In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice.

P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.

A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

Drug transporters on arachnoid barrier cells contribute to the blood-cerebrospinal fluid barrier.

The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.

The orphan nuclear receptor HNF4alpha determines PXR- and CAR-mediated xenobiotic induction of CYP3A4.

The drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is thought to be involved in the metabolism of nearly 50% of all the drugs currently prescribed. Alteration in the activity or expression of this enzyme seems to be a key predictor of drug responsiveness and toxicity. Currently available studies indicate that the ligand-activated nuclear receptors pregnane X receptor (PXR; NR1I2) and constitutive androstane receptor (CAR; NR1I3) regulate CYP3A4 expression. However, in cell-based reporter assays, CYP3A4 promoter activity was most pronounced in liver-derived cells and minimal or modest in non-hepatic cells, indicating that a liver-specific factor is required for physiological transcriptional response. Here we show that the orphan nuclear receptor hepatocyte nuclear factor-4alpha (HNF4alpha; HNF4A) is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3A4. We identified a specific cis-acting element in the CYP3A4 gene enhancer that confers HNF4alpha binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4alpha had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of Hnf4alpha had reduced basal and inducible expression of CYP3A. These data identify HNF4alpha as an important regulator of coordinate nuclear-receptor-mediated response to xenobiotics.

Methylation profiling reveals novel molecular classes of rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) represent a family of aggressive soft tissue sarcomas that present in both children and adults. Pathologic risk stratification for RMS has been based on histologic subtype, with poor outcomes observed in alveolar rhabdomyosarcoma (ARMS) and the adult-type pleomorphic rhabdomyosarcoma (PRMS) compared to embryonal rhabdomyosarcoma (ERMS). Genomic sequencing studies have expanded the spectrum of RMS, with several new molecularly defined entities, including fusion-driven spindle cell/sclerosing rhabdomyosarcoma (SC/SRMS) and MYOD1-mutant SC/SRMS. Comprehensive genomic analysis has previously defined the mutational and copy number spectrum for the more common ERMS and ARMS and revealed corresponding methylation signatures. Comparatively, less is known about epigenetic correlates for the rare SC/SRMS or PRMS histologic subtypes. Herein, we present exome and RNA sequencing, copy number analysis, and methylation profiling of the largest cohort of molecularly characterized RMS samples to date. In addition to ARMS and ERMS, we identify two novel methylation subtypes, one having SC/SRMS histology and defined by MYOD1 p. L122R mutations and the other matching adult-type PRMS. Selected tumors from adolescent patients grouped with the PRMS methylation class, expanding the age range of these rare tumors. Limited follow-up data suggest that pediatric tumors with MYOD1-mutations are associated with an aggressive clinical course.

Role of Vitamins A and D in BCR-ABL Arf-/- Acute Lymphoblastic Leukemia.

The effects of vitamin A and/or vitamin D deficiency were studied in an Arf-/- BCR-ABL acute lymphoblastic leukemia murine model. Vitamin D sufficient mice died earlier (p = 0.003) compared to vitamin D deficient (VDD) mice. Vitamin A deficient (VAD) mice fared worst with more rapid disease progression and decreased survival. Mice deficient for vitamins A and D (VADD) had disease progression similar to VAD mice. Regulatory T cells, previously shown to associate with poor BCR-ABL leukemia control, were present at higher frequencies among CD4+ splenocytes of vitamin A deficient vs. sufficient mice. In vitro studies demonstrated 1,25-dihydroxyvitamin D (1,25(OH)2VD3) increased the number of BCR-ABL ALL cells only when co-cultured with bone marrow stroma. 1,25(OH)2VD3 induced CXCL12 expression in vivo and in vitro in stromal cells and CXCL12 increased stromal migration and the number of BCR-ABL blasts. Vitamin D plus leukemia reprogrammed the marrow increasing production of collagens, potentially trapping ALL blasts. Vitamin A (all trans retinoic acid, ATRA) treated leukemic cells had increased apoptosis, decreased cells in S-phase, and increased cells in G0/G1. ATRA signaled through the retinoid X receptor to decrease BCR-ABL leukemic cell viability. In conclusion, vitamin A and D deficiencies have opposing effects on mouse survival from BCR-ABL ALL.

The major human pregnane X receptor (PXR) splice variant, PXR.2, exhibits significantly diminished ligand-activated transcriptional regulation.

The pregnane X receptor (PXR; PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR, lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXR response element (PXRE) in electrophoretic mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR ligands. Cotransfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, and UGT1A1) were higher than mock-transduced cells in the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the same host cells failed to induce target genes over levels in mock-transfected cells after drug treatment. Our homology modeling suggests that ligands bind PXR.1 more favorably, probably because of the presence of a key disordered loop region, which is missing in PXR.2. Yeast two-hybrid assays revealed that, even in the presence of ligand, the corepressors remain tightly bound to PXR.2, and coactivators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, corepressors remain tightly bound, and coactivators are not recruited to PXR.2.

A phosphomimetic mutation at threonine-57 abolishes transactivation activity and alters nuclear localization pattern of human pregnane x receptor.

The pregnane X receptor (PXR) plays crucial roles in multiple physiological processes. However, the signaling mechanisms responsible are not well defined; it is most likely that multiple functions of PXR are modulated by its phosphorylation. Therefore, we sought to determine whether mutation at a highly conserved Thr(57) affects human PXR (hPXR) function. Site-directed mutagenesis was performed to generate phosphorylation-deficient (hPXR(T57A)) and phosphomimetic (hPXR(T57D)) mutants. Gene reporter, Western blotting, immunocytochemistry, mammalian two-hybrid, and electrophoretic mobility shift assays were used to study cytochrome P450 3A4 (CYP3A4) promoter activation, protein levels, localization, cofactor interaction, and CYP3A4 promoter binding of the hPXR mutants, respectively. hPXR(T57D), but not hPXR(T57A), lost its transcriptional activity. Neither mutation altered hPXR's protein levels and interaction with steroid receptor coactivator-1. hPXR and hPXR(T57A) exhibited a homogenous nuclear distribution, whereas hPXR(T57D) exhibited a distinctive punctate nuclear localization pattern similar to that of hPXR mutants with impaired function that colocalize with silencing mediator of retinoid and thyroid receptors (SMRT), although silencing of SMRT did not rescue the altered function of hPXR(T57D). However, hPXR(T57D), but not hPXR(T57A), impaired hPXR's ability to bind to the CYP3A4 promoter, consistent with the mutant's transactivation function. Furthermore, the 70-kDa form of ribosomal protein S6 kinase (p70 S6K) phosphorylated hPXR in vitro and inhibited its transcriptional activity, whereas hPXR(T57A) partially resisted the inhibitory effect of p70 S6K. Our studies identify a functionally significant phosphomimetic mutant (hPXR(T57D)) and show p70 S6K phosphorylation and regulation of hPXR transactivation to support the notion that phosphorylation plays important roles in regulating hPXR function.

DNA Methylation Profiling Reveals Prognostically Significant Groups in Pediatric Adrenocortical Tumors: A Report From the International Pediatric Adrenocortical Tumor Registry.

PURPOSE: Pediatric adrenocortical carcinomas (ACCs) are aggressive; the overall survival of patients with ACCs is 40%-50%. Appropriate staging and histologic classification are crucial because children with incomplete resections, metastases, or relapsed disease have a dismal prognosis. The clinical course of pediatric adrenocortical tumors (ACTs) is difficult to predict using the current classification schemas, which rely on subjective microscopic and gross macroscopic variables. Recent advances in adult ACT studies have revealed distinct DNA methylation patterns with prognostic significance that have not been systematically interrogated in the pediatric population. PATIENTS AND METHODS: We performed DNA methylation analyses on 48 newly diagnosed ACTs from the International Pediatric Adrenocortical Tumor Registry and 12 pediatric adrenal controls to evaluate for distinct methylation groups. Pediatric methylation data were also compared systematically with the adult ACC cohort from The Cancer Genome Atlas (TCGA). RESULTS: Two pediatric ACT methylation groups were identified and showed differences in selected clinicopathologic and outcome characteristics. The A1 group was enriched for CTNNB1 variants and unfavorable outcome. The A2 group was enriched for TP53 germline variants, younger age at onset, and favorable outcome. Pediatric ACT methylation groups were maintained when International Pediatric Adrenocortical Tumor Registry cohort data were combined with TCGA cohort data. The CpG-island hypermethylator phenotype characterizing the TCGA cohort was not identified in the pediatric patients. When methylome findings were combined with independent histopathologic review using the Wieneke criteria, a high-risk population was identified with uniform fatal outcome. CONCLUSION: Our results indicate DNA methylation analysis can enhance current diagnostic algorithms. A combination of methylation and histologic classification produced the strongest prediction model and may prove useful in future risk-adapted therapeutic trials.

Expression of the pregnane X receptor in mice antagonizes the cholic acid-mediated changes in plasma lipoprotein profile.

OBJECTIVE: Modification of lipoprotein metabolism by bile acids has been mainly explained by activation of the farnesyl X receptor (FXR). The aim of the present study was to determine the relative contribution of the pregnane X receptor (PXR), another bile acid-activated nuclear receptor to changes in plasma lipoprotein profile. METHODS AND RESULTS: Wild-type mice, Pxr-deficient mice, and Pxr-null mice expressing human PXR (Pxr-null SXR-Tg mice) were fed a cholic acid-containing diet, and consequences on plasma lipoprotein profiles and target gene expression were assessed. Cholic acid produced significant decreases in high-density lipoprotein (HDL) cholesterol, plasma apolipoprotein (apo)A-I and hepatic apoA-I mRNA in wild-type mice. Interestingly, the effect of cholic acid was significantly more pronounced in Pxr-deficient mice, indicating that PXR contributes to the weakening of the effect of bile acids on lipoprotein metabolism. Reciprocally, changes in HDL/apoA-I profiles were abolished in Pxr-null SXR-Tg mice in which PXR-responsive genes, particularly those involved in bile acid detoxification were readily activated after cholic acid treatment. CONCLUSIONS: PXR expression in mice antagonizes the cholic acid-mediated downregulation of plasma HDL cholesterol and apoA-I, and magnification of PXR/SXR-mediated changes may constitute a new mean to counteract the effects of bile acids on plasma lipoproteins.