OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia.

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.

T cell-selective deletion of Oct1 protects animals from autoimmune neuroinflammation while maintaining neurotropic pathogen response.

BACKGROUND: Treatments for autoimmune diseases aim to dampen autoreactivity while preserving normal immune function. In CD4+ T cells, the transcription factor Oct1/Pou2f1 is a dispensable transcription factor for T cell development and response to primary infection, but promotes expression of target genes, including Il2 and Ifng, under conditions of antigen reencounter. As a result, they are more strongly expressed upon secondary stimulation. Such repeated antigen encounters occur in memory recall responses, in autoimmunity where self-antigen can be recognized multiple times, and in chronic infection where foreign antigen is persistent. Based on these previous findings, we hypothesized that Oct1 loss would protect animals from autoimmunity but maintain normal responses to pathogens in the CNS. OBJECTIVE: We used a conditional mouse Oct1 (Pou2f1) allele and a CD4-Cre driver to determine the effect of T cell-specific Oct1 loss on autoimmune- and viral-induced neuroinflammation using an autoantigen-driven EAE model of autoimmunity and a JHMV model of viral infection. RESULTS: Oct1 conditional deletion mitigated clinical scores and reduced infiltrating T cells and cytokine production in the EAE model. Consistently, Oct1-deficient CD4+ T cells stimulated in vitro showed increased expression of markers associated with T cell anergy, particularly in the absence of co-stimulatory signals. In contrast, anti-viral T cell effector functions are intact in the absence of Oct1, with no changes in neuroinflammation, infiltrating T cells or cytokine production. CONCLUSION: Our findings uncover a significant difference between the effect of Oct1 loss on autoimmune and anti-pathogen responses, which potentially could be exploited for therapeutic benefit.

Oct1/Pou2f1 is selectively required for colon regeneration and regulates colon malignancy.

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to establish organoids in vitro, but blocked the ability to recover from treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss-of-heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with differentially expressed genes between the two models. Oct1 ChIPseq using HCT116 colon carcinoma cells identifies target genes associated with mitotic stability, metabolism, stress response and malignancy. This set of gene targets overlaps significantly with genes differentially expressed in the two tumor models. These results reveal that Oct1 is selectively required for recovery after colon damage, and that Oct1 has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology.

OCT1 Deficiency Affects Hepatocellular Concentrations and Pharmacokinetics of Cycloguanil, the Active Metabolite of the Antimalarial Drug Proguanil.

Cycloguanil, the active metabolite of proguanil, acts on malaria schizonts in erythrocytes and hepatocytes. We analyzed the impact of the organic cation transporter OCT1 on hepatocellular uptake and pharmacokinetics of proguanil and cycloguanil. OCT1 transported both proguanil and cycloguanil. Common variants OCT1*3 and OCT1*4 caused a substantial decrease and OCT1*5 and OCT1*6 complete abolishment of proguanil uptake. In 39 healthy subjects, low-activity variants OCT1*3 and OCT1*4 had only minor effects on proguanil pharmacokinetics. However, both, cycloguanil area under the time-concentration curve and the cycloguanil-to-proguanil ratio were significantly dependent on number of these low-functional alleles (P = 0.02 for both). Together, CYP2C19, CYP3A5, OCT1 polymorphisms, and sex accounted for 61% of the variation in the cycloguanil-to-proguanil ratio. Most importantly, in vitro OCT1 inhibition caused a fivefold decrease of intracellular cycloguanil concentrations in primary human hepatocytes. In conclusion, OCT1-mediated uptake is a limiting step in bioactivation of proguanil, and OCT1 polymorphisms may affect proguanil efficacy against hepatic malaria schizonts.

Organic cation transporter 1 (OCT1) modulates multiple cardiometabolic traits through effects on hepatic thiamine content.

A constellation of metabolic disorders, including obesity, dysregulated lipids, and elevations in blood glucose levels, has been associated with cardiovascular disease and diabetes. Analysis of data from recently published genome-wide association studies (GWAS) demonstrated that reduced-function polymorphisms in the organic cation transporter, OCT1 (SLC22A1), are significantly associated with higher total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels and an increased risk for type 2 diabetes mellitus, yet the mechanism linking OCT1 to these metabolic traits remains puzzling. Here, we show that OCT1, widely characterized as a drug transporter, plays a key role in modulating hepatic glucose and lipid metabolism, potentially by mediating thiamine (vitamin B1) uptake and hence its levels in the liver. Deletion of Oct1 in mice resulted in reduced activity of thiamine-dependent enzymes, including pyruvate dehydrogenase (PDH), which disrupted the hepatic glucose-fatty acid cycle and shifted the source of energy production from glucose to fatty acids, leading to a reduction in glucose utilization, increased gluconeogenesis, and altered lipid metabolism. In turn, these effects resulted in increased total body adiposity and systemic levels of glucose and lipids. Importantly, wild-type mice on thiamine deficient diets (TDs) exhibited impaired glucose metabolism that phenocopied Oct1 deficient mice. Collectively, our study reveals a critical role of hepatic thiamine deficiency through OCT1 deficiency in promoting the metabolic inflexibility that leads to the pathogenesis of cardiometabolic disease.

Increased Systemic Exposure and Stronger Cardiovascular and Metabolic Adverse Reactions to Fenoterol in Individuals with Heritable OCT1 Deficiency.

Fenoterol is a widely used anti-asthmatic and tocolytic agent, but high plasma concentrations of fenoterol may lead to severe and even fatal adverse reactions. We studied whether heritable deficiency of the liver organic cation transporter 1 (OCT1), a trait observed in 3% of Europeans and white Americans, affects fenoterol plasma concentrations and toxicity. OCT1 transported fenoterol with high affinity, and OCT1 inhibition in human hepatocytes reduced fenoterol uptake threefold. After administration of 180 µg of fenoterol to 39 healthy individuals, the OCT1-deficient individuals (zero active OCT1 alleles; n = 5) showed 1.9-fold greater systemic fenoterol exposure (P = 4.0 × 10-5 ) and 1.7-fold lower volume of distribution (P = 8.0 × 10-5 ). Correspondingly, the OCT1-deficient individuals had a 1.5-fold stronger increase in heart rate (P = 0.002), a 3.4-fold greater increase in blood glucose (P = 3.0 × 10-5 ), and significantly lower serum potassium levels. In conclusion, heritable OCT1 deficiency significantly increases plasma concentrations of fenoterol and may be an important factor underlying the excess mortality associated with fenoterol.

The lack of the organic cation transporter OCT1 at the plasma membrane of tumor cells precludes a positive response to sorafenib in patients with hepatocellular carcinoma.

BACKGROUND: Sorafenib is the drug of choice in the treatment of advanced hepatocellular carcinoma (HCC). Beneficial effects are limited by mechanisms of chemoresistance, which include downregulation and/or impaired function of plasma membrane transporters accounting for drug uptake. The organic cation transporter 1 (OCT1) plays a major role in sorafenib uptake and decreased expression in HCC has been associated with poorer response. METHODS: The multicenter retrospective TRANSFER study involved tumor biopsies from 39 patients with advanced HCC and sorafenib therapy for ≥4 wk. Endpoint was the relationship between clinicopathological features and immunohistological result. Immunostaining was performed using specific primary anti-OCT1-head and anti-OCT1-tail antibodies. Tumors were classified according to a simplified staining score as absent, weak, moderate or strong, taking into account the localization of the staining at the plasma membrane as positive or negative. RESULTS: Results confirmed OCT1 downregulation in half of the cases investigated (10% absent, 38% weak). However, only one third of tumors expressing OCT1 displayed plasma membrane location (15% vs. 36% cytosolic expression). When comparing HCC with and without OCT1 expression, no different sorafenib response was found. When tumors expressing OCT1 at the plasma membrane were considered separately, a marked longer survival was found (Log Rank p<0.001). No association between OCT1 expression at the plasma membrane with tumor stage, previous treatment with TACE or radiological response was seen.In conclusion, these results indicate that the presence of OCT1 at the plasma membrane, rather than its expression levels, is related to better outcome of HCC patients treated with sorafenib.

Renoprotective Effects of Metformin are Independent of Organic Cation Transporters 1 &2 and AMP-activated Protein Kinase in the Kidney.

The type-2 diabetes drug metformin has proven to have protective effects in several renal disease models. Here, we investigated the protective effects in a 3-day unilateral ureteral obstruction (3dUUO) mouse model. Compared with controls, ureteral obstructed animals displayed increased tubular damage and inflammation. Metformin treatment attenuated inflammation, increased the anti-oxidative response and decreased tubular damage. Hepatic metformin uptake depends on the expression of organic cation transporters (OCTs). To test whether the effects of metformin in the kidney are dependent on these transporters, we tested metformin treatment in OCT1/2-/- mice. Even though exposure of metformin in the kidney was severely decreased in OCT1/2-/- mice when evaluated with [11C]-Metformin and PET/MRI, we found that the protective effects of metformin were OCT1/2 independent when tested in this model. AMP-activated protein kinase (AMPK) has been suggested as a key mediator of the effects of metformin. When using an AMPK-ß1 KO mouse model, the protective effects of metformin still occurred in the 3dUUO model. In conclusion, these results show that metformin has a beneficial effect in early stages of renal disease induced by 3dUUO. Furthermore, these effects appear to be independent of the expression of OCT1/2 and AMPK-ß1, the most abundant AMPK-ß isoform in the kidney.

Oct-1 regulates IL-17 expression by directing interchromosomal associations in conjunction with CTCF in T cells.

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive T cells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive T cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sites cooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2.

Oxaliplatin is an integral component of colorectal cancer therapy, but its clinical use is associated with a dose-limiting peripheral neurotoxicity. We found that the organic cation transporter 2 (OCT2) is expressed on dorsal root ganglia cells within the nervous system where oxaliplatin is known to accumulate. Cellular uptake of oxaliplatin was increased by 16- to 35-fold in cells overexpressing mouse Oct2 or human OCT2, and this process was associated with increased DNA platination and oxaliplatin-induced cytotoxicity. Furthermore, genetic or pharmacologic knockout of Oct2 protected mice from hypersensitivity to cold or mechanical-induced allodynia, which are established tests to assess acute oxaliplatin-induced neurotoxicity. These findings provide a rationale for the development of targeted approaches to mitigate this debilitating toxicity.

Ablation of both organic cation transporter (OCT)1 and OCT2 alters metformin pharmacokinetics but has no effect on tissue drug exposure and pharmacodynamics.

Organic cation transporter (OCT)1 and OCT2 mediate hepatic uptake and secretory renal clearance of metformin, respectively. Pharmacokinetic/pharmacodynamic (PK/PD) implications of simultaneous impairment of both transporters, such as by systemic pan-OCT inhibition, have not been studied directly. At present metformin PK/PD, distribution, and excretion were studied in Oct1/Oct2-knockout mice. Metformin clearance was reduced 4.5-fold from renal blood flow to unbound glomerular filtration rate, and volume of distribution was reduced 3.5-fold in Oct1/Oct2-knockout mice. Oral bioavailability was not affected (F = 64 ± 4 versus 59 ± 11; knockout versus wild type). Liver- and kidney-to-plasma concentration ratios were decreased in Oct1/Oct2-knockout mice 4.2- and 2.5-fold, respectively. The 2.9-fold increase in oral metformin exposure and reduced tissue partitioning yielded little to no net change in tissue drug concentrations. Absolute kidney exposure was unchanged (knockout/wild type = 1.1 ± 0.2), and liver exposure was only modestly decreased (knockout/wild type = 0.6 ± 0.1). Oral glucose area under the curve (AUC) lowering by metformin was not impaired in Oct1/Oct2-knockout mice at the five dose levels tested (ED50 = 151 versus 110 mg/kg; glucose lowering at highest dose = 42 ± 1 versus 39 ± 4%; knockout versus wild type); however, higher systemic metformin exposures were necessary in knockout mice to elicit the same effect (half-maximal efficacious AUC = 70 versus 26 µg x h/ml). Despite major changes in metformin clearance and volume of distribution in Oct1/Oct2-knockout mice, tissue drug exposure and PD were not affected. These findings challenge the presumption that systemic OCT inhibition will affect metformin pharmacology.

Dynamic regulation of Oct1 during mitosis by phosphorylation and ubiquitination.

BACKGROUND: Transcription factor Oct1 regulates multiple cellular processes. It is known to be phosphorylated during the cell cycle and by stress, however the upstream kinases and downstream consequences are not well understood. One of these modified forms, phosphorylated at S335, lacks the ability to bind DNA. Other modification states besides phosphorylation have not been described. METHODOLOGY/PRINCIPAL FINDINGS: We show that Oct1 is phosphorylated at S335 in the Oct1 DNA binding domain during M-phase by the NIMA-related kinase Nek6. Phospho-Oct1 is also ubiquitinated. Phosphorylation excludes Oct1 from mitotic chromatin. Instead, Oct1(pS335) concentrates at centrosomes, mitotic spindle poles, kinetochores and the midbody. Oct1 siRNA knockdown diminishes the signal at these locations. Both Oct1 ablation and overexpression result in abnormal mitoses. S335 is important for the overexpression phenotype, implicating this residue in mitotic regulation. Oct1 depletion causes defects in spindle morphogenesis in Xenopus egg extracts, establishing a mitosis-specific function of Oct1. Oct1 colocalizes with lamin B1 at the spindle poles and midbody. At the midbody, both proteins are mutually required to correctly localize the other. We show that phospho-Oct1 is modified late in mitosis by non-canonical K11-linked polyubiquitin chains. Ubiquitination requires the anaphase-promoting complex, and we further show that the anaphase-promoting complex large subunit APC1 and Oct1(pS335) interact. CONCLUSIONS/SIGNIFICANCE: These findings reveal mechanistic coupling between Oct1 phosphorylation and ubquitination during mitotic progression, and a role for Oct1 in mitosis.

Influence of Oct1/Oct2-deficiency on cisplatin-induced changes in urinary N-acetyl-beta-D-glucosaminidase.

PURPOSE: This study aimed to test the influence of functional renal organic cation transporters (OCT2 in humans, Oct1 and Oct2 in mice) on biomarkers of cisplatin nephrotoxicity, such as urinary activity of N-acetyl-beta-D-glucosaminidase (NAG). EXPERIMENTAL DESIGN: Temporal cisplatin-induced nephrotoxicity was assessed by histopathology and biomarkers. Cisplatin-mediated NAG changes and survival were determined in wild-type and Oct1/2(-/-) mice. Identification of OCT2 inhibitors was done in transfected 293Flp-In cells, and the NCI(60) cell line panel was used to assess contribution of OCT2 to cisplatin uptake in cancer cells. RESULTS: Classical biomarkers such as blood urea nitrogen and serum creatinine were not elevated until 72 hours after cisplatin administration and substantial kidney damage had occurred. Oct1/2(-/-) mice had 2.9-fold lower NAG by 4 hours (P < 0.0001) and 2.3-fold increased survival (P = 0.0097). Among 16 agents, cimetidine strongly inhibited uptake of tetraethylammonium bromide (P = 0.0006) and cisplatin (P < 0.0001), but did not have an influence on cisplatin uptake in SK-OV-3 cells, the cancer line with the highest OCT2 mRNA levels. In wild-type mice, cimetidine inhibited cisplatin-induced NAG changes (P = 0.016 versus cisplatin alone) to a degree similar to that seen in Oct1/2(-/-) mice receiving cisplatin (P = 0.91). Cumulative NAG activity of >0.4 absorbance units (AU) was associated with 21-fold increased odds for severe nephrotoxicity (P = 0.0017), which was linked with overall survival (hazard ratio, 8.1; 95% confidence interval, 2.1-31; P = 0.0078). CONCLUSIONS: Cimetidine is able to inhibit OCT2-mediated uptake of cisplatin in the kidney, and subsequently ameliorate nephrotoxicity likely with minimal effect on uptake in tumor cells.

Reduced-function SLC22A1 polymorphisms encoding organic cation transporter 1 and glycemic response to metformin: a GoDARTS study.

OBJECTIVE: Metformin is actively transported into the liver by the organic cation transporter (OCT)1 (encoded by SLC22A1). In 12 normoglycemic individuals, reduced-function variants in SLC22A1 were shown to decrease the ability of metformin to reduce glucose excursion in response to oral glucose. We assessed the effect of two common loss-of-function polymorphisms in SLC22A1 on metformin response in a large cohort of patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: The Diabetes Audit and Research in Tayside Scotland (DARTS) database includes prescribing and biochemistry information and clinical phenotypes of all patients with diabetes within Tayside, Scotland, from 1992 onwards. R61C and 420del variants of SLC22A1 were genotyped in 3,450 patients with type 2 diabetes who were incident users of metformin. We assessed metformin response by modeling the maximum A1C reduction in 18 months after starting metformin and investigated whether a treatment target of A1C <7% was achieved. Sustained metformin effect on A1C between 6 and 42 months was also assessed, as was the time to metformin monotherapy failure. Covariates were SLC22A1 genotype, BMI, average drug dose, adherence, and creatinine clearance. RESULTS: A total of 1,531 patients were identified with a definable metformin response. R61C and 420del variants did not affect the initial A1C reduction (P = 0.47 and P = 0.92, respectively), the chance of achieving a treatment target (P = 0.83 and P = 0.36), the average A1C on monotherapy up to 42 months (P = 0.44 and P = 0.75), or the hazard of monotherapy failure (P = 0.85 and P = 0.56). CONCLUSIONS: The SLC22A1 loss-of-function variants, R61C and 420del, do not attenuate the A1C reduction achieved by metformin in patients with type 2 diabetes.

Oct1 loss of function induces a coordinate metabolic shift that opposes tumorigenicity.

Cancer cells frequently undergo a shift from oxidative to glycolytic metabolism. Although there is interest in targeting metabolism as a form of cancer therapy, this area still remains in its infancy. Using cells, embryos and adult animals, we show here that loss of the widely expressed transcription factor Oct1 induces a coordinated metabolic shift: mitochondrial activity and amino acid oxidation are increased, while glucose metabolism is reduced. Altered expression of direct Oct1 targets encoding metabolic regulators provides a mechanistic underpinning to these results. We show that these metabolic changes directly oppose tumorigenicity. Collectively, our findings show that Oct1, the genes it regulates and the pathways these genes affect could be used as targets for new modes of cancer therapy.

Sox2 and Pou2f1 interact to control lens and olfactory placode development.

Sox2, which encodes an SRY-like HMG box transcription factor, is critical for vertebrate development. Sox2 mediates its transcriptional effects through the formation of complexes with specific co-factors, many of which are unknown. In this report, we identify Oct-1, encoded by the Pou2f1 gene, as a co-factor for Sox2 in the context of mouse lens and nasal placode induction. Oct-1, Sox2, and Pax6 are co-expressed during lens and nasal placode induction and during subsequent developmental stages. Genetic combination of Sox2 and Pou2f1 mutant alleles results in impaired induction of the lens placode, an ocular phenotype that includes anophthalmia, and a complete failure of nasal placode induction. These ocular and nasal phenotypes closely resemble those observed in Pax6 null embryos. Moreover, we identify DNA-binding sites that support the cooperative formation of a complex between Sox2 and Oct-1 and mediate Sox2/Oct-1-dependent transactivation of the Pax6 lens ectoderm enhancer in vitro. We demonstrate that the same Sox- and Octamer-binding sites are essential for Pax6 enhancer activity in the lens placode and its derivatives in transgenic mouse embryos. Collectively, these results indicate that Pou2f1, Sox2 and Pax6 are interdependent components of a molecular pathway utilized in both lens and nasal placode induction.

The octamer binding transcription factor Oct-1 is a stress sensor.

The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to gamma radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5' regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress.