Folylpoly-γ-glutamate Synthetase Research Articles

Folylpolyglutamate synthetase inactivation in relapsed ALL induces a druggable folate metabolic vulnerability

AimsThe antifolate methotrexate (MTX) is an anchor drug used in acute lymphoblastic leukemia (ALL) with poorly understood chemoresistance mechanisms in relapse. Herein we find decreased folate polyglutamylation network activities and inactivating FPGS mutations, both of which could induce MTX resistance and folate metabolic vulnerability in relapsed ALL. MethodsWe utilized integrated systems biology analysis of transcriptomic and genomic data from relapse ALL cohorts to infer hidden ALL relapse drivers and related genetic alternations during clonal evolution. The drug sensitivity assay was used to determine the impact of relapse-specific FPGS mutations on sensitivity to different antifolates and chemotherapeutics in ALL cells. We used liquid chromatography-mass spectrometry (LC-MS) to quantify MTX and folate polyglutamate levels in folylpoly-γ-glutamate synthetase (FPGS) mutant ALL cells. Enzymatic activity and protein degradation assays were also conducted to characterize the catalytic properties and protein stabilities of FPGS mutants. An ALL cell line-derived mouse leukemia xenograft model was used to evaluate the in vivo impact of FPGS inactivation on leukemogenesis and sensitivity to the polyglutamatable antifolate MTX as well as non-polyglutamatble lipophilic antifolate trimetrexate (TMQ). ResultsWe found a significant decrease in folate polyglutamylation network activities during ALL relapse using RNA-seq data. Supported by functional evidence, we identified multifactorial mechanisms of FPGS inactivation in relapsed ALL, including its decreased network activity and gene expression, focal gene deletion, impaired catalytic activity, and increased protein degradation. These deleterious FPGS alterations induce MTX resistance and inevitably cause marked intracellular folate shrinkage, which could be efficiently targeted by a polyglutamylation-independent lipophilic antifolate TMQ in vitro and in vivo. ConclusionsMTX resistance in relapsed ALL relies on FPGS inactivation, which inevitably induces a folate metabolic vulnerability, allowing for an efficacious antifolate ALL treatment strategy that is based upon TMQ, thereby surmounting chemoresistance in relapsed ALL.

SKA1 induces denovo MTX-resistance in osteosarcoma through inhibiting FPGS transcription.

Denovo methotrexate (MTX)-resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the denovo MTX-resistant OS cell line SF-86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to denovo MTX-resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with denovo MTX-resistance and poor 5-year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly-γ-glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX-PG. We further demonstrated that SKA1 interacts with DNA-directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX-sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF-86 cells due to the formation of an inhibitory SKA1-RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF-86. Collectively, our study has established the denovo MTX-resistant cell line SF-86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of denovo MTX-resistance in OS.

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.

The intricate role of Smads in the regulation of FPGS gene expression

Antifolates are important components of treatment regimens of hematological malignancies and solid tumors. Following cellular uptake, antifolates undergo polyglutamylation, catalyzed by the enzyme folylpoly-γ-glutamate synthetase (FPGS), which is crucial for their intracellular retention and cytotoxic activity. Hence, loss of FPGS activity results in diminished antifolate polyglutamylation and antifolate resistance. Aberrant TGF-β/Smad signaling is thought to be a major contributor to leukemogenesis, as members of this pathway are negative regulators of hematopoiesis. Recently we characterized the human antifolate resistant leukemia cell line MTA C-3 which lost 97% of its cellular FPGS activity and harbors a heterozygous point mutation in exon12 of FPGS. This resulted in the loss of 99% of the expression of the wild type FPGS allele, while the expression of the mutant allele was retained. Research into the molecular mechanism underlying the selective silencing of the wild type allele in these cells established exon12 of FPGS as an intragenic transcriptional regulator, with the ability to drive transcription in vitro . We further showed that exon12 can be occupied by many transcription factors and chromatin remodeling proteins (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo and that the expression of FPGS is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12, both in acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. Here we further demonstrate the possible roles of Smads in the regulation of FPGS gene expression. We found that treatment of MTA C-3 cells with 5-aza-deoxycytidine and trichostatin A results in repression of the mutant allele as well as in decreased expression of FPGS in drug sensitive parental CCRF-CEM cells. This repression correlated with an increased expression of Smad3 and the inhibitory Smad7. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.

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-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.

