Michal Stark

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.

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.

Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation

The human reduced folate carrier (hRFC) is the major uptake route for antifolates used in cancer chemotherapy. Here we explored the molecular basis for the decrease or loss of hRFC gene expression in seventeen tumor cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/AP-1-like element and GC-box in hRFC promoters A and B, respectively, as well as AP-2, Mzf-1 and E-box that are contained within or near four tandemly repeated sequences upstream of promoter A. Decreased or abolished binding either to [32P]GC-box, Mzf-1, AP-1, E-box, or CRE oligonucleotides was detected in ∼50-80% of antifolate-resistant cell lines. Strikingly, ∼80% of the cell lines displayed a simultaneously decreased binding to three or more of these hRFC promoter elements, whereas normal AP-2 binding was retained. The possible contribution of promoter methylation to hRFC gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed hRFC promoter methylation; consistently, MDA-MB-231 was the only cell line that retained binding to all six cis-acting elements. Western blot analysis demonstrated decreased expression of transcriptional activators (pCREB-1, pATF-1, USF-1, c-Fos, c-Jun, Sp1, and Sp3) and/or increased expression of repressors (short Sp3 isoforms), whereas normal AP2α levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and hRFC gene expression. This is the first report that transcriptional silencing of the hRFC gene in multiple tumor cell lines with resistance to various novel antifolates is a result of a simultaneous loss of function of multiple transcription factors but not promoter methylation.

Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers

Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction.

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.

Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest

Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.

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.

Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody

Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer. Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP‐binding cassette (ABC) superfamily including multidrug resistance protein 1 ‐MRP1 (ABCC1). To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope. Using a phage display approach, we have isolated a recombinant single‐chain Fv (scFv) antibody that specifically reacts with the extracellular N‐terminus of the human MRP1. Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable MRP1 expression levels but not to MRP1 null cells. Furthermore, this scFv antibody failed to react with tumor cells that overexpress other members of the MRP family that have an extracellular N‐terminus (MRP2 and MRP3) as well as with MRP4, MRP5, and breast cancer resistance protein. Flow cytometric analysis also showed a good correlation between the fluorescence intensity of the anti‐MRP1 scFv antibody and MRP1 levels in viable tumor cells. These findings constitute the first successful isolation of a small recombinant scFv antibody directed to an extracellular epitope of the MRP1 in viable malignant cells. These novel small Fv‐based recombinant antibodies that possess superior tumor penetration capabilities may possibly be used to selectively target drugs or tumor cells that express MRP‐1.

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.