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Pteridines

Official Journal of the International Society of Pteridinology

Editor-in-Chief: Fuchs, Dietmar


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Volume 26, Issue 2

Issues

Novel insights in folate receptors and transporters: implications for disease and treatment of immune diseases and cancer

Gerrit Jansen
  • Amsterdam Rheumatology and Immunology Center, VU University Medical Center, Cancer Center Amsterdam, PO Box 7057, 1007 MB Amsterdam, The Netherlands
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Godefridus J. Peters
  • Corresponding author
  • Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, PO Box 7057, 1007 MB Amsterdam, The Netherlands
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Published Online: 2015-04-21 | DOI: https://doi.org/10.1515/pterid-2015-0005

Abstract

Folate receptors and transporters as well as folate enzymes play an essential role in human disease and form important targets for the treatment of immune diseases and cancer. To discuss new developments in this area, every 2 years a multidisciplinary meeting is held, which aims to be an informal forum for fundamental scientists and clinicians. During this meeting, the regulation of folate transporters and folate enzymes is discussed at the level of expression, transcription, translation, post-translational modification, and splicing and enzyme regulation. Importantly, this knowledge is applied and translated into exciting clinical applications by clinicians with various backgrounds, such as surgeons, nephrologists, rheumatologists and oncologists. Moreover, the meeting provides an excellent forum for a scientific interaction between academia and industry.

Keywords: folate deficiency; folate imaging; folate receptor; proton-coupled folate transporter; reduced folate transporter

Introduction

The 5th International Symposium on Folate Receptors and Transporters brought together almost 60 distinguished scientists from eight nations to share the developments on their basic, translational and clinical research on folate receptors and transporters in multidisciplinary areas of health and disease. The biennial symposium, organized by the Folate Receptor Society (FRS), covers sessions on the biology of folate receptors and transporters, antifolates in cancer, antifolates in immune diseases, folate receptors and imaging, and folate receptors in reproduction, development and disease. This symposium report covers highlights of the indicated sessions.

In his introductory remarks, FRS President Philip Low (West Lafayette, IN, USA) summarized the progress that has been made over the past decade(s) and presented at previous symposia. A selection of these achievements is listed in Table 1.

Table 1

Key developments in folate research.

Philip Low anticipated an inspiring symposium where updates on these topics would be presented along with interactive discussions with speakers and poster presenters.

Barton Kamen lecture

As a tribute to one of the founding members of the FRS, Barton Kamen, who sadly passed away 2 years ago just prior to the 4th FRS Symposium, Manohar Ratnam (Detroit, MI, USA) delivered the Barton Kamen Lecture. Herein, he summarized Bart Kamen’s major contributions to the field of folate receptors, folate biology and pharmacology of classical antifolates, and to the improvement in the treatment of childhood acute lymphoblastic leukemia [particularly addressing methotrexate (MTX) toxicity]. Bart Kamen published a seminal work on the identification, isolation and cloning of folate receptors [34, 35]; receptor functioning and cycling via potocytosis [35–39]; and folate receptors in malignant tissues [40, 41]. Ratnam presented the recent research from his laboratory [42], which was initiated and inspired by discussions with Barton Kamen, asking the question whether innocuous transcription factor modulators alter tumor sensitivities to standard treatments. Take, for example, the glucocorticoid dexamethasone, which is commonly administered with chemotherapy regimens, will it directly sensitize or protect tumors by regulating key determinants of drug sensitivity? Indeed, in a variety of non-small cell lung cancer (NSCLC) cells, dexamethasone appeared to alter the expression of genes involved in the action of the antifolate pemetrexed (PMX)/Alimta/multitargeted antifolate. In fact, dexamethasone down-regulated thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expression in cells harboring high glucocorticoid receptor-α (GRα) expression levels to protect against pemetrexed activity. Therefore, examination of tumor GRα status seems indicated for stratification of patients for pemetrexed-based chemotherapies. Together, this is one example of Bart Kamen’s many original ideas that hold relevance for current antifolate chemotherapy.

Biology of folate transporters and enzymes: relation with disease

David Goldman (New York, NY, USA) discovered the proton-coupled folate transporter (PCFT) and established its physiological role in intestinal folate absorption and transport across the choroid plexus in cerebrospinal fluid (Figure 1) by demonstrating marked impairment of these processes in subjects with the rare autosomal recessive disorder hereditary folate malabsorption, in which there are loss-of-function PCFT mutations [16, 44]. Hence, PCFT-dependent intestinal absorption is required to sustain folate homeostasis in man. (For a list of folate-related disorders, see Table 2.) Dr. Goldman also suggested that, because of loss-of-function mutations of PCFT or folate receptor-α (FRα), each alone or both produce cerebral folate deficiency; PCFT may be necessary for folate export from FRα-containing endosomes at the choroid plexus. PCFT is also highly expressed in the liver, kidney, retina and placenta, but its functional role at these sites is not clear. Dr. Goldman also described (i) the very high affinity of PCFT for pemetrexed at its optimal pH of 5.5 and the relatively rapid and favored transport of this drug at pH levels characteristic of the tumor environment and (ii) the ubiquitous functional expression of PCFT in epithelial cancers [17].

Overview of folate transporters. PCFT (proton-coupled folate transporter/SLC46A1): pH optimum 5.5, expressed in epithelial tissues, ubiquitously expressed in epithelial tumors. RFC (reduced folate carrier/SLC19A1): pH optimum 7.4, organic phosphate (OP-) antiporter, ubiquitously expressed in normal and malignant cells. FR (folate receptor): four isoforms: FRα, FRβ, FRγ and FRδ; expressed in epithelial tissues and in epithelial and hematopoietic cancers. (Adapted with permission from Zhao et al. [43].)
Figure 1:

Overview of folate transporters. PCFT (proton-coupled folate transporter/SLC46A1): pH optimum 5.5, expressed in epithelial tissues, ubiquitously expressed in epithelial tumors. RFC (reduced folate carrier/SLC19A1): pH optimum 7.4, organic phosphate (OP-) antiporter, ubiquitously expressed in normal and malignant cells. FR (folate receptor): four isoforms: FRα, FRβ, FRγ and FRδ; expressed in epithelial tissues and in epithelial and hematopoietic cancers. (Adapted with permission from Zhao et al. [43].)

Table 2

Folate disorders and clinical symptoms.

Robert Steinfeld (Göttingen, Germany) was the first to describe loss-of-function mutations of FRα as a basis for cerebral folate deficiency [52]. Recently, Dr. Steinfeld provided evidence for a novel mechanism by which folates are transported in the nervous system: (i) Folate transport across the choroid plexus is mediated by folate receptor-mediated transcytosis. (ii) Folate-receptor exosomes are released at the apical membrane into the cerebrospinal fluid. (iii) The exosomes are then absorbed across the ependymal cells into the brain, after which they diffuse to, and enter, the neural cells. Dr. Steinfeld suggested that the role of PCFT at the choroid plexus is in the provision of folates across the basolateral membrane into the cytoplasm, which is necessary for the normal function of these cells [53].

The resolution of the crystal structure of the FRα by Karsten Melcher (Grand Rapids, MI, USA) provided further insight into the role of this transporter in folate malabsorption into the brain [8]. FRα and FRβ are highly glycosylated glycosylphosphatidylinisotol-anchored membrane proteins that hamper their purification and crystallization [8, 9, 54]. Dr. Melcher described how folic acid fits in the binding pocket of FRα and how the space of this pocket can be used for FRα- and FRβ-targeting antifolates. A slight acidification would destabilize the FR-folic acid complex.

Asok Anthony (Indianapolis, IN, USA) described a mechanism for the translational up-regulation of FRα expression as a candidate sensor of physiological folate deficiency, e.g., increased plasma homocysteine levels. Notably, homocysteine appeared to promote interactions between heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and the 18-base FRα mRNA cis-element to promote the synthesis of FRα under folate-deficient conditions [55]. Additionally, homocysteinylated hnRNP-E1 serves as a master regulator in response to the restoration of nutritional folate deficiency.

Richard Finnell (Austin, TX, USA) developed a number of mouse knock-out models to understand the physiological consequence of a deficiency in several of these transporters during embryonic development [3, 56]. Knock-out of the FRα (Folr1) will lead to developmental disorders, which can be prevented by supplementing the diet with leucovorin. This knock-out model can also affect the development of the heart and aorta. Interestingly, the model also identified some folic acid response targets such as the Wnt pathway, which is lost in knock-out mice. These studies also revealed that FRα may translocate to the nucleus and act as a transcription factor. Reduced folate carrier (RFC) knock-out mice do not come to a full development and need folic acid supplementation to develop and survive. There is a failure of chorioallantoic fusion in RFC null mice such that the neural tube will not close and there are cardiac malformations. The folate deficiency also leads to a depletion of S-adenosyl-homocysteine, reducing the methylation of DNA and histones. The decreased methylation also affects the hedgehog pathway, e.g., leading to a depletion of Gli3. In contrast to the FR- and RFC1- mice, PCFT knock-out mice do not present neural tube defects, but have profound neurological defects, anemia and thrombocytopenia; they will not survive beyond 6 weeks, but betaine supplementation enables survival to adulthood. In this mouse model of HFM, the folate depletion is accompanied by a massively increased homocysteine, the highest seen in disease.

Another novel folate disorder was reported by Henk Blom (Freiburg, Germany), who described an association between folate depletion and a marked (>99%) decrease in the activity of DHFR [57, 58]. The patients in that report displayed normal folate and homocysteine in plasma, but had decreased total folate content in the red blood cells and cerebrospinal fluid (CSF), especially that of 5-methyl-THF. Clinical symptoms consisted of low hemoglobin, cerebral folate deficiency, megaloblastic anemia and epilepsy. Symptoms (folates and clinical) improved partially on treatment with folic acid and improved further on folinic acid. For an overview of folate metabolism, including the role of DHFR, see Figure 2.

Schematic representation of the folate supply (via diet or food supplements) and metabolism. Depending on the source, folates will enter the folate cycle directly (from food as 5-methyl tetrahydrofolate) or indirectly (from supplements, folic acid). Folic acid will be reduced by DHFR to dihydrofolate and tetrahydrofolate. An increase of 5-methyl-tetrahydrofolate will reduce homocysteine levels since the methyl group is used to convert homocysteine to methionine [catalyzed by methionine synthase (MS) with vitamin B12 as a co-factor], which, after conversion to S-adenosylmethionine, will serve as a methyl donor for methylation reaction catalyzed by DNA methyltransferase (DNMT). TS, thymidylate synthase; FPGS, folylpolyglutamate synthetase; MTHFR, methylene tetrahydrofolate reductase. (Adapted with permission from Peters et al. [59].)
Figure 2:

Schematic representation of the folate supply (via diet or food supplements) and metabolism. Depending on the source, folates will enter the folate cycle directly (from food as 5-methyl tetrahydrofolate) or indirectly (from supplements, folic acid). Folic acid will be reduced by DHFR to dihydrofolate and tetrahydrofolate. An increase of 5-methyl-tetrahydrofolate will reduce homocysteine levels since the methyl group is used to convert homocysteine to methionine [catalyzed by methionine synthase (MS) with vitamin B12 as a co-factor], which, after conversion to S-adenosylmethionine, will serve as a methyl donor for methylation reaction catalyzed by DNA methyltransferase (DNMT). TS, thymidylate synthase; FPGS, folylpolyglutamate synthetase; MTHFR, methylene tetrahydrofolate reductase. (Adapted with permission from Peters et al. [59].)

Folates in cancer

The specific properties of PCFT and FRα were applied by Larry Matherly (Detroit, MI, USA), who described the tissue distribution of the three main transporters: PCFT, RFC and FRα [60, 61]. PCFT has a high expression in many tumor cells, which, together with its low pH optimum, makes it the most important folate transporter for many tumors in vivo. Dr. Matherly, together with Dr. Aleem Gangjee, designed and tested a series of tumor-targeted antifolates with dual PCFT and FRα cellular uptake, independent of RFC-mediated uptake, leading to selective folate-based therapy. 6-Substituted pyrrolo[2,3-d]pyrimidines with a thiophene for benzene replacement and 3-carbon bridge seem to be the most potent analogs, with FRα and PCFT selectivity. Some of these analogs are potent inhibitors of the purine de novo nucleotide synthesis at the glycinamide ribonucleotide transformylase (GARFTase) level. Since purine nucleotides are essential for cancer cell survival, these compounds form a new class of drugs that can specifically target cancer cells.

Aleem Gangjee (Pittsburgh, PA, USA) described the medicinal chemistry part of the synthesis of these targeted antifolates. The newly synthesized compounds were characterized for their substrate specificity for the transporters RFC, PCFT, FRα and FRβ, and for their cell growth inhibitory capacities [18]. The chemical structure of one of these compounds, the parent compound AG94, was also superimposed with 5,10-dideazatetrahydrolic acid and docked in GARFTase. Small changes in the structure improved potency and transporter selectivity.

Frits Peters (Amsterdam, The Netherlands) described the relative roles of the three folate transporters, PCFT, FR (with high specificity for the oxidized folate, folic acid) and the classical RFC (with high specificity for reduced folates and MTX), for their differential ability to transport PMX, folic acid, stereoisomers of the reduced folate precursor leucovorin and the novel antifolate pralatrexate (PLX). Leucovorin is extensively used in combination with 5-fluorouracil (5FU) in the treatment of colon cancer, wherein leucovorin potentiates the 5FU-induced inhibition of the folate enzyme TS. TS is the only de novo source for thymidine nucleotides (Figure 2), and its inhibition in tumors leads to a so-called thymine-less death. Earlier data published by Dr. Peters showed a correlation between the inhibition of TS and the response of patients to this treatment [62, 63]. His present data showed that the pure natural stereoisomeric form of leucovorin, Fusilev, has some advantages over a racemic mixture in some cell lines in potentiating either the cytotoxicity of 5FU in colon cancer cell lines or the in situ inhibition of TS, and in intact cellular assays. Leucovorin and the oxidized folate, folic acid, appeared as bona fide substrates for PCFT at acidic pH (5.5), but at neutral pH, PCFT did not play a relevant role in their cellular uptake nor in the cytotoxicity of the antifolates PMX and PLX. PLX was only a substrate for RFC and displayed a very poor binding affinity for FRα. The data also showed that, for protection by leucovorin (including Fusilev) of PMX and PLX cytotoxicity, a sufficient expression of RFC is necessary, providing a window for selectivity since in normal tissues the near-neutral pH environment ensures the efficient uptake of leucovorin.

Elisa Giovannetti (Amsterdam, The Netherlands) provided a nice translation of the experimental data on PMX. Since the promoter of PCFT can be highly methylated [64], this can hamper PCFT functional activity and confer antifolate resistance. Furthermore, treatment with a demethylating agent increased antifolate uptake. Hence determination of PCFT methylation or of PCFT expression may help to select patients potentially sensitive to PMX. PCFT expression (protein and mRNA) was determined in cell lines derived from mesothelioma (MPM) and NSCLC, which showed a large variation, which was also found in human tumor specimens. MPM patients treated with a combination of PMX and carboplatin, and having a high PCFT expression (both mRNA and protein), exhibit a longer overall survival upon treatment than patients with a low PCFT expression in their tumor. No correlation was found for RFC, but the combination of a low TS expression and a high PCFT expression provided the strongest predictive combination of biomarkers [65].

Since folates are anionic molecules, they need specific transporters for their cellular entry. Cellular extrusion of folates and antifolates can, in part, be also mediated by the RFC, which serves as a bidirectional carrier. However, the dominant efflux route is mediated by specific members of the multidrug resistance protein (MRP) family, in particular ABCC1-5 and ABCG2 [23, 24]. As part of cellular homeostasis (Figure 2), efflux of (anti)folates can be prevented by a cellular process called polyglutamylation, wherein the enzyme folylpolyglutamate synthetase (FPGS) adds up to six to seven glutamate residues to the molecules, leading to a polyanionic molecule that is retained in cells for a long period, even lasting weeks to months in some tissues. In a study described by Rick Moran (Richmond, VA, USA), the relative roles of cytosolic and mitochondrial localization of FPGS, species differences and tissue-specific promoters were explored [66]. Murine liver and kidney utilize two FPGS promoters (P1 and P2), whereas tumors and other normal tissues use just one [67, 68]. In contrast, human tissues and tumors have only one promoter, which is similar to the mouse P2. Expression of the two forms of FPGS in mouse (one in normal tissues and the other in the tumor and normal dividing tissues) is regulated by different transcription factors; the two forms also differ in feedback inhibition by polyglutamates, which are more efficient in proliferating tissues. One of the promoters in mice (P2) has a high density of CpG islands, and the other is CpG sparse; DNA methylation is a factor only for the CpG sparse P1 and does not play a role in tumor cell expression from P2. However, as mice do not show any difference in folate metabolism when P1 is knocked out, FPGS-P1 knock-outs can serve as a model for humans. These animals can be made available upon request. Dr. Moran also showed that the mitochondrial and cytosolic polyglutamate pools do not mix.

Another regulatory mechanism of FPGS expression is alternative splicing, which was summarized by Jacqueline Cloos (Amsterdam, The Netherlands). Earlier studies on FPGS showed a discrepancy between FPGS enzyme activity and gene expression, both in clinical samples [69, 70] and in MTX-resistant cell lines [71, 72], which could be explained by the presence of various spliced forms of FPGS [73]. In particular, exon 10 skipping was associated with decreased FPGS activity and, hence, resistance to antifolates. In childhood ALL, the frequency of a partial retention of intron 8 was observed rather frequently and appeared to be related to decreased FPGS activity and resistance to MTX in model systems. Also, in lung cancer samples, alternative splicing patterns were found, but they appeared distinct from leukemia, e.g., the intron 8 partial retention variant was not found. Thus, aberrant pre-mRNA splicing warrants an exploration in the context of antifolate resistance in leukemias and solid tumors [74].

Folate receptor targeting

The high expression of folate receptors in certain tumors and immune cells (see below) makes this receptor ideal for specific tumor targeting, e.g., by using FR-targeted antibody drug conjugates (ADC). Hence antibodies have been developed that bind to the receptor. Yinghui Zhou (Waltham, MA, USA) described an approach to direct these drug conjugates specifically to ovarian cancer and NSCLC. Dr. Zhou described the optimization of an ADC conjugate composed of an antibody, a cytotoxic agent [maytansine (DM-1), a potential anti-microtubule agent] and a disulfide linker that enables specific cleavage at the tumor site. A successful example is Kadcyla, a conjugate of trastuzumab (targeted against HER2) and DM-4 [75]. IMGN853 is being developed for ovarian and endometrial cancer, and is a conjugate of a humanized FRα antibody (M9436A) with optimal linker properties. It is now in clinical development with a companion diagnostic for detection and selection of patients with a FRα-positive tumor.

Daniel Powell (Philadelphia, PA, USA) presented an approach for FRβ targeting to develop specific treatment for acute myeloid leukemia blasts [76] with a chimeric antigen receptor (CAR) expressing T cells, similar to that previously described for FRα-targeted CAR T cells [77]. In this approach, isolated T cells are transduced with a single-chain fragment variable (scFv) anti-FRβ CAR-encoding lentivirus. Two FRβ-specific CARs were constructed and characterized, one with a humanized FRβ antibody (m909) [78] and the other with a higher affinity scFv variant (m923). Particularly, the latter construct had potent therapeutic activity against preclinical AML models with minimal toxicity.

Keith Knutson (Port Saint Lucie, FL, USA) presented an update of an ongoing alternative FR-targeted immunotherapeutic approach for ovarian and breast cancer, i.e., generation of T-cell immunity to the FRα protein after active immunization with a FRα peptide-based vaccine [79]. Multiple peptides were evaluated for immunization. Results showed that the vaccine was immunogenic and induced high levels of peripherally circulating FRα-specific T cells. Antibody responses to the peptides were minimal. Ongoing clinical trials revealed low-grade (1 and 2) toxicities with FRα vaccinations. Future directions are to make FRα peptides more immunogenic, modify vaccine formulations and/or combine them with checkpoint blockade chemotherapeutics. Additionally, FRα vaccinations will be explored for ovarian cancer prevention studies (phase II clinical trial).

Chris Leamon (Endocyte, Inc., West Lafayette, IN, USA) presented an overview of ongoing FR-based small-molecule drug conjugate (SMDC) developments at Endocyte. The company’s molecular design includes a high-affinity/small-size ligand (folic acid), a hydrophilic spacer, a releasable linker and a highly potent drug. A folic acid vinca alkaloid SMDC (EC145, vintafolide) displayed potent antitumor activity, and when used in combination with 99Tc-etarfolatide (EC20) SPECT scans, it could identify what tissues could accumulate [10, 80, 81]. Indeed, in a randomized phase II trial (PRECEDENT study) comparing vintafolide (EC145) and pegylated liposomal doxorubicin (PLD) in combination vs. PLD alone in patients with platinum-resistant ovarian cancer, vintafolide plus PLD demonstrated an improvement over standard therapy [82]. However, in a phase III confirmatory clinical trial (PROCEED), one of the primary end points of improved progression-free survival in patients with 100% FR-positive ovarian cancer was not met and accrual of patients was stopped. Possible contributing factors to this failure are currently under investigation. Another clinical trial (TARGET) of vintafolide plus docetaxel for the treatment of FR-positive NSCLC is currently ongoing. Initial results showed clinically meaningful improvement across all efficacy end points over single-agent docetaxel. Finally, an update was presented of ongoing studies with a folate-tubylisin SMDC (EC1456). EC1456 has potent activity against tumors with low FR expression, is not a substrate for the drug efflux transporter Pgp (unlike vinca alkoids) and retains activity against EC145-resistant tumors [83]. Moreover, EC1456 consistently produces greater tumor activity than EC145 in various tumor models, including multidrug-resistant ones. A phase I clinical trial with EC1456 was initiated in March 2014.

Antifolates in immune diseases

Macrophages play a central role in physiology and pathology. Dysregulation of their polarized state is associated with a broad spectrum of diseases [84]. M1-type macrophages producing pro-inflammatory mediators are thought to be the major drivers of chronic inflammatory diseases such as rheumatoid arthritis (RA). Conversely, in tumor microenvironment, M2-type macrophages produce immunosuppressive mediators that can promote tumor growth [85]. Similarly, myeloid-derived suppressor cells (MDSC) elicit factors that may enhance tumor growth [86]. The notion that activated macrophages and MDSC express functional FRβ makes them attractive targets for small-molecule (antifolate) targeting or immunotherapy approaches.

For many decades, the folate antagonist MTX is the anchor drug for RA treatment, but its exact mechanism of action remains elusive [87]. Gerrit Jansen (Amsterdam, The Netherlands) reported on the expression profiles of folate transporters and folate metabolic genes in blood cells of three groups; healthy controls, early-onset RA patients starting MTX treatments and RA patients clinically resistant to MTX. Interestingly, basal folate metabolism gene expression appeared up-regulated under chronic inflammatory conditions in early RA and MTX suppressed these up-regulated folate pathway genes. Moreover, clinical MTX resistance in RA was not associated with an impaired ability of MTX to inhibit the folate metabolic pathway [88]. This study also revealed targets in folate metabolism, e.g., GARFTase, being druggable by novel generation folate antagonists [89].

A study that aimed to predict MTX response and adverse effects in RA and JIA patients was presented by Robert de Jonge (Rotterdam, The Netherlands). With the use of a personalized-medicine approach, genetic variations (SNPs) and metabolites (folate, MTX-polyglutamates) within the MTX pathway (one-carbon fingerprint) were analyzed to predict the outcome of MTX treatment in RA. Beyond clinical parameters (body mass index, smoking) and baseline disease severity, genetic factors that were associated with unresponsiveness to MTX included SNPs in the efflux transporters ABCB1 (rs1045642 G>A) and ABCC3 (rs4793665 T>C), and methionine synthesis (MTRR, rs1801394 A>G) [90, 91]. Furthermore, low baseline erythrocyte folate and erythrocyte MTX-polyglutamate levels after 3 months of treatment were predictive for a decreased MTX response [92]. None of these biomarkers was associated with adverse effects of MTX treatment.

Amaya Puig-Krüger (Madrid, Spain) addressed the topic of macrophage polarization in RA based on initial observations in her laboratory that FRβ expression appeared more restricted to M2-type (anti-inflammatory/tumor-promoting) macrophages than to M1-type (pro-inflammatory/tumor-suppressor) macrophages [93]. Ex vivo isolated RA synovial fluid CD163+ macrophages were almost devoid of FRβ but stained positive for the folate transporter RFC, whose expression marks pro-inflammatory macrophages in vivo including RA synovial tissue and ulcerative colitis [94]. Interestingly, the addition of MTX to human CD14+ monocytes had a greater impact (increased expression of CCL20) on M1-type skewing than on M2-type skewing. The latter appeared associated with a marked increase in TS expression in GM-CSF skewed macrophages compared to M-CSF skewed macrophages. These data suggest that MTX uptake via the classical folate transporter RFC, differentially expressed in M1-type macrophages [94], is functionally more efficient than FRβ in M2-type macrophages.

Taku Nagai (Kagoshima City, Japan) described the immunohistochemical staining of the synovial tissue of RA and osteoarthritis (OA) patients [95]. FRβ-positive macrophages were identified in the synovial lining and sublining of RA and OA patients. In a rat arthritis model, FRβ-positive macrophages could be targeted with FRβ antibody-conjugated immunotoxins, leading to reduced joint swelling [96]. Beyond this, these immunotoxins also had a tumor-suppressive effect in a rat C6 glioma tumor xenograft model [97]. Recently, they developed a monoclonal antibody (anti-FRαβ) reactive to both the FRα and the FRβ isoform. This antibody reacted with both FRα-positive tumor cells and FRβ-positive tumor-associated macrophages (TAMs), paving the way for new immunotherapeutic approaches. Notably, anti-FRαβ displayed complement- and antibody-dependent cytotoxicity to FRα- and FRβ-positive cells. Likewise, anti-FRαβ-based immunotoxins demonstrated cytotoxic effects.

June Lu (Endocyte, Inc., West Lafayette, IN, USA) reported on small-molecule approaches of exploiting functional FRβ for targeting tumor-associated macrophages. Preliminary data with a novel folate-conjugated DNA alkylating agent showed promising selectivity of targeting and inducing apoptosis in TAMs in a breast cancer xenograft model. This concept deserves further exploration among other anti-TAM strategies that are currently being investigated [98].

Timothy Ratliff (West Lafayette, IN, USA) characterized FRβ expression on MDSC, a myeloid subset capable of suppressing T-cell activity and inhibiting anti-tumor immunity or regulating autoimmunity/inflammation. In mouse LyC6 monocytic MDSC [99], suppressor activity was fully confined to FRβ-positive cells as judged by elevated iNOS and arginase expression and increased NO production. Of interest, suppression activity was increased under hypoxic over normoxic condition and could also be modulated by extracellular folate levels, i.e., folate depletion attenuated the suppressive activity of FRβ expression in LyC6 MDSC by down-regulating iNOS function and NO production under hypoxic conditions.

Philip Low presented an overview of FRβ expression on activated macrophages in tumor tissues (lung) and tissues related to (chronic) inflammatory diseases (e.g., RA, lupus, psoriasis) with folate-conjugated fluorescent probes and a humanized FRβ antibody (m909) [78]. Furthermore, detailed analysis of peripheral blood monocyte subfractions revealed that functional (folate-fluorescein binding) FRβ was present on pro-inflammatory CD14high/CD16low cells [100]. Hence, FRβ targeting may allow the prevention of infiltration of these cells at inflammatory sites.

Folate-receptor imaging

Beyond therapeutic targeting of FRs by small-molecule drugs or anti-FR immunoconjugates, the receptor is also highly suitable for imaging modalities in disease monitoring and guided surgery.

Optical imaging-guided surgery with folate-conjugated fluorescent probes has proven its value in identifying FR-positive ovarian carcinoma lesions [30] or lung cancer [31].

Alex Vahrmeijer (Leiden, The Netherlands) presented new developments in these optical imaging techniques by using novel folate-conjugated fluorescent probes in the infrared wavelength range [101, 102], which are currently being tested in surgical practice.

Qingshou Chen (West Lafayette, IN, USA) reported on the synthesis of a novel FR-targeted positron emission tomography (PET) imaging agent (folate-PEG-NOTA-Al-18F) with potential for applications in cancer and autoimmune diseases. This PET tracer could be rapidly synthesized (<45 min) with good radiochemical yield (40–50%) and high specific activity. Micro-PET imaging studies showed high and specific uptake of this tracer in KB (FRα+) mice tumor models. Dosimetry evaluations indicated that the tracer is suitable for repeated imaging of patients.

Conny van der Laken (Amsterdam, The Netherlands) reviewed the current (macrophage-targeted) PET tracers that are presently being evaluated for disease monitoring in RA [103]. A major challenge is to monitor early-onset arthritis as well as therapy monitoring with non-invasive PET. Van der Laken reported on the preclinical characterization of an FR-targeted PET tracer, [18F]-PEG-folate, in an arthritic rat model [104, 105]. [18F]PEG-folate imaging demonstrated good and specific visualization of inflamed joints in arthritic rats and had a lower background uptake than other common macrophage tracers, e.g., [11C]PK11195. [18F]-PEG-folate is currently being processed for clinical evaluation in RA patients.

Christina Müller (PSI, Villigen, Switzerland) presented a comprehensive overview of the development and optimization of FR-targeted radionuclides for cancer treatment [11]. Specific issues that were addressed included the prevention of undesired radio-nephrotoxicity due to targeting of kidney FR. For a conventional FR-targeted radioconjugate (e.g., 177Lu-EC0800), this could be achieved by blocking FR-mediated radiofolate uptake via pre-exposure to the antifolate PMX [106]. A good alternative approach appeared to be 177Lu-DOTA-folate conjugate (cm09) with an albumin binding entity, which showed an almost 10-fold better tumor/kidney ratio than 177Lu-EC0800 owing to the enhanced blood circulation time [107]. Recent studies also showed that the albumin-binding folate conjugate labeled with novel radionuclides such as the “matched pair” 44Sc/47Sc holds great promise for both PET imaging (44Sc-folate) and radionuclide therapy (47Sc-folate) of FR-positive cancers [108].

Poster presentations

Poster presentations form an integral part of FRS meetings. They provide an informal international forum to present data and often lead to intensive discussion. Posters were presented by various (mostly young) investigators and were discussed intensively during breaks and the separate poster sessions. The subjects of the poster followed the general outline of the meeting.

Anna Wojtuszkiewicz (Amsterdam, The Netherlands) described how impaired FPGS mRNA splicing is responsible for the loss of FPGS activity and might be a relevant MTX resistance mechanism in vivo. Moreover, novel strategies were described on how to interfere with aberrant splicing that may restore sensitivity to MTX.

Erik Meijer (Amsterdam, The Netherlands) performed a proteome analysis on colon tumor samples from patients taken 48 h after receiving a standard dose of 5FU, with or without leucovorin. This revealed changes in a number of previously unidentified proteins, such as an increase in the isoform NELF-D of negative elongation factor C (NELFCD), AMP deaminase 3 (AMPD) and myeloblastin (PRTN3), and a decrease in neudesin (NENF), antigen KI-67 (MKI67) and HERC4. These proteins may function as biomarkers for treatment decision making in the future.

Mike Wilson and Zhanjun Hou (Detroit, MI, USA) presented two back-to-back posters describing the mRNA expression of RFC, PCFT and FRα, and the intracellular targets for PMX (TS and GARFTase) in normal lung and lung tumors, and in ovarian cancer. Normal lung and lung tumors show a comparable expression for PCFT, whereas RFC and FRα were decreased in lung tumor compared to normal lung tissue, and both TS and GARFTase were increased in lung tumors. RFC, PCFT and FRα were all expressed in ovarian cancer, with FRα increasing with stage, whereas PCFT remained constant. Normal and engineered cell lines from both tumor types were used to test the cell growth inhibitory potency of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates, and it was concluded that these compounds may offer significant promise for treating NSCLC and ovarian cancer, reflecting their selective membrane transport by PCFT (and FRα in ovarian cancer) over RFC and inhibition of de novo purine biosynthesis.

Gaetano Marverti (Ferrara, Italy) described the development of a bioconjugate compound with folic acid named FA-LR, made from folic acid and a lead candidate that inhibits hTS (the LR peptide). This conjugate can specifically bind the FRα on ovarian cancer cells and can thus be internalized. Ovarian cancer cell lines expressing high/moderate FRα levels, such as IGROV-1 and OAW28, proved to be the best candidates for targeting studies with the bioconjugate.

Joseph Reddy (West Lafayette, IN, USA) described the preclinical anti-tumor activity of EC1456 and its tolerability. Preliminary results in the ongoing phase I clinical evaluation showed a similar tendency.

Iontcho Vlahov (West Lafayette, IN, USA) described how antifolates can be used as FR-targeting ligands for delivering cytotoxic agents and, at the same time, as therapeutic agents within a conjugate construct

Davide Bernareggi (Milan, Italy) described the development of immunocompetent mice models as a novel approach to study anti-cancer αFR reagents.

Chantal Scheepshouwer (Amsterdam, The Netherlands) described the use of optical imaging to visualize tumors. Bioluminescence, high-frequency ultrasound and photo-acoustic oxygen-saturation imaging have complementing features and can provide an accurate, noninvasive visualization of the functional and molecular characteristics of bladder tumors, which is essential for the evaluation of new potential therapeutics.

Shirley Albano-Aluquin (Hershey, PA, USA) reported that activated macrophages in giant cell arteritis selectively expressed folate β receptors. Further quantification of these receptors and their antifolate-binding properties in future studies can guide targeted therapy with antifolates.

Durga Chandrupatla (Amsterdam, The Netherlands) described the development and optimization of a rat model of RA, which was validated for prolonged articular inflammation to widen the therapeutic window for the evaluation of novel therapeutic agents and response monitoring by (folate-based) PET tracers.

Concluding remarks

Philip Low thanked the speakers and participants for their constructive and inspiring contributions by presenting their most recent results in basic, translational and clinical research on rolate receptor and transporter area. Despite the impressive progress, obviously there are things left to be done. Learning from this symposium, we present the following agenda and directions for future research:

  • Better understanding of FR-α, β, γ and δ functions and regulation of their gene expression

  • Better understanding of folate transporters at the plasma membrane and cell organelle levels

  • Better FR-targeted therapeutics including drugs, antibodies, CAR T cells and CAR NK cells

  • Characterization of FR signaling and trafficking pathways

  • Development of FR isotype-specific ligands

  • Better characterization of FR-β involvement in autoimmune, inflammatory and malignant diseases.

  • Optimization of folate-based PET and optical imaging ligands for diagnosis and selection of patients

  • Immunohistochemistry for transporters and receptors to select patients for FR-targeting drugs

  • Targeting of FR, RFC and PCFT in pathogens

Acknowledgments

The authors appreciate the help and input of the board of the Folate Receptor Society with the organization of the meeting and the design of the program. The board includes the president, Philip S. Low, Ph.D., the executive secretary, Manohar Ratnam, Ph.D., and the following FRS board members: Yehuda G. Assaraf, Ph.D., Silvana Canevari, Ph.D., Mariangela Figini, Ph.D., I. David Goldman, M.D., Ann Jackman, Ph.D., Gerrit Jansen, Ph.D., Chris Leamon, Ph.D., Larry Matherly, Ph.D., and Takami Matsuyama, MD. Saskia Broekman is acknowledged for excellent secretarial assistance. The meeting would not have been possible without the generous support of Endocyte, Inc., Visual Sonics/Fuji Film, Immunogen, Inc., Eli Lilly & Co., Barbara Ann Karmanos Cancer Center, The Folate Receptor Society and the Royal Netherlands Academy of Arts and Sciences. The venue for the 6th International Symposium of Folate Receptors and Transporters is scheduled for September 2016 in the Rocky Mountains area, Colorado, USA.

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Article note

This paper is a report on the 5th International Symposium on Folate Receptors and Transporters (Heemskerk, The Netherlands, September 30–October 4, 2014); http://folatesymposium2014.wix.com/5thfolatesymposium#!

About the article

Corresponding author: Godefridus J. Peters, Department of Medical Oncology, VU University Medical Center, Cancer Center Amsterdam, PO Box 7057, 1007 MB Amsterdam, The Netherlands, E-mail:


Received: 2015-03-05

Accepted: 2015-03-12

Published Online: 2015-04-21

Published in Print: 2015-06-01


Citation Information: Pteridines, Volume 26, Issue 2, Pages 41–53, ISSN (Online) 2195-4720, ISSN (Print) 0933-4807, DOI: https://doi.org/10.1515/pterid-2015-0005.

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