Folylpoly-Glutamate Synthetase Expression Is Associated with Tumor Response and Outcome from Pemetrexed-Based Chemotherapy in Malignant Pleural Mesothelioma

Pemetrexed-based chemotherapy represents the standard of care in first-line treatment of advanced malignant pleural mesothelioma (MPM). However, there are no established predictors of clinical benefit. Pemetrexed inhibits multiple enzymes involved in pyrimidine and purine synthesis, but the main target is thymidylate synthase (TS). After cellular uptake pemetrexed is converted into more effective polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS). We hypothesized that FPGS and TS protein expressions are associated with clinical outcome after pemetrexed-based chemotherapy. Pretreatment tumor samples from 84 patients with histologically confirmed MPM, who received pemetrexed combined with platinum (79 of 84) or single-agent pemetrexed (5 of 84) as first-line treatment, were retrospectively analyzed. FPGS and TS protein expressions were semiquantitatively assessed by using the Hybrid (H)-scoring system (range, 0-300). H-scores were correlated with radiological response according to modified Response Evaluation Criteria in Solid Tumors, progression-free survival (PFS) and overall survival (OS). Median H-score of the entire cohort was 230 for FPGS (range, 100-300), and 210 for TS (range, 100-300). High FPGS protein expression was significantly associated with longer PFS (pCOX = 0.0337), better objective tumor response (partial response versus stable disease + progressive disease; pKW = 0.003), and improved disease-control rate (partial response + stable disease versus progressive disease; pKW = 0.0208), but not with OS. In addition, high TS protein expression was associated with progressive disease under pemetrexed-based therapy (p = 0.0383), and shorter OS (pCOX = 0.0071), but no association with PFS was observed. FPGS and TS expressions were associated with clinical response and outcome to pemetrexed-based first-line chemotherapy in MPM. Prospective evaluation of FPGS and TS expressions and their prognostic/predictive power in MPM patients is warranted.

Abstract B6: Folylpoly-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in non-small cell lung cancer (NSCLC)

Abstract Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC. Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with pemetrexed combined with platinum (74 pts (46%)) or as single agent (82 pts (51%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0–300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0–100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1). Results: Median pretreatment H-scores were 180 for FPGS (range: 0–280) and 205 (range: 120–290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs. 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs. 210 for non-responders, P=0.03). Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of pemetrexed-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from pemetrexed-based therapy in advanced NSCLC. Further prospective validation studies are warranted.

Predictive value of thymidylate synthase and folylpoly-glutamate synthetase for clinical benefit from pemetrexed in malignant pleural mesothelioma.

7024 Background: The antifolate pemetrexed (PMX) targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, PMX is converted to more potent polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict response and outcome following PMX treatment of patients (pts) with malignant pleural mesothelioma (MPM). Methods: Pre-treatment tumor samples from 64 pts with MPM, treated with PMX combined with platinum (58/64) or as single agent (6/64), were retrospectively analyzed. TS and FPGS protein expression levels were evaluated by immunohistochemistry using the H-Scoring system (ranging from 0 to 300), which relies on the product of intensity of specific tumor cell immunoreactivity (range 0 to 3) and the percentage of positive tumor cells. Radiologic evaluation of response was performed according to RECIST. Results: Median pre-treatment H-scores were 220 (range: 50-300) for TS and 240 for FPGS (range: 110-300). Using the log-rank test and the median H-score as cut-off, we found a significant association of improved progression-free survival (PFS) with low TS protein expression (median PFS of 223 vs 181 days; hazard ratio [HR] 0.574, 95% CI, 0.330 to 0.997; P=0.049). High FPGS protein expression was associated with objective response (mean H-score 254 for responders vs 233 for non-responders, P=0.026) as well with absolute clinical benefit meaning objective response and disease control (mean H-score 248 for responders and stable disease pts vs 220 for progressive disease pts, P=0.005). We found an association of high FPGS protein expression with prolonged PFS (median PFS of 199 vs 97 days; HR 0.487, 95% CI, 0.093 to 1.068; P=0.013), additionally. Conclusions: This is the first report of FPGS examination in MPM. Based on our retrospective study, base line determination of TS and FPGS expression may be predictive for clinical benefit of PMX-based therapy in MPM. Confirmation of the prognostic and predictive value of TS and FPGS expression in MPM by prospective studies is warranted.

Abstract 79: The compartmentalization of folate metabolism depends on two isoforms of folylpoly-γ-glutamate synthetase and the mitochondrial folate transporter

Abstract The reactions of folate metabolism are distributed between the cytosolic and mitochondrial compartments of mammalian cells and both components are essential for mammalian cell survival. The enzyme folylpoly-γ-glutamate synthetase (FPGS) has been shown to be essential for the intracellular retention of folates as polyglutamate derivatives. The fpgs gene produces two transcripts, one for an isoform with a leader peptide targeting it to the mitochondrial matrix, and the other is transcribed without this sequence, so that the encoded isoform remains in the cytosol. Transfer of folates into the mitochondrial matrix is mediated by an inner mitochondrial membrane transport protein, the mitochondrial folate transporter (MFT). Either deletion of the MFT or of mitochondrial FPGS is cytotoxic and renders the cell auxotrophic for glycine; the mechanism of this effect is far from apparent. To better understand this compartmentalization, we generated cell lines that express only the cytosolic or mitochondrial isoform of FPGS under tetracycline-inducible control in FPGS-null AuxB1 cells. Western blot analysis of the FPGS protein in these cell lines indicate that both FPGS isoforms are expressed near endogenous levels in the presence of doxycycline and that each cell line solely expresses the FPGS isoform in the respective compartment. Clonogenic survival of each doxycycline-induced cell line demonstrates a cellular requirement for each FPGS isoform. Additional studies showed that cells complemented with cytosolic FPGS only accumulated and retained folates in the cytosol but not mitochondria, leading us to conclude that folate polyglutamates are not substrates for the MFT. In addition, expression of only the mitochondrial form of FPGS resulted in cells that progressively trapped folates in the mitochondria, leading us to the concept that folate polyglutamates do not escape the mitochondria. However, cytosolic folates were observed in cells expressing only mitochondrial FPGS. We are currently using these cell lines to study the transfer of folates across the mitochondrial membrane and polyglutamation in each compartment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 79.

Histone Deacetylase Inhibitors Induce FPGS mRNA Expression and Intracellular Accumulation of Long-Chain Methotrexate Polyglutamates in Childhood Acute Lymphoblastic Leukemia: Implications for Combination Therapy

Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. MTX is retained within cells as long-chain polyglutamates (MTX-PGs) after metabolism by the enzyme folylpoly-γ -glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX.

Recent Advances in Classical and Non-Classical Antifolates as Antitumor and Antiopportunistic Infection Agents: Part II

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) are newer antifolates that specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Recently, classical antifolates that do not need to be polyglutamylated have also been developed and include plevitrexed (6) and GW1843 (7). Nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)} are nonclassical antifolates for antitumor and parasitic chemotherapy that passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Structural requirements for inhibition with antifolates have been studied extensively and novel agents that exploit key interactions in the active site of TS, DHFR, FPGS, and RFC have been proposed. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates were discussed in Part I. The 6-6 bicyclic and tricyclic antifolates will be discussed here in Part II.

Histone Deacetylase Inhibitor Induces FPGS mRNA Expression in Childhood B-Precursor and T Acute Lymphoblastic Leukemia: Implication of Combination Therapy with Methotrexate To Enhance Cytotoxicity.

Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.

Recent Advances in Classical and Non-Classical Antifolates as Antitumor and Antiopportunistic Infection Agents: Part I

Antifolates that inhibit the key enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR) have found clinical utility as antitumor and antiopportunistic agents. Methotrexate {MTX, (1)} and 5-fluorouracil (5-FU) were among the first clinically useful DHFR and TS inhibitors, respectively. The development of resistance to 5-FU, its occasional unpredictable activity and toxicity resulted in the search of novel antifolates. Pemetrexed (4) and raltitrexed (5) specifically inhibit TS, and are clinically useful as antitumor agents. A major mechanism of tumor resistance to clinically useful antifolates is based on their need for polyglutamylation via the enzyme folylpoly-gamma-glutamate synthetase (FPGS). Novel antifolates have been developed that do not need to be polyglutamylated and include plevitrexed (6) and GW1843 (7). Nonclassical antifolates for antitumor and parasitic chemotherapy, such as nolatrexed (8), trimethoprim {TMP, (11)} and piritrexim {PTX, (12)}, can passively diffuse into cells and hence do not have to depend on FPGS or the reduced folate carrier (RFC). Variations in the structures of antifolates have helped delineate the structural influence on the interaction with TS, DHFR, FPGS, and RFC utilization. The differences in the active site of human and pathogen DHFR have also been exploited. The literature contains excellent reviews on the design and synthesis of antifolates prior to 1996. This two-part review discusses the design, synthesis and structural requirements for TS and DHFR inhibition and their relevance to antitumor and parasitic chemotherapy, since 1996. Monocyclic and 6-5 fused bicyclic antifolates will be discussed in Part I, while 6-6 bicyclic and tricyclic antifolates will be discussed in Part II.

Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

BackgroundExpression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells.MethodsTo determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA.ResultsFPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage.ConclusionWe demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

TEL/AML1 and E2A/PBX1 Tranlsocations Lead to Altered Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in ALL.

Expression of the human folylpoly-γ-glutamate synthetase (FPGS) gene is controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2–3 fold higher in B-precursor (Bp) ALL cell lines and primary cells when compared to T-lineage ALL. These differences correlate with intracellular accumulation of long chain methotrexate (MTX) polyglutamates (PG), and more important with clinical sensitivity to MTX. However, significant heterogeneity of FPGS expression exists within hematopoietic cells of the same lineage, suggesting additional factors influence FPGS expression in Bp- and T-ALL. cDNA microarray data was analyzed for expression of folate-related genes (DHFR, FPGS, RFC, MTHFR, and γ-GGH) in six prognostic leukemia subtypes (T-ALL, hyperdiploid (> 50 chromosomes), BCR-ABL, E2A-PBX1, TEL-AML1, and MLL) (Yeoh et al, Cancer Cell, 2002). It was found that only the FPGS gene exhibited significant heterogeneity of expression compared the others analyzed. Therefore, we investigated the effect of distinct non-random genomic translocations on FPGS gene expression to determine whether gene fusions may be responsible for the heterogeneity of FPGS expression in ALL cells. Using quantitative fluorescence real-time RT-PCR, we first detected reduced levels of FPGS mRNA expression in the RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) human leukemia cell lines expressing non-random chromosomal gene fusions when compared to control (NALM6). To determine if expression of fusion proteins encoded by the E2A-PBX1 and TEL-AML1 translocations lead to decrease FPGS promoter activity and mRNA expression, we used a FPGS-luciferase reporter gene assay. The CHO FPGS null mutant cell line AUXB1, was triple transfected with constructs expressing luciferase (pFPGS-luc), β-galactosidase (pCMVβ), and E2A-PBX1 (pCMVKJ7) or TEL-AML1 (pTEL-AML1) and assayed for luciferase and β-galactosidase activity. Expression of E2A-PBX1 and TEL-AML1, determined by quantitative RT-PCR and Western blot, resulted in 45% and 30% decrease in the level of normalized FPGS-luciferase activity, respectively. These data indicate that FPGS gene transcription is decreased by both translocations, either by direct binding to regulatory elements or indirectly via downstream pathways influenced by E2A-PBX1 and TEL-AML1. Therefore, to confirm these findings and to identify genes or pathways in Bp-ALL regulated by E2A-PBX1 and TEL-AML1 gene fusions, we constructed NALM6 stable cell lines expressing E2A-PBX1 or TEL-AML1 using vectors pCI-neo/E2A-PBX1 and pCI-neo/TEL-AML1. In both NALM6 stable cell lines the level of FPGS mRNA expression was decreased by more than 50% compared to wild type (NALM6). Total RNA was then extracted from NALM6, NALM6/E2A-PBX1, and NALM6/TEL-AML1 cells and analyzed by cDNA microarray. Gene expression profile analysis from existing databases and our NALM6/E2A-PBX1 and NALM6/TEL-AML1 stable cell lines identified genes differentially expressed compared to NALM6 control. These included transcription factors (CSDA, AKAP12, ARL2, PBX1, TEL), cell cycle regulators (CDK2P1), signal transduction (PIK3C3), proliferation and cell migration (PTPRK), zinc finger binding protein (PHF2), among others. This study is the first to demonstrate the causal relationship between the presence of E2A-PBX1 and TEL-AML1 gene fusions and altered FPGS expression and may explain the differential sensitivity to MTX exhibited by these childhood ALL phenotypes.

Cytotoxic effects of pemetrexed in gastric cancer cells

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium-based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT-PCR analysis of thymidylate synthase (TS), folylpoly-gamma-glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5-fluorouracil, with IC50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC50 for 5-fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes.

