Solute Carrier Transporters Research Articles

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Solute carrier nutrient transporters in rheumatoid arthritis fibroblast-like synoviocytes.

Metabolomic studies show that rheumatoid arthritis (RA) is associated with metabolic disruption. Metabolic changes in fibroblast-like synoviocytes (FLS) likely contribute to FLS abnormal response and strongly contribute to joint destruction. These changes often involve increased expression of nutrient transporters to meet a high demand for energy or biomolecules. The solute carrier (SLC) transporter families are nutrient transporters and serve as 'metabolic gates' for cells by mediating the transport of several different nutrients such as glucose, amino acids, vitamins, neurotransmitters, and inorganic/metal ions. In RA FLS SLC-mediated transmembrane transport was one pathway associated with different epigenetic landscape between RA and osteoarthritis (OA) FLS. These highlight that transporters from the SLC family offer unique targets for further research and offer the promise of future therapeutic targets for RA.

Blood-Brain Barrier Solute Carrier Transporters and Motor Neuron Disease.

Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood–brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC50) analysis of [3H]choline uptake indicated that choline is sensitive in TR-BBB cells, whereas amiloride is most sensitive in ALS cell lines. Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.

Whole-Exome Sequencing Revealed New Candidate Genes for Human Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a complex disease affecting young adults. It is a pathological condition impairing myocardium activity that leads to heart failure and, in the most severe cases, transplantation, which is currently the only possible therapy for the disease. DCM can be attributed to many genetic determinants interacting with environmental factors, resulting in a highly variable phenotype. Due to this complexity, the early identification of causative gene mutations is an important goal to provide a genetic diagnosis, implement pre-symptomatic interventions, and predict prognosis. The advent of next-generation sequencing (NGS) has opened a new path for mutation screening, and exome sequencing provides a promising approach for identifying causal variants in known genes and novel disease-associated candidates. We analyzed the whole-exome sequencing (WES) of 15 patients affected by DCM without overloading (hypertension, valvular, or congenital heart disease) or chronic ischemic conditions. We identified 70 pathogenic or likely pathogenic variants and 1240 variants of uncertain clinical significance. Gene ontology enrichment analysis was performed to assess the potential connections between affected genes and biological or molecular function, identifying genes directly related to extracellular matrix organization, transcellular movement through the solute carrier and ATP-binding cassette transporter, and vitamin B12 metabolism. We found variants in genes implicated to a different extent in cardiac function that may represent new players in the complex genetic scenario of DCM.

Identification of neurotoxic compounds in cyanobacteria exudate mixtures

Release of toxic cyanobacterial secondary metabolites threatens biosecurity, foodwebs and public health. Microcystis aeruginosa (Ma), the dominant species in global freshwater cyanobacterial blooms, produces exudates (MaE) that cause adverse outcomes including nerve damage. Previously, we identified > 300 chemicals in MaE. It is critical to investigate neurotoxicity mechanisms of active substances among this suite of Ma compounds. Here, we screened 103 neurotoxicity assays from the ToxCast database to reveal targets of action of MaE using machine learning. We then built a potential Adverse Outcome Pathway (AOP) to identify neurotoxicity mechanisms of MaE as well as key targets. Finally, we selected potential neurotoxins matched with those targets using molecular docking. We found 38 targets that were inhibited and eight targets that were activated, collectively mainly related to neurotransmission (i.e. cholinergic, dopaminergic and serotonergic neurotransmitter systems). The potential AOP of MaE neurotoxicity could be caused by blocking calcium voltage-gated channel (CACNA1A), because of antagonizing neurotransmitter receptors, or because of inhibiting solute carrier transporters. We identified nine neurotoxic MaE compounds with high affinity to those targets, including LysoPC(16:0), 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, egonol glucoside, polyoxyethylene (600) monoricinoleate, and phytosphingosine. Our study enhances understanding of neurotoxicity mechanisms and identifies neurotoxins in cyanobacterial bloom exudates, which may help identify priority compounds for cyanobacteria management.

Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.

Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathwaybut inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.

Gain-of-function genetic screens in human cells identify SLC transporters overcoming environmental nutrient restrictions.

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of SLCs in response to environmental stresses remain poorly elucidated. To infer functional relationships between SLCs and metabolites, we developed a strategy to identify SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from growth-limiting metabolic bottlenecks. Among the transporters identified, we characterized the cationic amino acid transporter SLC7A3 as a gene that, when up-regulated, overcame low availability of arginine and lysine by increasing their uptake, whereas SLC7A5 was able to sustain cellular fitness upon deprivation of several neutral amino acids. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells uncovered functional transporter-nutrient relationships and revealed that transport activity up-regulation may be sufficient to overcome environmental metabolic restrictions.

GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis.

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

Genetic Variants of ABC and SLC Transporter Genes and Chronic Myeloid Leukaemia: Impact on Susceptibility and Prognosis.

Solute carrier (SLC) and ATP-binding cassette (ABC) transporters comprise a variety of proteins expressed on cell membranes responsible for intrusion or extrusion of substrates, respectively, including nutrients, xenobiotics, and chemotherapeutic agents. These transporters mediate the cellular disposition of tyrosine kinase inhibitors (TKIs), and their genetic variants could affect its function, potentially predisposing patients to chronic myeloid leukaemia (CML) and modulating treatment response. We explored the impact of genetic variability (single nucleotide variants—SNVs) of drug transporter genes (ABCB1, ABCG2, SLC22A1, and SLC22A5) on CML susceptibility, drug response, and BCR-ABL1 mutation status. We genotyped 10 SNVs by tetra-primers-AMRS-PCR in 198 CML patients and 404 controls, and assessed their role in CML susceptibility and prognosis. We identified five SNVs associated with CML predisposition, with some variants increasing disease risk, including TT genotype ABCB1 (rs1045642), and others showing a protective effect (GG genotype SLC22A5 rs274558). We also observed different haplotypes and genotypic profiles associated with CML predisposition. Relating to drug response impact, we found that CML patients with the CC genotype (rs2231142 ABCG2) had an increased risk of TKI resistance (six-fold). Additionally, CML patients carrying the CG genotype (rs683369 SLC22A1) presented a 4.54-fold higher risk of BCR-ABL1 mutations. Our results suggest that drug transporters’ SNVs might be involved in CML susceptibility and TKI response, and predict the risk of BCR-ABL1 mutations, highlighting the impact that SNVs could have in therapeutic selection.

The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells.

Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1β production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1β production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1β levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.

Structural basis for proton coupled cystine transport by cystinosin

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

Reimagining the Framework Supporting the Static Analysis of Transporter Drug Interaction Risk; Integrated Use of Biomarkers to Generate Pan-Transporter Inhibition Signatures.

Solute carrier (SLC) transporters present as the loci of important drug-drug interactions (DDIs). Therefore, sponsors generate in vitro half-maximal inhibitory concentration (IC50 ) data and apply regulatory agency-guided "static" methods to assess DDI risk and the need for a formal clinical DDI study. Because such methods are conservative and high false-positive rates are likely (e.g., DDI study triggered when liver SLC R value ≥ 1.04 and renal SLC maximal unbound plasma (Cmax,u )/IC50 ratio ≥ 0.02), investigators have attempted to deploy plasma- and urine-based SLC biomarkers in phase I studies to de-risk DDI and obviate the need for drug probe-based studies. In this regard, it was possible to generate in-house in vitro SLC IC50 data for various clinically (biomarker)-qualified perpetrator drugs, under standard assay conditions, and then estimate "% inhibition" for each SLC and relate it empirically to published clinical biomarker data (area under the plasma concentration vs. time curve (AUC) ratio (AUCR, AUCinhibitor /AUCreference ) and % decrease in renal clearance (ΔCLrenal )). After such a "calibration" exercise, it was determined that only compounds with high R values (> 1.5) and Cmax,u /IC50 ratios (> 0.5) are likely to significantly modulate liver (AUCR > 1.25) and renal (ΔCLrenal > 25%) biomarkers and evoke DDI risk. The % inhibition approach supports integration of liver and renal SLC data and allows one to generate pan-SLC inhibition signatures for different test perpetrators (e.g., SLC % inhibition ranking). In turn, such signatures can guide the selection of the most appropriate individual (or combinations of) biomarkers for testing in phase I studies.

Abstract P3109: Identification Of Drug Transporter Genomic Variants And Inhibitors That Protect Against Doxorubicin Induced Cardiotoxicity

Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. To answer these questions, six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human-induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. We demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. The role of SLC28A3 in DIC was then confirmed using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes. To identify the potential causal variant, Nanopore-based fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing. An extended in silico haplotype and functional analysis followed by base editing, identified a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus. Next, using high-throughput drug screening of 1200 drugs in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. Finally, using eight cancer cell lines, we demonstrate that desipramine does not impede doxorubicin cytotoxicity.

Systematic in silico discovery of novel solute carrier-like proteins from proteomes

Solute carrier (SLC) proteins represent the largest superfamily of transmembrane transporters. While many of them play key biological roles, their systematic analysis has been hampered by their functional and structural heterogeneity. Based on available nomenclature systems, we hypothesized that many as yet unidentified SLC transporters exist in the human genome, which await further systematic analysis. Here, we present criteria for defining “SLC-likeness” to curate a set of “SLC-like” protein families from the Transporter Classification Database (TCDB) and Protein families (Pfam) databases. Computational sequence similarity searches surprisingly identified ~120 more proteins in human with potential SLC-like properties compared to previous annotations. Interestingly, several of these have documented transport activity in the scientific literature. To complete the overview of the “SLC-ome”, we present an algorithm to classify SLC-like proteins into protein families, investigating their known functions and evolutionary relationships to similar proteins from 6 other clinically relevant experimental organisms, and pinpoint structural orphans. We envision that our work will serve as a stepping stone for future studies of the biological function and the identification of the natural substrates of the many under-explored SLC transporters, as well as for the development of new therapeutic applications, including strategies for personalized medicine and drug delivery.

Polymorphisms in solute carrier genes (SLC19A1, SLCO1B1, and SLCO1B3) predicts survival and toxicity in North Indian lung cancer patients undergoing platinum-based doublet chemotherapy.

Solute Carrier (SLC) transporters are known mediators of drug disposition that facilitate the influx of substrates and various chemotherapeutic agents into cells. Polymorphisms in the SLC19A1, SLCO1B1, and SLCO1B3 gene influence the prognosis in the cancer patients, but little is known about their role in lung cancer in Asians. So, the current study aims to investigate the polymorphisms in SLC19A1, SLCO1B1, and SLCO1B3 genes in Northern Indian lung cancer patients. Patients with lung cancer who had a confirmed histology and cytology diagnosis were enrolled in the study. SLC polymorphisms were assessed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) for variations in SLC19A1 (G80 A), SLCO1B1 (A388 G, T521 C), and SLCO1B3 (A1683-5676 G). Our results showed that mutant genotype for SLC19A1 G80 A polymorphism had higher median survival time (MST) compared to wild genotype. ADCC patients with mutant genotype showed better survival compared to wild genotype for SLC19A1 G80 A. SCLC patients G80 A polymorphism showed increased survival in patients with mutant genotype (p=0.04). In SLCO1B3, A1683-5676 G patients carrying heterozygous alleles and administered with platinum and docetaxel showed inferior survival (p=0.006). In T521 C variant, patients with carrier genotype had reduced chances of developing anaemia (p=0.04). Patients with SLC19A1 and SLCO1B3 variants showed lower incidence of thrombocytopenia and nephrotoxicity. Our findings imply that Solute Carrier gene polymorphisms modulate the overall survival in lung cancer patients undergoing platin-based doublet chemotherapy, also these polymorphisms have a modifying impact on the associated adverse events/toxicity.

Transport Mechanisms at the Blood-Brain Barrier and in Cellular Compartments of the Neurovascular Unit: Focus on CNS Delivery of Small Molecule Drugs.

Ischemic stroke is a primary origin of morbidity and mortality in the United States and around the world. Indeed, several research projects have attempted to discover new drugs or repurpose existing therapeutics to advance stroke pharmacotherapy. Many of these preclinical stroke studies have reported positive results for neuroprotective agents; however, only one compound (3K3A-activated protein C (3K3A-APC)) has advanced to Phase III clinical trial evaluation. One reason for these many failures is the lack of consideration of transport mechanisms at the blood–brain barrier (BBB) and neurovascular unit (NVU). These endogenous transport processes function as a “gateway” that is a primary determinant of efficacious brain concentrations for centrally acting drugs. Despite the knowledge that some neuroprotective agents (i.e., statins and memantine) are substrates for these endogenous BBB transporters, preclinical stroke studies have largely ignored the role of transporters in CNS drug disposition. Here, we review the current knowledge on specific BBB transporters that either limit drug uptake into the brain (i.e., ATP-binding cassette (ABC) transporters) or can be targeted for optimized drug delivery (i.e., solute carrier (SLC) transporters). Additionally, we highlight the current knowledge on transporter expression in astrocytes, microglia, pericytes, and neurons with an emphasis on transport mechanisms in these cell types that can influence drug distribution within the brain.

Abstract 1843: Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB)

Abstract Glucose transporters at the blood brain barrier (BBB) maintain the continuous high glucose and energy demands of the brain. These transporters have been targets for the creation of therapeutic molecules as a means to cross the BBB. The BBB has been a major limitation for the successful development of treatments for brain tumors. To date, only a limited number of lipophilic small chemical entities, which can cross the BBB, have been approved for the treatment of GBM. These include the nitrosoureas such as carmustine (BCNU) and lomustine (CCNU) and temozolomide (TMZ). VAL-083 is a di-epoxide substituted hexitol sugar (galactitol). It has a molecular weight of 146 and is highly water soluble. VAL-083 is active against several GBM cell lines and animal models and acts as a DNA targeting agent rapidly forming cross-links at the N7 position of guanine, resulting in lethal DNA double strand breaks and cancer cell death. Its is active in GBM patients, both newly diagnosed and recurrent. Furthermore, its activity is independent of the DNA repair enzyme MGMT which limits the effectiveness of O6 guanine alkylating agents such as the nitrosoureas and TMZ.In clinical pharmacology studies, VAL-083 rapidly accumulates in the cerebrospinal fluid (CSF) and brain tumor tissue. Studies with radiolabeled VAL-083 have shown a half-life of ~ 20 h in CSF vs 2h for plasma post-IV infusion. Recent clinical studies in newly diagnosed GBM patients demonstrated levels of VAL-083 in CSF at least as high as in plasma two hours post infusion (ratio 1.24 ± 0.35). In comparison, peak TMZ concentrations in the CSF are ~30% of that of the plasma.Due to its structural similarity with glucose, we have initiated in vitro glucose transporter studies to identify if SGLTs (sodium dependent glucose cotransporters) or GLUTs (glucose transporters) are contributing to brain penetration. Our initial studies evaluated VAL-083 as a substrate of the human SGLT1 and SGLT2 SLC (solute carrier) transporters. HEK293 cells expressing either the SGLT1 or SGLT2 transporter were exposed to VAL-083 at 3 and 15 µM for 2 and 20 min. The probe substrate methyl-α-D- glucopyranoside (AMG) was used as a positive control, as well as comparison against the reference inhibitors phloridzin dihydrate (100 µM, 20 min) for SGLT1 and dapagliflozin (0.3 µM, 10 min) for SGLT2. Surprisingly, no transporter-specific accumulation of VAL-083 by SGLT1 or SGLT2 was observed. Ongoing studies in CaCo-2 cells are investigating whether VAL-083 is transported via other glucose transporters including GLUT1. GLUT1 is expressed on erythrocytes and the endothelial cells of the BBB and may be highly relevant to the uptake of VAL-083 into brain tissue. The results of these studies will be reported at the meeting. Citation Format: John M. Langlands, Sarath Kanekal, Anne Steino, Armando Mendez, Zoltan Karman, Beata Martonne Toth, Krisztina Heredi-Szabo, Dennis Brown. Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1843.

Abstract 5358: Involvement of solute carrier transporters SLC25A17 and SLC27A6 in metabolic reprograming of castration-resistant prostate cancer

Abstract Enzalutamide (XTANDI®), an antiandrogen, is used for the treatment of advance-stage prostatecancer. Approximately, 60% of patients receiving enzalutamide show initial remission followedby disease relapse with the emergence of highly aggressive castration-resistant prostate cancer.Solute carrier (SLC) transporters play a critical role in cancer drug resistance by altering cellularmetabolism. We have recently reported the predominance of SLC25A17 and SLC27A6 duringenzalutamide resistance (Cells 9(12):2535, 2020) by generating enzalutamide resistant (C4-2B-Enzu) cells exposing human prostate cancer C4-2B cells in the presence of increasingconcentration of enzalutamide (1-20 µM) and maintained in the same condition for six months.However, the role of these molecules during metabolic reprogramming have not beenelucidated. Knockdown of SLC25A17 and SLC27A6 gene by sgRNA transfection suppressedcell proliferation, and migration in C4-2B-Enzu cells. Analysis of cell cycle showed that in theabsence of SLC25A17 and SLC27A6 there is induction of G1/S cell cycle arrest and reductionin the protein expression of checkpoint factors viz. Cyclin D1, CDK6, CDK4, and CDK2.SLC25A17 and SLC27A6 knockdown resulted in downregulation of acetyl-CoA carboxylase(ACC), fatty acid synthase (FASN) and Acyl-CoA synthetase long chain family member 5(ACSL5) with simultaneous decrease in the levels of lactic acid, triglyceride and citric acid in C4-2B-Enzu cells. In addition, SLC25A17 knockdown resulted in marked increase in apoptosis asevident by increase in protein expression of RIPK3 and BAX, compared to SLC27A6 knockdownin C4-2B-Enzu cells. Taken together, our results demonstrate that solute carriers SLC25A17and SLC27A6 play an important role in the development of enzalutamide resistance throughmetabolic reprogramming and loss of these molecules results in cell death. Thus, SLC25A17and SLC27A6 may serve as a therapeutic target for enzalutamide resistant prostate cancer. Citation Format: Prem P. Kushwaha, Shiv S. Verma, Eswar Shankar, Spencer Lin, Sanjay Gupta. Involvement of solute carrier transporters SLC25A17 and SLC27A6 in metabolic reprograming of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5358.

Transporters and Toxicity: Insights From the International Transporter Consortium Workshop 4.

Over the last decade, significant progress been made in elucidating the role of membrane transporters in altering drug disposition, with important toxicological consequences due to changes in localized concentrations of compounds. The topic of "Transporters and Toxicity" was recently highlighted as a scientific session at the International Transporter Consortium (ITC) Workshop 4 in 2021. The current white paper is not intended to be an extensive review on the topic of transporters and toxicity but an opportunity to highlight aspects of the role of transporters in various toxicities with clinically relevant implications as covered during the session. This includes a review of the role of solute carrier transporters in anticancer drug-induced organ injury, transporters as key players in organ barrier function, and the role of transporters in metal/metalloid toxicity.