Substrates For Reduced Folate Carrier Research Articles

Schedule-Dependent Synergy Between the Histone Deacetylase Inhibitor Belinostat and the Dihydrofolate Reductase Inhibitor Pralatrexate in T-and B-cell Lymphoma Cells in vitro.

Pralatrexate (Folotyn; PLX) and belinostat (Beleodaq; BLS) are registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and are being considered for other lymphomas. In this study we investigated whether BLS had the ability to potentiate the cytotoxicity of PLX. A panel of lymphoma cell lines was used for the combination studies: the B-cell SUDHL-4, SUDHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. Uptake of PLX was mediated by the reduced folate carrier (RFC). PLX showed a 6-fold better RFC substrate affinity compared to methotrexate, and 2-fold better than levoleucovorin (l-LV). Sensitivity expressed as the concentration that resulted in 50% growth inhibition (IC50) after 72 hr exposure to PLX varied from 2.8 to 20 nM and for BLS from 72 to 233 nM, independent of the background of the cell lines. The interaction between BLS and PLX was studied using the median-drug effect analysis. At a fixed molar ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: around 1.0). In three selected cell lines (SUDHL-4, SUDHL-5, and HT) sequential exposure (24 h pretreatment with BLS, followed by 48 h to PLX + BLS), did not improve interaction (CI: 0.9–1.4). As an alternative approach a non-fixed ratio was used by exposing SUDHL-4, SUDHL-5, and HT cells to IC25 concentrations of either BLS or PLX in combination with the other drug. Exposure to IC25 of PLX did not decrease the IC50 for BLS (CI from 0.6–1.2), but exposure to IC25 of BLS markedly increased PLX sensitivity (low CIs from 0.40 to 0.66). Mechanistic studies focused on induction of apoptosis, and showed cleavage of predominantly caspase-9 in HT and SUDHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of PLX and BLS showed additivity in various lymphoma cell lines, with a schedule-dependent synergism in B-cell lymphoma. Based on these data, proficient inhibition of HDAC activity by BLS holds promise in sensitization of tumor cells to PLX.

Abstract 3239: Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution

Abstract Three specialized systems exist for membrane transport of folates and antifolates across biological membranes. The reduced folate carrier (RFC) is ubiquitously expressed in tumors and normal tissues. Folate receptors (FR) α and β, and the proton-coupled folate receptor (PCFT) exhibit narrow patterns of tissue expression and serve specialized physiologic roles. FRs are expressed in a number of cancer cells (e.g., FRα in ovarian cancer and non-small cell lung cancer; FRβ in acute myelogenous leukemia), whereas PCFT is expressed in a large number of human solid tumors but not leukemias. FRs are either inaccessible to the circulation (FRα) or are nonfunctional (FRβ) in normal tissues. PCFT transport is limited in most normal tissues, given the requirement of acidic pH for optimal activity. These properties facilitate tumor targeting of cytotoxic compounds with specificities for PCFT or/and FR transport. Clinically used antifolates such as methotrexate and pemetrexed are substrates for RFC and their uptake by both normal tissues and tumors confers dose-limiting toxicity due to limited tumor selectivity. We previously reported a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate with a 4-carbon bridge and a phenyl glutamate side chain (AGF127). Previous studies of related 6-substituted pyrrolo[2,3-d]pyrimidines established that the nature and length of the bridge region plays an important role in determining tumor cell potency and transport selectivity. Thus, it was of interest to vary the length and insert heteroatoms into the side chain -(CH2)4- of AGF127, including -(CH2)n-S- (compound 1, n= 3 and compound 2, n = 4) and -(CH2)n-O- (compound 3, n= 3 and compound 4, n = 4), to determine the impact on drug potency and transport selectivity. The novel analogs were tested as growth inhibitors against engineered Chinese hamster ovary (CHO) cells expressing human FRα (RT16), RFC (PC43-10), and PCFT (R2/PCFT4). AGF127 showed potent inhibition of CHO cells expressing FRα (IC50 = 8.6 nM), and reduced activity toward CHO cells expressing RFC (57 nM) or PCFT (840 nM). Compounds 1, 2, and 3, showed reduced inhibitions toward FRα-expressing RT16 cells (4-35-fold) compared to AGF127; however, compound 4 was equipotent to AGF127. Like AGF127, none of the series were effective PCFT substrates. Compound 3, like AGF127, inhibited RFC-expressing PC43-10 cells (IC50 = 37 nM); RFC activity was reduced 6- to >20-fold for compounds 1, 2, and 4. Toward FRα-, RFC-, and PCFT-expressing KB human tumor cells, the order of potency was 4 = AGF127 > 2 = 3 = 1. Compound 4 was also more active (15-fold) than pemetrexed with KB cells. Collectively, the potent and selective activity of compound 4 toward FRα-expressing tumor cells and limited activity toward RFC-expressing cells offers significant advantages over AGF127 and clinically used non-targeted antifolates, suggesting that further preclinical evaluation is warranted. Citation Format: Aleem Gangjee, Rishabh Mohan, Manasa Ravindra, Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Larry H. Matherly. Selective tumor targeting with 5-substituted pyrrolo[2,3-d]pyrimidines with heteroatom bridge substitution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2017-3239