Differences in Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in Childhood ALL Result from Cell Lineage of Origin and Presence of Non-Random Translocations.

Methotrexate (MTX) is a universal component of chidlhood ALL therapies and its conversion to long chain polyglutamates (PG) by folylpoly-γ-glutamate synthetase (FPGS) is essential for its antileukemic acitivity. Expression of FPGS appears to be controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2-3 fold higher in B-precursor (Bp) ALL cells when compared to T-lineage ALL, and these differences correlate with intracellular accumulation of long chain MTX-PG and lymphoblast sensitivity to MTX. To characterize these lineage differences in FPGS expression between B and T lymphoblasts we examined its mRNA expression in Nalm6 (Bp-ALL) and CCRF-CEM (T-ALL) cells during cellular growth and cell cycle checkpoints. During early exponential growth (24 hrs), FPGS expression was 6-fold higher in Nalm6 when compared to CCRF-CEM but decreased significantly after 72 hrs while it was unchanged in CCRF-CEM cells. During G1/G0 phase we found that FPGS expression was 15-fold higher in Nalm6 when compared to CCRF-CEM cells. Taken together, these data suggest that during proliferation and cell cycle progression FPGS gene expression is regulated differently in Bp-ALL and T-ALL cells. To determine whether this lineage-specific regulation occurs at the transcriptional level we performed nuclear run-on assays. We found that FPGS mRNA transcription initiation rate was 1.7-fold higher in Nalm6 when compared to CCRF-CEM cells, indicating that differences in promoter regulation lead to the observed lineage differences in FPGS expression in Bp- vs. T-ALL. We then used a methylation specific PCR assay to investigate the methylation status of the FPGS proximal promoter region from both Nalm6 and CCRF-CEM cells. Our data indicate that the FPGS proximal promoter region is unmethylated in both cell lines and therefore can not explain the observed lineage-specific differences. 5′-RACE experiments demonstrated that Nalm6 and CCRF-CEM have the same FPGS transcriptional start sites, but a reporter gene assay indicated that the minimal promoter region that directs FPGS transcription in CCRF-CEM cells is insufficient to drive FPGS mRNA transcription in NALM6 cells. In order to identify potential regulatory regions directing FPGS transcription in Bp-lymphoblasts, we used DNaseI hypersensitivity assays. We identified a hypersensitive region located 8.5 kbp upstream to exon 1 in Nalm6 cells suggesting that tissue-specific regulatory elements responsible for lineage-specific FPGS expression in Bp-ALL cells may be localized within this region. Finally, we detected reduced levels of FPGS mRNA expression in RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) cells expressing chromosomal translocated fusions when compared to control (Nalm6). To characterize the molecular basis of the E2A-PBX1 and TEL-AML1 interactions with FPGS mRNA expression in Bp lymphoblasts we used antisense and RNAi technology to downregulate these two genetic fusions. Our data lead us to hypothesize that in addition to lineage-specific regulatory differences in FPGS expression, molecular mechanisms associated with non-random translocations may alter FPGS mRNA expression and influence MTX sensitivity in Bp-ALL lymphoblasts.

Effects of Antisense-based Folylpoly-γ-glutamate Synthetase Down-regulation on Reduced Folates and Cellular Proliferation in CCRF-CEM Cells

The effect of down-regulation of folylpoly-γ-glutamate synthetase (FPGS) activity on intracellular reduced folate accumulation and cellular proliferation was examined, using an inducible antisense expression system in the human T-lymphoblastic leukemia cell line CCRF-CEM. FPGS catalyzes the addition of γ-glutamyl residues to natural folates and classical antifolates, which results in their enhanced cellular retention and increased cytotoxicity. As such, this enzyme has become a focus as a potential anticancer drug target. However, direct evidence to support this concept has been elusive. Hence, a study was undertaken using an antisense-based expression system to down-regulate FPGS activity. This inducible expression system was used to demonstrate that lower FPGS activity can lead to substantially lower intracellular folate content, which coincides with suppression of thymidylate synthesis and inhibition of cellular proliferation.

Molecular cloning of murine folylpoly-γ-glutamate synthetase

Folylpoly-γ-glutamate synthetase (FPGS) is essential for mammalian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites. Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells. The amino acid sequence of murine FPGS is 82% identical to human FPGS [1] with identical discrete regions of up to 41 residues. Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells [2]. Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS. The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations.