit impaired splicing of FPGS mRNA based on intron retention and/or exon skipping, thereby resulting in loss of FPGS function due to premature translation termination. Furthermore, analysis of FPGS transcripts in blood or bone marrow specimens from patients with acute lymphoblastic leukemia revealed exon 12 skipping, both at diagnosis and at relapse, the latter of which occurs after high-dose MTX-containing chemotherapy. These results constitute the first demonstration of the loss of FPGS function via aberrant mRNA splicing, thereby resulting in loss of antifolate retention and drug resistance. The clinical ramifications of these novel findings are discussed.

Sex-specific differences in peripheral microvascular blood flow in preterm infants.

Microvascular blood flow is related to physiologic instability in newborn preterm infants. We investigated sex-specific differences in basal microvascular blood flow and the ability of the microvasculature to respond to vasoactive stimuli following preterm birth. Ninety-six infants in two gestational age groups (24-28 and 29-36 wk) were studied on days 1-5 of life. Laser Doppler flowmetry was used to measure baseline microvascular blood flow and vasodilatation in response to acetylcholine and local warming. A significant interaction of gestational age and sex was observed for baseline flow at 24 h of age. In the 24-28 wk group, male infants had higher baseline flow than females. Male, but not female, infants born at 24-28 wk exhibited a significant relationship between baseline flow and vasodilatory response to acetylcholine at 24 h of age. By 120 h of age, both sexes exhibited similar responses. Infants born at 24-28 wk exhibited greater vasodilatation in response to local warming than those born at 29-36 wk at 24, 72, and 120 h of age. Sex-specific differences in microvascular blood flow and vasodilatory capacity in the immediate newborn period may affect the transitional circulation, contributing to excess of morbidity and mortality in preterm males.