Abstract 5123: Targeted thieno[2,3-d]pyrimidines with fluorinated phenyl side chains as antitumor agents

Abstract The reduced folate carrier (RFC) is ubiquitously expressed in tissues and tumors and is the major tissue transporter for folate cofactors. Folate receptor (FR) α and β, as well as the proton-coupled folate transporter (PCFT), exhibit narrower patterns of tissue expression and are likely to serve more specialized physiologic roles. FRs are expressed in a subset of cancer cells (e.g., ovarian and non-small cell lung cancer for FRα, acute myelogenous leukemia for FRβ), whereas PCFT is expressed in a wide range of human solid tumors but not leukemias. Earlier generations of glycinamide ribonucleotide formyltransferase (GARFTase) and de novo purine nucleotide biosynthesis (e.g., Lometrexol) have shown promise as antitumor drugs. However, these compounds are excellent substrates for RFC and thus are non-selective for tumors, leading to dose-limiting toxicities. We previously reported a novel class of 6-substituted thieno[2,3-d] pyrimidines with a phenyl side chain and 3- and 4-carbon bridge lengths (AGF17 and AGF23, respectively) which selectively targeted FRs but not RFC or PCFT. To increase antitumor efficacy, we synthesized fluorinated analogs of AGF17 and AGF23, designated as AGF309 and AGF304, respectively. AGF309 and AGF304 potently inhibited proliferation of Chinese hamster ovary (CHO) cell lines engineered to individually express human FRα (IC50s of 1.08 and 2.27 nM, respectively) or FRβ (IC50s 0.48 and 1.14 nM, respectively). AGF304 and AGF309 showed nominal activity toward RFC- and PCFT-expressing CHO cells. Both AGF309 and AGF304 were also potently inhibitory toward FRα-expressing human KB human tumor cells (IC50s of 7.19 and 5.27nM, respectively). By analogy with previous iterations of 6-substituted thieno [2,3-d] pyrimidine compounds, growth inhibitory activity by AGF309 and AGF304 likely involves inhibition of de novo purine biosynthesis at GARFTase and potentially a secondary intracellular target. Collectively, our in vitro findings of the potent and selective antitumor activity and FR selectivity suggest that further preclinical evaluation of AGF309 and AGF304 as potential antitumor agents is warranted. Citation Format: Nian Tong, Aleem Gangjee, Adrianne Wallace Povrik, Carrie O’Connor, Aamod Aamod Dekhneb, Zhanjun Hou, Larry H. Matherly. Targeted thieno[2,3-d]pyrimidines with fluorinated phenyl side chains as antitumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5123. doi:10.1158/1538-7445.AM2017-5123

Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

Schedule-Dependent Synergy Between Belinostat and Pralatrexate in T-and B-Cell Lymphoma Cells in Vitro

Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) are two new agents that have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have also shown promising activity in other types of lymphoma. FOL is a folate analogue and a potent inhibitor of dihydrofolate reductase (DHFR), designed to accumulate in cancer cells preferentially via the reduced folate carrier (RFC) and retained therein via efficient polyglutamylation. Inhibition of DHFR leads to an imbalance of deoxynucleotides (e.g. depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that broadly inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. We investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether the toxicity of FOL can be controlled by levo-leucovorin (Fusilev), the natural stereoisomer of leucovorin, and whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL.A panel of lymphoma cell lines was used for the combination studies including: the B-cell lymphoma cell lines SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell lymphoma cell lines Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL was determined in competition uptake experiments with [3H] Methotrexate (MTX), revealing a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin. FOL displayed very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than levo-leucovorin). FOL had a low substrate affinity for PCFT (>10-fold lower than folic acid and levo-leucovorin in [3H] leucovorin uptake competition experiments). Levo-leucovorin could completely protect toxicity by FOL, but had no effect on BEL toxicity. Sensitivity of lymphoma cell lines (IC50 concentrations after 72 hrs drug exposure) to FOL drug varied from 2.8 nM (Hut-78), 5.5 nM (SU-DHL4 and 5), 7.4 nM (HT) to 20 nM (Karpas-299 and Jeko-1) while IC50 values for BEL were in the range of 100 nM (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT).The interaction between BEL and FOL was studied using the median-drug effect analysis with Calcusyn software. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all the lymphoma cell lines revealed an additive effect (CI: all around 1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure to the drugs (24 hr pretreatment with either BEL or FOL) followed by 48 hr to the combination, did not improve the results with CI values varying between 0.9 and 1.4. As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 concentrations of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 concentrations of BEL markedly increased the sensitivity to FOL as reflected by the low CIs varying from 0.40 to 0.66. Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest.In conclusion, the combination of FOL and BEL showed additive activity in various lymphoma cell lines, while a schedule-dependent synergism was observed. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Furthermore, toxicity of FOL could be completely protected by levo-leucovorin. DisclosuresReddy:spectrum: Employment, Equity Ownership.

Abstract C69: Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro

Abstract Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have promising activity in other lymphoma. FOL is a folate analog and a potent dihydrofolate reductase (DHFR) inhibitor, designed to accumulate in cancer cells via the reduced folate carrier (RFC) and retained via efficient polyglutamylation. DHFR inhibition leads to an imbalance of deoxynucleotides (depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. In this study we investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL. A panel of lymphoma cell lines was used for the combination studies: the B-cell SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL (in competition with [3H]-methotrexate), showed a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin (l-LV). FOL had a very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than l-LV) and a low affinity for PCFT (>10-fold lower than folic acid and l-LV in [3H]-LV uptake competition experiments). Sensitivity (IC50 concentrations after 72 hr exposure) to FOL varied from 2.8 (Hut-78), 5.5 (SU-DHL4 and 5), 7.4 (HT) to 20 nM (Karpas-299 and Jeko-1) and for BEL from 100 (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT). The interaction between BEL and FOL was studied using the median-drug effect analysis. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all cell lines showed additivity (CI: ±1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure (24 hr pretreatment with either BEL or FOL, followed by 48 hr to FOL + BEL), did not improve interaction (CI: 0.9-1.4). As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 of BEL markedly increased FOL sensitivity (low CIs from 0.40-0.66). Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest. In conclusion, the combination of FOL and BEL showed additivity in various lymphoma cell lines, with a schedule-dependent synergism. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Citation Format: Godefridus J. Peters, Frank H. Van Gemert, I. Kathmann, Saskia A. Cillessen, Gerrit Jansen, Guru Reddy. Schedule-dependent synergy between the histone deacetylase inhibitor belinostat and the dihydrofolate reductase inhibitor pralatrexate in T-and B-cell lymphoma cells in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C69.

Abstract 873: Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation.

Abstract The reduced folate carrier (RFC) is the major tissue folate transporter in mammalian cells. RFC is essential for providing folates for biosynthetic pathways leading to nucleotides and amino acids. Clinically relevant antifolates including methotrexate (Mtx), pemetrexed, and pralatrexate are all substrates for RFC and RFC levels and function are key determinants of antitumor activities. While recent reports have described the development of novel 6-pyrrolo[2,3-d]pyrimidine antifolates with selectivity for cellular uptake and tumor targeting by the proton-coupled folate transporter, RFC levels are nonetheless important determinants of antitumor activities for these agents, by impacting cellular folate pools which compete for polyglutamylation and binding to intracellular enzyme targets. Human RFC is subject to complex transcriptional and posttranscriptional controls. RFC forms homo-oligomers, providing another potential level of regulation, as a dominant-phenotype results with mutant-wild type RFC mixtures. This reflects impaired intracellular trafficking of oligomeric RFC from the endoplasmic reticulum to the plasma membrane surface. To explore the question of whether folates can acutely regulate RFC levels or activity, RFC-null HeLa cells were stably transfected with RFCMyc-his10 in pCDNA3 plasmid. Transfectants were cultured without folate (10 d), then with (6R,S)5-formyltetrahydrofolate (leucovorin (LCV)) from 0.5 to 200 nM (5 d). Cells were assayed for RFC transcripts and total cellular RFC proteins. Membrane surface RFC was measured by sulfo-N-hydroxysuccinimide-SS-biotin labeling and the formation of homo-oligomers was assessed by cross-linking with 1,1-methanediyl bismethanethiosulfonate. Finally, RFC transport was assayed with [3H]Mtx at neutral pH. Levels of RFC transcripts and total cell RFC protein progressively increased with increasing LCV up to 200 nM. Conversely, surface RFC and [3H]Mtx transport were maximal at ∼ 2 nM LCV, then deceased (∼2-3-fold) with increasing LCV. These effects were maximal at 20 nM LCV. RFC plasma membrane targeting was confirmed by immunofluorescence staining and confocal microscopy. RFC protein was completely localized to the plasma membrane at low (≤ 2 nM) LCV concentrations, however, at higher concentrations, significant intracellular RFC was detected. There were no changes in ratios of RFC homo-oligomer over monomer in response to changes in extracellular LCV. Our results suggest a novel acute regulation of RFC in response to changes in extracellular folate concentrations involving (i) increased RFC transcripts and proteins, possibly due to differences in transcript stability, and (ii) increased cytosolic RFC due to impaired intracellular trafficking and membrane targeting. The net effect would be enhanced RFC-mediated folate and antifolate accumulations under conditions of folate deprivation. Citation Format: Zhanjun Hou, Steve Orr, Erika Etnyre, Christina Cherian, Larry Matherly. Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 873. doi:10.1158/1538-7445.AM2013-873

Abstract 1364: Design, synthesis and evaluation of potent antiproliferative and cytotoxic antifolates with selective FRs and PCFT transport over RFC and potent GARFTase inhibition

Abstract Lack of selectivity for folate transporters is one of the major causes of toxicity and chemotherapy failure with clinically used antifolates. Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT), account for most of the folate influx activity in mammalian cells. We recently reported potent inhibition of cell proliferation by a classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine with a thienoyl for benzoyl-substituted side chain and a 4-carbon bridge (AG71). AG71 was selective for transport by FRs and PCFT over RFC and inhibited glycinamideribonucleotide formyl transferase (GARFTase) in de novo purine biosynthesis. In SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. A series of five thienoyl regioisomers of AG71 with [1,3] (AG117 and AG118) and [1,2] (AG114, AG115, AG116) thienoyl substitutions by the 4-carbon bridge and the L-glutamate were synthesized. Analogs were evaluated for anti-proliferative activities in engineered Chinese hamster ovary (CHO) cells expressing RFC, FR, or PCFT to determine the optimum structures for selective transport by FRs and/or PCFT. Additional studies were performed in KB and IGROV1 tumor cells, using inhibition of cell proliferation or colony formation as metrics. Like AG71, none of the novel thienyol regioisomers were substrates for RFC. AG117 and AG118 were potently active toward FR- and PCFT-expressing CHO cells and toward KB and IGROV1 tumor cells, equivalent or exceeding levels for AG71. In colony-forming assays in KB cells, AG117 and AG118 were cytotoxic. AG114, AG115, and AG116 were substantially less active or completely inert. AG117 and AG118, like AG71, were high affinity ligands for FRα by competitive ligand binding with 3H-folic acid, and were potent substrates for PCFT by direct competition 3H-methotrexate transport. By protection assays, AG71 and AG117 preserved potent anti-proliferative activity even in the presence of 50 nM leucovorin; adenosine and 5-imidazole-4-carboxamide were completely protective, establishing the de novo purine biosynthetic pathway and GARFTase as the principal intracellular targets. GARFTase inhibition was confirmed by in situ GARFTase metabolic assays with 14C-glycine as a radiotracer. Our results identify 6-subsituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG71 as potent anti-proliferative and cytotoxic antifolates, reflecting selective transport by PCFT and FR over RFC and inhibition of GARFTase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1364. doi:10.1158/1538-7445.AM2011-1364

Abstract 4546: Expression profiling of the major folate facilitative transporters in human tumors and normal tissues

Abstract Membrane transport of folates into mammalian cells involves major facilitative transporters including the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Classical antifolates such as methotrexate (Mtx), pemetrexed, or raltitrexed are all substrates for RFC and PCFT such that the cytotoxic effects of these drugs are determined by levels of these transporters in tumors and normal tissues. As PCFT is a folate-proton symporter with an acidic pH optimum, extracellular pH represents another key determinant of transport, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments. We recently identified novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selective transport substrates for PCFT over RFC and that potently inhibit cell proliferation by targeting glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. To better exploit this approach for selectively targeting tumors versus normal tissues, we systematically determined expression profiles for PCFT and RFC in human malignancies and normal tissues. Real-time reverse transcription PCR (qRT-PCR) was used to determine transcript levels in 57 human solid tumor and 27 leukemia cell lines. Both RFC and PCFT transcripts were highly expressed in the majority of solid tumor cell lines whereas only RFC was expressed appreciably in the leukemia cell lines. For 10 solid tumor cell lines, PCFT and RFC transcripts were accompanied by corresponding levels of respective proteins on western blots. Functional validation of transporter expression in solid tumor cell lines was determined by transport of [3H]Mtx at pH 5.5 for PCFT, and at pH 7.2 for RFC. Tumor uptake of [3H]Mtx correlated closely with transcript and protein levels for both PCFT and RFC. qRT-PCR of cDNA arrays from 48 normal human tissues measured high levels of PCFT expression in the small intestine, kidney, liver, and the adrenal and pituitary glands. On cDNA arrays of human primary tumors, significant albeit variable levels of PCFT transcripts were detected in breast, colon, kidney, liver, lung, ovary, and prostate tumors. Patterns of transporter expression in primary tumors were confirmed by immunohistochemistry of tissue arrays probed with transporter-specific antibodies. In conclusion, we have established the expression patterns and pH-specific transport for PCFT and RFC in human solid tumor cell lines, primary tumors and select normal tissues. This study represents the first comprehensive analysis of PCFT expression in tumors and normal tissues and how this relates to RFC. Our finding of widespread PCFT expression in human solid tumors paired with our previous discovery of novel PCFT-specific antifolates offers exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors based on their acidic microenvironments. 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 4546.

Folate-based thymidylate synthase inhibitors in cancer chemotherapy.

Understanding the relationship between chemical structure and biological properties of folate analogs, particularly their interactions with the target enzymes, transport proteins and folate-metabolizing enzyme, folylpolyglutamate synthetase (FPGS), has enabled the rational design and development of the selective thymidylate synthase (TS) inhibitors with folate-based structures for clinical uses. These compounds specifically inhibit TS devoid of concomitant effects at other loci, unlike 5-fluorouracil (5-FU). ZD1694 ('Tomudex') was designed as a non-nephrotoxic and highly active analog of N10-propargyl-5,8-dideazafolic acid (CB3717), which is a potent TS inhibitor but had unacceptable nephrotoxicity caused by its poor water solubility. The potent cytotoxic activity of ZD1694 is dependent upon active uptake into cells via the reduced folate carrier (RFC), and subsequent rapid and extensive metabolism to polyglutamate forms inside cells. Marked enhancement of the TS inhibitory activity has been noted as the glutamate chain is elongated. Polyglutamation is critical to the biological activity of ZD1694 against tumor and normal proliferating tissues. The retentive property of ZD1694 polyglutamates inside cells led to a single, infrequent administration schedule in clinical studies. ZD1694 has completed phase I and phase II evaluation with activity observed in several tumor types, particularly in colorectal cancer with a 26% objective response rate. A recent European phase III study of ZD1694, randomized against a 5-FU plus leucovorin regimen, demonstrated an equivalent response rate for advanced colorectal cancer (complete or partial responses; 20 versus 17%) and less toxicity than seen with the latter regimen. The newer selective TS inhibitors, which retain potency for TS inhibition but are not substrates for RFC and/or FPGS, are currently under clinical evaluation. These classes of compound may have benefits for circumvention of resistance by virtue of alterations in these protein functions and for the management of toxicity.