Rachel Eclov

ABCG2 encodes the mitoxantrone resistance protein (MXR, BCRP), an ATP-binding cassette (ABC) efflux membrane transporter. Computational analysis of the ~300 kb region of DNA surrounding ABCG2 (chr4:88911376-89220011, hg19) identified 30 regions with potential cis-regulatory capabilities. These putative regulatory regions were tested for their enhancer and suppressor activity in a human liver cell line using luciferase reporter assays. The in vitro enhancer and suppressor assays identified four regions that decreased gene expression and five regions that increased expression >1.6-fold. Four of five human hepatic in vitro enhancers were confirmed as in vivo liver enhancers using the mouse hydrodynamic tail vein injection assay. Two of the in vivo liver enhancers (ABCG2RE1 and ABCG2RE9) responded to 17β-estradiol or rifampin in human cell lines, and ABCG2RE9 had ChIP-seq evidence to support the binding of several transcription factors and the transcriptional coactivator p300 in huma...

Author(s): Eclov, Rachel | Advisor(s): Kroetz, Deanna L | Abstract: ABCG2 encodes for a multidrug efflux transporter called the mitoxantrone resistance protein (MXR, BCRP) that mediates the efflux of substrates out of the cell and is important in detoxification. The present study was focused on the expression and function of MXR amino acid variants, activity of the ABCG2 promoter and promoter variants, characterization of ABCG2 locus cis-regulatory elements and variant enhancers, and examination of DNA methylation around ABCG2. MXR expression, localization and activity were characterized using whole cells and inside-out vesicles. The Q141K variant had reduced expression. MXR I206L had increased efflux of pheophorbide A and both V12M and D620N had increased ATPase activity. The activity of ABCG2 regulatory elements was tested in in vitro and in vivo luciferase assays. Two promoter SNPs (rs76656413 and rs59370292) had decreased in vivo liver enhancer activity. Six regions with in vivo...

BackgroundRegulatory T cells (Tregs) have been a therapeutic target of interest since early pre-clinical work revealed that their depletion led to enhanced tumor control. Despite continuing advances in the development of novel cellular-, antibody- and chemotherapeutic-based strategies to increase anti-tumor immunity, Treg presence and activity within the tumor microenvironment remains a complicating factor to their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg deletional strategies, we investigated the ability to target FOXP3, the master regulator of Treg development, maintenance, and suppressive function using hydrocarbon stapled alpha-helical peptides (SAHs). We developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain, in an effort to impede FOXP3 transcriptional function. SAHs overcome three major protein-protein interaction (PPI) therapeutic hurdles, namely: cellular penet...

Severe Congenital Adrenal Hyperplasia (CAH) is most commonly caused by genetic defects in the CYP21A2 gene, which leads to a deficiency of 21-hydroxylase enzyme and disruption in the biosynthesis of Adrenal corticosteriods. Despite treatment with corticosteroids, patients remain at significant risk for adrenal crisis, experiencing a 3-fold higher mortality rate than age matched controls. They also suffer from significant infertility, bone, metabolic, and cardiovascular disease, and hyperandrogenism in women leading to genital abnormalities, hirsutism, and other complications. We are developing an AAV5- based gene therapy (BBP-631) that will provide a functional copy of the CYP21A2 gene to the adrenal glands of CAH patients. To determine the durability of this therapy we treated cynomolgus monkeys with increasing doses of BBP-631 via intravenous injection. At 4-, 12- and 24-weeks post treatment, expression of hCYP21A2 mRNA and vector genome copies (VGC) in the adrenals and other peri...

The expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays. Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs...

encodes the breast cancer resistance protein (BCRP), an efflux membrane transporter important in detoxification of xenobiotics. In the present study, the basal activity of thepromoter in liver, kidney, intestine and breast cell lines was examined using luciferase reporter assays. The promoter activity of reference and variantsequences were compared in HepG2, HEK293T, HCT116 and MCF-7 cell lines. Thepromoter activity was strongest in the kidney and intestine cell lines. Four variants in the basalpromoter (rs76656413, rs66664036, rs139256004 and rs59370292) decreased the promoter activity by 25-50% in at least three of the four cell lines. The activity of these four variants were also examinedusing the hydrodynamic tail vein assay, and two SNPs (rs76656413 and rs59370292) significantly decreasedliver promoter activity by 50-80%. Electrophoretic mobility shift assays confirmed a reduction in nuclear protein binding to the rs59370292 variant probe, while the rs76656413 probe had a shift...

The mitoxantrone resistance protein (MXR; BCRP; ABCG2), referred to here as MXR, is an efflux membrane transporter expressed apically in several tissues with a broad range of both exogenous and endogenous substrates. The transport activity, tissue distribution and cellular localization of MXR suggest that it plays a pivotal role in endogenous substrate disposition as well as the protection and detoxification of the body from xenobiotics. Overexpression of MXR is associated with drug resistance to a variety of anticancer drugs, and it has been linked with decreased disease-free survival in several cancers. An individual’s susceptibility to certain drug-induced side effects has also been linked to MXR expression or nonsynonymous single nucleotide polymorphisms (SNPs) in the MXR gene ABCG2. Therefore, it is important to not only characterize the functional effects of ABCG2 SNPs, but to also look at other mechanisms which alter MXR expression and function. We hypothesize that there are ...

Sensory peripheral neuropathy is a common and sometimes debilitating toxicity associated with paclitaxel therapy. This study aims to identify genetic risk factors for the development of this toxicity. A prospective pharmacogenetic analysis of patients with primary breast cancer, randomized to the paclitaxel arm of CALGB 40101, was used to identify genetic predictors of the onset and severity of sensory peripheral neuropathy. A genome-wide association study in 855 subjects of European ancestry was conducted and findings were replicated in additional European (n = 154) and African American (n = 117) subjects. A single nucleotide polymorphism in FGD4 was associated with the onset of sensory peripheral neuropathy in the discovery cohort [rs10771973; HR, 1.57; 95% confidence interval (CI), 1.30-1.91; P = 2.6 × 10(-6)] and in a European (HR, 1.72; 95% CI, 1.06-2.80; P = 0.013) and African American (HR, 1.93; 95% CI, 1.13-3.28; P = 6.7 × 10(-3)) replication cohort. There is also evidence t...