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I. David Goldman - One of the best experts on this subject based on the ideXlab platform.

  • Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer.
    Molecular Pharmacology, 2018
    Co-Authors: Rongbao Zhao, Mitra Najmi, Srinivas Aluri, David C. Spray, I. David Goldman
    Abstract:

    The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers Antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and Antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This Antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An Antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.

  • Determinants of the activities of Antifolates delivered into cells by folate-receptor-mediated endocytosis
    Cancer Chemotherapy and Pharmacology, 2015
    Co-Authors: Rongbao Zhao, Michele Visentin, I. David Goldman
    Abstract:

    Purpose Elements in the endocytic process that are determinants of the activities of Antifolates delivered by folate-receptor alpha (FRα) were explored. Methods Antifolate growth inhibition was assessed with a 1- or 5-day exposure in reduced folate carrier-null HeLa cell lines that express a high level of FRα in the presence or absence of the proton-coupled folate transporter (PCFT). pH-dependent rates of dissociation from FRα were also determined. Results With a 1-day drug exposure which is relevant to the pulse clinical administration of these drugs, FRα expression enhanced raltitrexed activity and modestly enhanced ZD9331 activity, but did not significantly augment the activity of pemetrexed or lomotrexol. With a 5-day drug exposure, FRα-mediated growth inhibition was increased for raltitrexed and ZD9331 and emerged for lomotrexol. While the FRα-augmented activity of lomotrexol and raltitrexed did not require PCFT, augmentation of ZD9331 activity required the co-expression of PCFT with both 1- and 5-day exposures. In contrast, there was no augmentation of pemetrexed activity by FRα under any condition. The activities of these agents correlated with their rate of dissociation from the receptor at acidic pH: raltitrexed > ZD9331 > lomotrexol > pemetrexed consistent with insufficient pemetrexed release from FRα for export from the endosomes. Conclusions FRα is unlikely to contribute to the pharmacological activity of Antifolates, such as pemetrexed, that bind tightly to, and dissociate slowly from, the receptor particularly when the exposure time is brief. While PCFT was required for FRα-mediated ZD9931 activity, the activities of the other Antifolates was independent of PCFT.

  • The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro
    Cancer Chemotherapy and Pharmacology, 2014
    Co-Authors: Michele Visentin, Ersin Selcuk Unal, I. David Goldman
    Abstract:

    Purpose To investigate the impact of 5-formyltetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro. Methods Cells were exposed to (6 S )5-formyltetrahydrofolate (5-formylTHF) for 24 h, before or after a 6-h exposure to Antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells. Results A 24-h delay between a 6-h exposure to Antifolates and a subsequent 24-h exposure to 4 μM 5-formylTHF sustained the full activities of both Antifolates. A 72-h interval was required between a single exposure of up to 4 μM 5-formylTHF and subsequent exposure to drugs to sustain activities of the Antifolates. When cells were incubated with 4 μM 5-formylTHF for 24 h weekly, for 4 weeks, there was no significant increase in the IC_50 for pralatrexate, but the MTX IC_50 increased 2.5-fold as compared to cells growing continuously in 25 nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16 %, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15 %. An extracellular concentration of MTX 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate. Conclusions Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical “rescue” regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of Antifolate.

  • Resistance to Antifolates
    Oncogene, 2003
    Co-Authors: Rongbao Zhao, I. David Goldman
    Abstract:

    The Antifolates were the first class of antimetabolites to enter the clinics more than 50 years ago. Over the following decades, a full understanding of their mechanisms of action and chemotherapeutic potential evolved along with the mechanisms by which cells develop resistance to these drugs. These principals served as a basis for the subsequent exploration and understanding of the mechanisms of resistance to a variety of diverse antineoplastics with different cellular targets. This section describes the bases for intrinsic and acquired Antifolate resistance within the context of the current understanding of the mechanisms of actions and cytotoxic determinants of these agents. This encompasses impaired drug transport into cells, augmented drug export, impaired activation of Antifolates through polyglutamylation, augmented hydrolysis of Antifolate polyglutamates, increased expression and mutation of target enzymes, and the augmentation of cellular tetrahydrofolate-cofactor pools in cells. This chapter also describes how these insights are being utilized to develop gene therapy approaches to protect normal bone marrow progenitor cells as a strategy to improve the efficacy of bone marrow transplantation. Finally, clinical studies are reviewed that correlate the cellular pharmacology of methotrexate with the clinical outcome in children with neoplastic diseases treated with this Antifolate.

  • Marked suppression of the activity of some, but not all, Antifolate compounds by augmentation of folate cofactor pools within tumor cells.
    Biochemical pharmacology, 2001
    Co-Authors: Rongbao Zhao, Feng Gao, I. David Goldman
    Abstract:

    Folates have been co-administered with some Antifolates to diminish host toxicity; however, the extent to which this will reduce antitumor activity is not known. To further clarify this issue, studies were undertaken to characterize and quantitate the impact of alterations in intracellular folate levels on the activities of a variety of Antifolates in L1210 murine leukemia cells. Intracellular folate cofactor levels increased almost in proportion to the increase in extracellular 5-formyltetrahydrofolate (5-CHO-THF) over a concentration range that encompassed physiological levels of 5-methyltetrahydrofolate. This resulted in a spectrum of increases in the ic50 values of Antifolates upon continuous exposure to drugs [Lometrexol (DDATHF) (70x) > trimetrexate (TMQ) (30x), multitargeted Antifolate, LY231514 (ALIMTA) (30x) > Raltitrexed, Tomudex (ZD1694) (10x), 6R-2',5'-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) (10x) > methotrexate (MTX) (6x) > (2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido]-4-(tetrazol-5-yl) butyric acid (ZD9331) (3x), N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) (3x)]. Upon a 4-hr pulse exposure to drug, the ic50 values for DDATHF and ALIMTA were increased > 180- and 5-fold, respectively, with only a 2.5-fold increase in the extracellular 5-CHO-THF level within the physiological range. The reductions in drug sensitivities could be attributed to decreases in accumulation of polyglutamate derivatives of ALIMTA and DDATHF. Hence, in these studies, natural folates diminished the activity of agents that undergo polyglutamation by suppression of the formation of these active congeners at the level of folylpolyglutamate synthetase. For inhibitors of dihydrofolate reductase, the suppressive effect of endogenous folates appears to be due to competition between the Antifolate and dihydrofolate at the level of the target enzyme. These data should be carefully considered in the design of regimens with Antifolates, which incorporate co-administration of folates.

Yehuda G. Assaraf - One of the best experts on this subject based on the ideXlab platform.

  • folylpoly γ glutamate synthetase a key determinant of folate homeostasis and Antifolate resistance in cancer
    Drug Resistance Updates, 2016
    Co-Authors: Shachar Raz, Michal Stark, Yehuda G. Assaraf
    Abstract:

    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.

  • 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
    Oncotarget, 2014
    Co-Authors: Shachar Raz, Michal Stark, Yehuda G. Assaraf
    Abstract:

    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-β/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.

  • Antifolates in cancer therapy structure activity and mechanisms of drug resistance
    Drug Resistance Updates, 2012
    Co-Authors: Nitzan Gonen, Yehuda G. Assaraf
    Abstract:

    In the past 65 years, Antifolates targeting folate metabolism played a pivotal role in drug treatment of malignant, microbial, parasitic and chronic inflammatory diseases. Drug discovery of novel Antifolates with improved properties and superior activities remains an attractive strategy both in academia and in the pharmaceutical industry. Among novel Antifolates are pemetrexed which primarily targets thymidylate synthase as well as pralatrexate which blocks dihydrofolate reductase, and displays enhanced transport and cellular retention properties. The present review describes the evolution and pharmacological activity of Antifolates and prospects for the development of the next generation Antifolates. Pre-clinical and clinical studies identified a plethora of mechanisms of Antifolate resistance that are a primary hindrance to curative cancer chemotherapy; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of Antifolates and in folate-dependent enzymes. Current advances including for example the deciphering of the dominant folate transporter proton-coupled folate transporter (PCFT/SLC46A1) facilitated the synthesis of experimental Antifolates aimed at selectively targeting solid tumor cells, which reside in an acidic microenvironment where PCFT supposedly functions optimally. Moreover, drugs that are structurally and mechanistically distinct from folates were conjugated to folic acid (e.g. Vintafolide/EC145, a folic acid desacetylvinblastine conjugate) to facilitate endocytosis via the folate receptor (FR) which is markedly overexpressed in various solid tumors. In an alternative approach, novel Antifolates selectively targeting the FR but not other folate transporters are being developed (e.g. BGC 945). Hence, targeting mechanisms of Antifolate-resistance could facilitate the development of rationally-based novel Antifolates and strategies that overcome chemoresistance.

  • Aberrant splicing of folylpolyglutamate synthetase as a novel mechanism of Antifolate resistance in leukemia
    Blood, 2009
    Co-Authors: Michal Stark, Chen Wichman, Irit Avivi, Yehuda G. Assaraf
    Abstract:

    Folylpoly-γ-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 γ-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.

  • Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis
    Biochemical Pharmacology, 2009
    Co-Authors: Alan Kambiz Fotoohi, Gerrit Jansen, Godefridus J Peters, Yehuda G. Assaraf, Ali Moshfegh, Jamileh Hashemi, Catharina Larsson, Freidoun Albertioni
    Abstract:

    treatment of human T-cell leukemia cells with 7-hydroxymethotrexate, the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of Antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate.. To identify the genes associated with methotrexate and 7-hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and Antifolates, were down-regulated >2-fold in methotrexate -resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate - resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine β synthase, as well as the ATP-dependent folate exporters / and /. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate -resistant subline, whereas in 7-hydroxymethotrexate -resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an Antifolate resistance modality that are distinct from its parent drug methotrexate.

Sarah S Bacus - One of the best experts on this subject based on the ideXlab platform.

  • kras mutation status is associated with enhanced dependency on folate metabolism pathways in non small cell lung cancer cells
    Molecular Cancer Therapeutics, 2014
    Co-Authors: Diarmuid M. Moran, Patricia B Trusk, David Sidransky, Sarah S Bacus
    Abstract:

    KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non–small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism– and purine synthesis–related pathways in KRAS -mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS -mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other Antifolates in KRAS -mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS -mutant cells by Antifolates, which may also contribute to higher efficacy of Antifolates in KRAS -mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS -mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 ( MTHFD2 ), and Antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher Antifolate activity in this subtype of NSCLC. Mol Cancer Ther; 13(6); 1611–24. ©2014 AACR .

  • KRAS Mutation Status Is Associated with Enhanced Dependency on Folate Metabolism Pathways in Non–Small Cell Lung Cancer Cells
    Molecular cancer therapeutics, 2014
    Co-Authors: Diarmuid M. Moran, Patricia B Trusk, Karen J Pry, K. Paz, David Sidransky, Sarah S Bacus
    Abstract:

    KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non-small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism- and purine synthesis-related pathways in KRAS-mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS-mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other Antifolates in KRAS-mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS-mutant cells by Antifolates, which may also contribute to higher efficacy of Antifolates in KRAS-mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS-mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and Antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher Antifolate activity in this subtype of NSCLC.

  • abstract lb 449 kras mutation and amplification status predicts sensitivity to Antifolate therapies in non small cell lung cancer
    Cancer Research, 2012
    Co-Authors: Diarmuid M. Moran, Patricia B Trusk, Scott A Shell, Ross D Camidge, Robert C Doebele, Eamon M Berge, Mark Vincent, Sarah S Bacus
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to poor prognosis and resistance to various targeted therapeutics in Non Small Cell Lung Cancer (NSCLC). Therapeutic strategies that target tumors harboring these mutations represent an unmet medical need. In this study, we investigated the relationship between Antifolate sensitivity and KRAS mutation/amplification status in NSCLC. Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed (PEM) and assayed for proliferation. In these studies, KRASwt (wildtype) and KRASmut (mutant) amplified cells showed resistance to MTX treatment (IC50 >10μM). In contrast, growth of all KRASmut non-amplified cell lines studied was inhibited with MTX treatment (IC50 <100nM). Similar effects were observed for PEM in this study. Interrogation of the NCI Developmental Therapeutics Program drug screen database for the relationship between KRAS mutation status and drug efficacy also revealed a similar trend in other NSCLC cell lines for MTX and other anti-folates. qPCR analysis demonstrated a dramatic downregulation of KRAS gene expression in KRASwt and KRASmut cells with Antifolate treatment. However, KRAS gene expression was less affected in Antifolate treated KRASmut amplified cells. Co-treatment of KRASmut cells with Antifolates and hypoxanthine/thymidine (which compensate for folate pathway inhibition) prevented downregulation of KRAS gene expression and rescued KRASmut cells. qPCR array analysis of miRNA expression in Antifolate treated cells revealed increased expression of specific miRNAs, including miR-181c, with treatment compared to untreated controls. Transfection of a miR-181c mimic led to downregulation of KRAS gene expression in cells. Furthermore, antagomirs targeting miR-181c partially inhibited the downregulation of KRAS by Antifolates. Importantly, we present clinical data describing rapid and durable radiographic responses in KRAS mutant NSCLC cancer patients. Collectively, these studies identify higher sensitivity to Antifolates in KRASmut non-amp NSCLC cell lines. Antifolate therapies decrease KRAS gene expression in KRASwt and KRASmut but do not do so in KRASmut amplified cells. We propose that decreased KRAS gene expression is detrimental to KRASmut cells due to their dependency on this survival pathway. We also propose that decreases in KRAS gene expression are mechanistically linked to stress (folate inhibition) induced miRNA expression which target KRAS gene expression. Overall, Antifolates represent a novel method to target KRAS and as such should be investigated further for use in this subtype of NSCLC. As clinical evidence emerges, both KRAS mutation and amplification status should be incorporated for patient stratification prior to Antifolate treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-449. doi:1538-7445.AM2012-LB-449

  • abstract pr 2 kras mutation and amplification status predicts sensitivity to Antifolate therapies in non small cell lung cancer
    Molecular Cancer Therapeutics, 2011
    Co-Authors: Sarah S Bacus
    Abstract:

    Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to poor prognosis and resistance to various targeted therapeutics in Non Small Cell Lung Cancer (NSCLC). Therapeutic strategies that target tumors harboring these mutations represent an unmet medical need. In this study, we investigated the relationship between Antifolate sensitivity and KRAS mutation/amplification status in NSCLC. Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed (PEM) and assayed for proliferation after 72h. In these studies, 5 out of 7 KRASwt (wildtype) cells and all KRASmut (mutant) amplified cells showed resistance to MTX treatment (IC50 >10μM). In contrast, growth of all KRASmut non-amplified cell lines studied was inhibited with MTX treatment (IC50 Collectively, these studies identify increased sensitivity to Antifolates in KRASmut non-amp NSCLC cell lines. Anti-folate therapies decrease KRAS gene expression in KRASwt and KRASmut cells but do not do so in KRASmut amplified cells. We propose that decreased KRAS gene expression is detrimental to KRASmut cells due to their dependency on this pathway for survival. We also propose that decreases in KRAS gene expression are mechanistically linked to stress (folate inhibition) induced miRNA expression which target KRAS gene expression. Overall, Antifolates represent a novel method to target KRAS and as such should be investigated further for use in this subtype of NSCLC. As clinical evidence emerges, it is apparent that both KRAS mutation and amplification status should be considered for patient stratification prior to Antifolate treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-2.

Richard L. Seither - One of the best experts on this subject based on the ideXlab platform.

David I Goldman - One of the best experts on this subject based on the ideXlab platform.

  • pemetrexed biochemical and cellular pharmacology mechanisms and clinical applications
    Molecular Cancer Therapeutics, 2007
    Co-Authors: Shrikanta Chattopadhyay, Richard G Moran, David I Goldman
    Abstract:

    Pemetrexed is a new-generation Antifolate, approved for the treatment of mesothelioma and non–small cell lung cancer, currently being evaluated for the treatment of a variety of other solid tumors. This review traces the history of Antifolates that led to the development of pemetrexed and describes the unique properties of this agent that distinguish it from other Antifolates. These include ( a ) its very rapid conversion to active polyglutamate derivatives in cells that build to high levels and are retained for long intervals to achieve prolonged and potent inhibition of its major target enzyme thymidylate synthase, ( b ) its high affinity for three folate transporters, and ( c ) its marked sensitivity to the level of physiologic folates in cells. The latter results in the unique and paradoxical finding that when transport mediated by the major folate transporter (the reduced folate carrier) is impaired, pemetrexed activity is preserved. This is due to concurrent contraction of competing cellular physiologic folates and utilization of a novel second transport carrier for which pemetrexed has high affinity, recently identified as the proton-coupled folate transporter (PCFT). Laboratory studies are reviewed that raise the possibility of new approaches to the use of folic acid supplementation in clinical regimens with pemetrexed. [Mol Cancer Ther 2007;6(2):404–17]

  • Antifolate resistance in a hela cell line associated with impaired transport independent of the reduced folate carrier
    Cancer Research, 2005
    Co-Authors: Rongbao Zhao, Shrikanta Chattopadhyay, Marie Hanscom, David I Goldman
    Abstract:

    3301 Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low-pH optimum in a variety of human solid tumor cell lines (Clinical Cancer Research, 2004, 10: 718). In HeLa cells, derived from human cervical cancer, this low pH activity has high affinity (Kt=45 nM) for pemetrexed (PMX) and is RFC-independent, since it is not diminished in an RFC-null subline (R5) (Clinical Cancer Research, 2004, 10: 6256). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH (Cancer Research, 2004, 64: 3313). The current study addresses whether this low pH folate transport activity can be down-regulated under Antifolate pressure and the consequence of this down-regulation on the activity of Antifolates. An R5 subline, R1, was selected under MTX selective pressure in a medium with a modestly reduced pH (pH 6.9). In R1 cells influx of MTX, PMX and folic acid at pH 5.5 was markedly and equally decreased as compared to R5 cells. PMX influx at pH 7.4 in R1 cells was also markedly reduced while MTX influx at this pH was decreased to a lesser extent. When MTX was removed from the growth medium there was a slow return of transport activity in R1 cells, and when MTX was added back, there was loss of transport at both pHs within eight weeks. PMX influx in R5 cells at both acidic and neutral pH was increased by preloading cells with 5-formyltetrahydrofolate (trans-stimulation), consistent with a process mediated by a facilitative carrier. In R1 cells there was marked decrease in accumulation of PMX, MTX, and folic acid at both pHs as compared to R5 cells. At both pHs R1 cells were more resistant than R5 cells to Antifolates, which require a facilitative process to gain entry into cells, although the extent of resistance was different for different Antifolates (PMX>MTX>tomudex). These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contribute to Antifolate activity, in particular, to PMX activity, and can be diminished by Antifolate selective pressure and, (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process. This study was supported by a grant from the National Institutes of Health, CA-82621, along with a grant from the Eli Lilly Company.

  • interconversion of tetrahydrofolate cofactors to dihydrofolate induced by trimetrexate after suppression of thymidylate synthase by fluorodeoxyuridine in l1210 leukemia cells
    Biochemical Pharmacology, 1992
    Co-Authors: Richard L. Seither, Tracy J. Rape, David I Goldman
    Abstract:

    Previous studies from this laboratory demonstrated that marked suppression of thymidylate synthase activity is required to slow the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate when dihydrofolate reductase is blocked by an Antifolate. This finding is due to the high catalytic activity of thymidylate synthase within cells in comparison to the tetrahydrofolate cofactor pool size. In the present study, we assessed the rate of resumption of thymidylate synthase catalytic activity in terms of [3H]deoxyuridine incorporation into DNA and dihydrofolate generation from tetrahydrofolate cofactors following exposure of cells to fluorodeoxyuridine. Log phase L1210 leukemia cells, incubated with fluorodeoxyuridine to abolish thymidylate synthase catalytic activity, were suspended into drug-free medium. Resumption of [3H]deoxyuridine incorporation into DNA was negligible; by 4 hr enzyme activity was still inhibited by approximately 98%. However, this was sufficient to interconvert all available tetrahydrofolate cofactors to dihydrofolate (T1/2 approximately 2 hr) when dihydrofolate reductase was inhibited by the lipophilic Antifolate trimetrexate. Interconversion of tetrahydrofolate cofactors to dihydrofolate correlated with a decline, then cessation, of purine synthesis as measured by the incorporation of [14C]formate into purine bases. These data suggest that an earlier than previously expected depletion of tetrahydrofolate cofactors with consequent inhibition of purine and other folate-dependent synthetic processes is likely to occur when Antifolates are administered after a fluoropyrimidine.