PBX1 Research Articles

Mediation by Placental DNA Methylation of the Association of Prenatal Maternal Smoking and Birth Weight.

Prenatal maternal smoking is a risk factor for lower birth weight. We performed epigenome-wide association analyses of placental DNA methylation (DNAm) at 720,077 cytosine-phosphate-guanine (CpG) sites and prenatal maternal smoking among 441 mother-infant pairs (2010-2014) and evaluated whether DNAm mediates the association between smoking and birth weight using mediation analysis. Mean birth weight was 3,443 (standard deviation, 423) g, and 38 mothers (8.6%) reported smoking at a mean of 9.4 weeks of gestation. Prenatal maternal smoking was associated with a 175-g lower birth weight (95% confidence interval (CI): -305.5, -44.8) and with differential DNAm of 71 CpGs in placenta, robust to latent-factor adjustment reflecting cell types (Bonferroni-adjusted P < 6.94 × 10-8). Of the 71 CpG sites, 7 mediated the association between prenatal smoking and birth weight (on MDS2, PBX1, CYP1A2, VPRBP, WBP1L, CD28, and CDK6 genes), and prenatal smoking × DNAm interactions on birth weight were observed for 5 CpG sites. The strongest mediator, cg22638236, was annotated to the PBX1 gene body involved in skeletal patterning and programming, with a mediated effect of 301-g lower birth weight (95% CI: -543, -86) among smokers but no mediated effect for nonsmokers (β = -38 g; 95% CI: -88, 9). Prenatal maternal smoking might interact with placental DNAm at specific loci, mediating the association with lower infant birth weight.

Long non-coding RNA HEIH promotes breast cancer development via negative modulation of microRNA-200b.

This study aimed to investigate the effects and regulatory mechanism of long non-coding RNA HEIH in the development of breast cancer. The expression of HEIH in breast tumor tissues and breast cancer cells was determined, followed by investigating the effects and regulatory mechanism of HEIH dysregulation on breast cancer cell viability, apoptosis, migration and invasion. The expression of HEIH was upregulated in breast cancer tissue samples and cell lines. Suppression of HEIH inhibited cell viability, promoted cell apoptosis, and decreased migration and invasion in MDA-MB-231 cells. Moreover, a negative relationship existed between HEIH and miR-200b, and HEIH regulated breast cancer development via regulating miR-200b. Pre-leukemia transcription factor 3 (PBX3) was verified as a functional target of miR-200b, and miR-200b regulated the malignant behaviors of breast cancer cells through targeting PBX3. Furthermore, suppression of HEIH inhibited the activation of Wnt/β-catenin pathway, which was remarkably reversed after suppression of HEIH and inhibition of miR-200b synchronously. Our results reveal that HEIH may contribute to breast cancer development via modulation of microRNA-200b/axis and inducing the activation of Wnt/β-catenin pathway. Further studies are still required to confirm our findings.

MiR-302a inhibits human HepG2 and SMMC-7721 cells proliferation and promotes apoptosis by targeting MAP3K2 and PBX3

Hepatocellular carcinoma (HCC) is the most common liver cancer and has a poor prognosis. miR-302a is an important regulator of tumor occurrence and deterioration, while MAP3K2 and PBX3 genes are involved in cancer cell proliferation and apoptosis. In this study, the expression of miR-302a and MAP3K2/PBX3 were evaluated by qPCR in liver cancer cell lines. Next, the target relationship between miR-302a and MAP3K2/PBX3 was verified using luciferase assays. Meanwhile, the expression correlation between miR-302a and target genes was analyzed in cancer tissue and para-cancerous tissue. In addition, an increased miR-302a level in HepG2 cells and SMMC-7721 cells were achieved through transfection with miR-302a mimics, and the effects on HepG2 cell and SMMC-7721 cell proliferation, apoptosis and MAPK pathways were determined using MTT, flow cytometry, qPCR and western blot assays. The results showed that liver cancer cell lines exhibited low miR-302a expression and MAP3K2 and PBX3 were confirmed to be the target genes of miR-302a. Meanwhile, the HE results showed that cells became enlarged with loose cytoplasm and formed balloon-like lesions in HCC specimens and we found a significant negative correlation between miR-302a and MAP3K2/PBX3 expression. In addition, treatment with miR-302a mimics inhibited HepG2 cells and SMMC-7721 cells proliferation and increased the apoptosis rate. Further research revealed that the MAPK key factors p-p38, p-ERK1/2 and p-JNK were significantly reduced in miR-302a transfected cells and MAP3K2/PBX3 silenced cells. Besides, MAP3K2 and PBX3 overexpression in miR-302a mimics-treated cells exerted the opposite effects. In conclusion, miR-302a inhibited proliferation and promoted apoptosis in human hepatoma cells by targeting MAP3K2 and PBX3.

FEATURES OF THE TETRATHALIUM(I) TRITIOSTANATE(II) SINGLE CRYSTAL GROWTH

Thermoelectric materials provide a direct transformation of heat into electric energy. Improving the efficiency of thermoelectric material is a complex and individual task for concrete material. Ternary chalcogenides type Tl4Sn(Pb)X3 (X–S,Se,Te) are perspective thermoelectric materials. To improve the thermoelectric figure of merit of a material the reduction of the phonon thermal is used what can be reached by using polycrystalline or single crystal samples.This work is devoted to the development of technological conditions for the crystal growth of Tl4SnS3 compound. Tl4SnS3 are formed in the Tl2S–SnS quasibinary system by peritectic reaction L + Tl2S↔Tl4SnS3 at 626 K and is characterized by polymorphic transformation at 600 K. The low temperature modification of Tl4SnS3 are crystallized in tetragonal system, SG P4/ncc.For the current investigation two samples with ratio 1) 66.67 mol.% Tl2S : 33.33 mol.% SnS (1) and 52 mol.% Tl2S : 48 mol.% SnS (2) were obtained. The 1 sample correspond to stoichiometric composition of Tl4SnS3 compound. The 2 sample correspond to nonstoichiometric composition what lies on the branch of the primary crystallization of high temperature modification of Tl4SnS3.The both synthesis were carried out from high purity elementary components in vacuumed to 0.13 Pa quartz ampoules by one-temperature direct method. The maximal temperature of synthesis 698 K was obtained with heating rate 50 K per hour. Cooling to an experimentally selected annealing temperature of 480 K was carried out with rate 30 K / h. The annealing was carried out during 336 h.The crystals growth of the both samples was carried using "the solution-melt" method in a two-zone furnace (temperature of the melt zone – 698 K, annealing zone – 480 K) in a quartz container with a cone-shaped lower part. Obtained single crystals were without cracks.Results of the microstructural analysis (MSA) indicates that the grown crystals do not contain inhomogeneities. Comparison of the experimentally obtained powder diffraction data with theoretically calculated indicate that a both crystals are single-phase. The calculated cell parameters of the 1 and 2 samples are close and agree with the theoretical data.

Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families.

The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome‐Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0‐fold (P = 0.036). Pre‐B‐cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3‐fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild‐type homozygote genotype had overexpression of PBX1. There is a 3‐fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

Functional testing of a human PBX3 variant in zebrafish reveals a potential modifier role in congenital heart defects.

ABSTRACTWhole-genome and exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs) are a prominent example of a human disorder with complex genetics. An inherited sequence variant in the human PBX3 gene (PBX3 p.A136V) has previously been shown to be enriched in a CHD patient cohort, indicating that the PBX3 p.A136V variant could be a modifier allele for CHDs. Pbx genes encode three-amino-acid loop extension (TALE)-class homeodomain-containing DNA-binding proteins with diverse roles in development and disease, and are required for heart development in mouse and zebrafish. Here, we used CRISPR-Cas9 genome editing to directly test whether this Pbx gene variant acts as a genetic modifier in zebrafish heart development. We used a single-stranded oligodeoxynucleotide to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene (pbx4 p.A131V). We observed that zebrafish that are homozygous for pbx4 p.A131V are viable as adults. However, the pbx4 p.A131V variant enhances the embryonic cardiac morphogenesis phenotype caused by loss of the known cardiac specification factor, Hand2. Our study is the first example of using precision genome editing in zebrafish to demonstrate a function for a human disease-associated single nucleotide variant of unknown significance. Our work underscores the importance of testing the roles of inherited variants, not just de novo variants, as genetic modifiers of CHDs. Our study provides a novel approach toward advancing our understanding of the complex genetics of CHDs.

Association of internal smoking dose with blood DNA methylation in three racial/ethnic populations

BackgroundLung cancer is the leading cause of cancer-related death. While cigarette smoking is the primary cause of this malignancy, risk differs across racial/ethnic groups. For the same number of cigarettes smoked, Native Hawaiians compared to whites are at greater risk and Japanese Americans are at lower risk of developing lung cancer. DNA methylation of specific CpG sites (e.g., in AHRR and F2RL3) is the most common blood epigenetic modification associated with smoking status. However, the influence of internal smoking dose, measured by urinary nicotine equivalents (NE), on DNA methylation in current smokers has not been investigated, nor has a study evaluated whether for the same smoking dose, circulating leukocyte DNA methylation patterns differ by race.MethodsWe conducted an epigenome-wide association study (EWAS) of NE in 612 smokers from three racial/ethnic groups: whites (n = 204), Native Hawaiians (n = 205), and Japanese Americans (n = 203). Genome-wide DNA methylation profiling of blood leukocyte DNA was measured using the Illumina 450K BeadChip array. Average β value, the ratio of signal from a methylated probe relative to the sum of the methylated and unmethylated probes at that CpG, was the dependent variables in linear regression models adjusting for age, sex, race (for pan-ethnic analysis), and estimated cell-type distribution.ResultsWe found that NE was significantly associated with six differentially methylated CpG sites (Bonferroni corrected p < 1.48 × 10−7): four in or near the FOXK2, PBX1, FNDC7, and FUBP3 genes and two in non-annotated genetic regions. Higher levels of NE were associated with increasing methylation beta-valuesin all six sites. For all six CpG sites, the association was only observed in Native Hawaiians, suggesting that the influence of smoking dose on DNA methylation patterns is heterogeneous across race/ethnicity (p interactions < 8.8 × 10−8). We found two additional CpG sites associated with NE in only Native Hawaiians.ConclusionsIn conclusion, internal smoking dose was associated with increased DNA methylation in circulating leukocytes at specific sites in Native Hawaiian smokers but not in white or Japanese American smokers.

LncRNA-uc.40 silence promotes P19 embryonic cells differentiation to cardiomyocyte via the PBX1 gene.

Uc.40 is a long noncoding RNA that is highly conserved among different species, although its function is unknown. It is highly expressed in abnormal human embryonic heart. We previously reported that overexpression of uc.40 promoted apoptosis and inhibited proliferation of P19 cells, and downregulated PBX1, which was identified as a potential target gene of uc.40. The current study evaluated the effects of uc40-siRNA-44 (siRNA against uc.40) on the differentiation, proliferation, apoptosis, and mitochondrial function in P19 cells, and investigated the relationship between uc.40 and PBX1 in cardiomyocytes. The uc.40 silencing expression was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Observation of morphological changes in transfected P19 cells during different stages of differentiation revealed that uc40-siRNA-44 increased the number of cardiomyocyes. There was no significant difference in the morphology or time of differentiation between the uc40-siRNA-44 group and the control group. uc40-siRNA-44 significantly promoted proliferation of P19 cells and inhibited serum starvation-induced apoptosis. There was no significant difference in mitochondrial DNA copy number or cellular ATP level between the two groups, and ROS levels were significantly decreased in uc40-siRNA-44-transfected cells. The levels of PBX1 and myocardial markers of differentiation were examined in transfected P19 cells; uc40-siRNA-44 downregulated myocardial markers and upregulated PBX1 expression. These results suggest that uc.40 may play an important role during the differentiation of P19 cells by regulation of PBX1 to promote proliferation and inhibit apoptosis. These studies provide a foundation for further study of uc.40/PBX1 in cardiac development.

Transcription Factors in Regulatory and Protein Subnetworks during Generation of Neural Stem Cells and Neurons from Direct Reprogramming of Non-fibroblastic Cell Sources

Direct reprogramming of non-fibroblastic cells to the neuronal cell types including induced neurons (iNs) and induced neural stem cells (iNSCs) has provided an alternative approach for the direct reprogramming of fibroblasts to those cells. However, to increase the efficiency of the reprogramming process the underlying mechanisms should be clarified. In the current study, we analyzed the gene expression profiles of five different cellular conversions to understand the most significant molecular mechanisms and transcription factors (TFs) underlying each conversion. For each conversion, we found the list of differentially expressed genes (DEGs) and the list of differentially expressed TFs (DE-TFs) which regulate expression of DEGs. Moreover, we constructed gene regulatory networks based on the TF-binding sites’ data and found the most central regulators and the most active part of the networks. Furthermore, protein complexes were identified from constructed protein–protein interaction networks for DE-TFs. Finally, we proposed a list of main regulators for each conversion; for example, in the direct conversion of epithelial-like cells (ECs) to iNSCs, combination of centrality with active modules or protein complex analyses highlighted the role of POU3F2, BACH1, AR, PBX1, SOX2 and NANOG genes in this conversion. To the best of our knowledge, this study is the first one that analyzed the direct conversion of non-fibroblastic cells toward iNs and iNSCs and we believe that the expression manipulation of identified genes may increase efficiency of these processes.

Inactivation of PBX3 and HOXA9 by down-regulating H3K79 methylation represses NPM1-mutated leukemic cell survival.

Acute myeloid leukemia (AML) with an NPM1 mutation (NPMc+) has a distinct gene expression signature and displays molecular abnormalities similar to mixed lineage leukemia (MLL), including aberrant expression of the PBX3 and HOXA gene cluster. However, it is unclear if the aberrant expression of PBX3 and HOXA is essential for the survival of NPM1-mutated leukemic cells.Methods: Using the gene expression profiling of TCGA and E-MTAB-3444 datasets, we screened for high co-expression of PBX3 and HOXA9 in NPMc+ leukemia patients. We performed NPMc+ depletion and overexpression experiments to examine aberrant H3K79 methylation through epigenetic regulation. Through RNA interference technology and small-molecule inhibitor treatment, we evaluated the effect of methyl-modified H3K79 on cell survival and explored the possible underlying mechanism.Results: We showed that NPMc+ increased the expression of PBX3 and HOXA9, which are both poor prognosis indicators in AML. High PBX3 and HOXA9 expression was accompanied by increased dimethylated and trimethylated H3K79 in transgenic murine Lin-Sca-1+c-Kit+ cells and human NPMc+ leukemia cells. Using chromatin immunoprecipitation sequencing (ChIP-seq) assays of NPMc+ cells, we determined that hypermethylated H3K79 was present at the expressed HOXA9 gene but not the PBX3 gene. PBX3 expression was positively regulated by HOXA9, and a reduction in either PBX3 or HOXA9 resulted in NPMc+ cell apoptosis. Importantly, an inhibitor of DOT1L, EPZ5676, effectively and selectively promoted NPMc+ human leukemic cell apoptosis by reducing HOXA9 and PBX3 expression.Conclusion: Our data indicate that NPMc+ leukemic cell survival requires upregulation of PBX3 and HOXA9, and this action can be largely attenuated by a DOT1L inhibitor.

MP12-10 SNP RS17383719 IN THE PBX1 GENE IS ASSOCIATED WITH CYSTINURIA

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology I1 Apr 2017MP12-10 SNP RS17383719 IN THE PBX1 GENE IS ASSOCIATED WITH CYSTINURIA Sabrina Reis, Ronaldo Guimarães, Nayara Viana, Katia Leite, Giovanni Marchini, Fabio Torricelli, William Nahas, Miguel Srougi, and Eduardo Mazzucchi Sabrina ReisSabrina Reis More articles by this author , Ronaldo GuimarãesRonaldo Guimarães More articles by this author , Nayara VianaNayara Viana More articles by this author , Katia LeiteKatia Leite More articles by this author , Giovanni MarchiniGiovanni Marchini More articles by this author , Fabio TorricelliFabio Torricelli More articles by this author , William NahasWilliam Nahas More articles by this author , Miguel SrougiMiguel Srougi More articles by this author , and Eduardo MazzucchiEduardo Mazzucchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.432AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cystinuria is a recessive disorder characterized by impaired tubular reabsorption of cystine and dibasic amino acids. Some studies in the literature have shown mutations in the SLC3A1 gene associated with type A cystinuria that follows a complete recessive inheritance in half of the cases. The other half has been related to mutations in the SLC7A9 gene considered responsible for cystinuria type B presumably inherited by an incomplete dominant manner. Considering the paucity of studies on the genetic basis of cystinuria, we performed genome-wide human SNP array, aiming to find some single nucleotide polymorphisms (SNPs) related to cystinuria development. To confirm the microarray results we performed PCR (Polymerase Chain Reaction) searching for five SNPs that we found related to the disease and two others reported in the literature. METHODS DNA samples from peripheral white blood cells were extracted from eight patients with cystinuria and 10 healthy subjects with no renal calculi that composed the control group. The SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. RESULTS The homozygote polymorphic genotype of SNP rs17383719 in gene PBX1 was significantly more frequent among cystinuric patients (p=0.015). The occurrence of the polymorphic allele for this SNP was associated with a 3-fold increased risk of cystinuria (p=0.036). The polymorphic alleles of SNPs rs913034 and rs7096453 (SVIL) were more frequent in the control group (p=0.04 and p=0.08, respectively). We did not detect polymorphic homozygote genotype in the SNPs for SLC7A9 (rs140134166) and SLC3A1 (rs200248046) genes. CONCLUSIONS This is the first study describing genetic variations in patients with cystinuria. The most important finding was the 3-fold increased risk for the development of the disease for the SNP in the PBX1 gene. PBX1 (Pre-B-cell-leukemia transcription factor 1) is a member of a transcription factors family associated with kidney development, but there are no studies in the literature associating this gene with nephrolithiasis. We postulate that the presence of SNP may change the gene expression of PBX1 affecting the renal absorption of cystine and other amino acids predisposing to nephrolithiasis. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e148 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Sabrina Reis More articles by this author Ronaldo Guimarães More articles by this author Nayara Viana More articles by this author Katia Leite More articles by this author Giovanni Marchini More articles by this author Fabio Torricelli More articles by this author William Nahas More articles by this author Miguel Srougi More articles by this author Eduardo Mazzucchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

PBX1 haploinsufficiency leads to syndromic congenital anomalies of the kidney and urinary tract (CAKUT) in humans

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) represent a significant healthcare burden since it is the primary cause of chronic kidney in children. CNVs represent a recurrent molecular...

PBX3 is Essential for Leukemia Stem Cell Maintenance in MLL-Rearranged Leukemia

Acute myeloid leukemia (AML) with MLL translocation is recognized as a group of aggressive hematopoietic malignancies with poor prognosis. An atypical homeodomain protein family (TALE family, three-amino-acid loop extension) has been reported to be involved in leukemogenesis and leukemia progression. For example, MEIS1 and PBX3 were abnormally expressed in examined AML patient samples when compared with their normal counterparts. While MEIS1 has been studied extensively, the precise role of PBX3 in leukemia development is still largely unknown.In this study, we explored the epigenetic regulatory network of PBX3 and the specific role of PBX3 in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 iscorrelated with poor prognosis of AML patients. In MLL-fusion induced AML mouse models that were established by retrovirus-mediated ectopic expression of MLL-AF9 or MLL-NRIP3 genes in murine BM progenitor cells (c-Kit+). PBX3 also showed high expression level in leukemia stem cells (LSC) compared with normal HSPCs. Unlike other homeobox genes such as HOXA9 and MEIS1 that were both highly expressed in LSKs and LSCs, PBX3 only showed extremely high expression in LSCs in comparison with HSPCs or other normal hematopoietic cells. To explore the role of PBX3 in leukemia, we analyzed the epigenetic modification patterns along PBX3 gene. By analyzing the public ChIP-seq database, we found that increased activation mark H3K79me2 and decreased repressive mark H3K27me3 exhibited on PBX3 in LSCs. Additionally, transcription activating modifications H3K79 hypermethylation of PBX3 was only observed in LSCs but not LSKs or GMPs, consistent with increased PBX3 expression level in LSCs compared with HSPCs. To further confirm this, we assessed H3K79me2 level of PBX3 in purified c-Kit+ cells which are enriched with stem cells from leukemic or control mice by ChIP-qPCR. Consistently, ChIP-qPCR results revealed the similar trend with dataset analysis. Moreover, we also detected the transcription repressive mark H3K9me3 on PBX3 in c-Kit+ cells and found a dramatic decrease of H3K9me3 in LSCs in comparison with HSPCs. Thus, our results suggested that high expression level of PBX3 in LSCs might be caused by elevated activation mark H3K79me2 and depressed repressive mark H3K27me3 and H3K9me3.Considering that high expression level of PBX3 in LSCs may contribute to leukemic progression, we used the CRISPR/Cas9 system to delete PBX3 to examine its potential role in MLL-AF9 mediated AML development. We found that PBX3 inactivation significantly prolonged the survival of leukemic mice (median survival was 25 days of control, 31 days of SgPBX3-a, 30 days of SgPBX3-b, 10 mice per group). Morphological analysis revealed more leukemic blast cells with segment nuclei exhibited in PBX3 inactivation group, suggesting that PBX3 deficiency is associated with increased leukemic cell differentiation. Since LSC was considered to be sufficient to drive leukemogenesis and maintain leukemia progression, we investigated the impact of PBX3 inactivation on LSC capability. In vitro colony forming assay showed that PBX3 inactivation group formed significantly fewer colonies of total numbers and type A clones. Flow cytometric analysis showed decreased LSCs frequency in murine leukemia cells of PBX3 inactivation group. Moreover, limiting dilution transplantation assays revealed 4-7 fold decreased of functional LSCs after PBX3 inactivation with the 1/102 of LSCs in control leukemic bone marrow in comparison with 1/442 in SgPBX3-a and 1/741 in SgPBX3-b respectively. Further analysis suggested that depletion of functional LSCs in PBX3 inactivation group mainly through accelerating apoptosis of LSCs, evidenced by both increased Annexin V positive cell frequency of whole BM blasts and LSCs in PBX3 inactivation group.In conclusion, we found PBX3 is epigenetically dis-regulated in LSCs of MLL-r AMLs and is essential for leukemia development. Inactivation of PBX3 could suppress leukemia progression by reducing LSCs frequency and impairing its function. More importantly, the differential expression of PBX3 in normal and malignant hematopoietic cells provides itself potentially as a prognostic marker and therapeutic target for MLL-rearranged leukemia.HD.G and YJ.C contributed equally to this work DisclosuresNo relevant conflicts of interest to declare.

ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL

Introduction: Acute lymphoblastic leukemia is the most common form of pediatric cancer, and a translocation between chromosomes 1 and 19 represents one of the most frequent cytogenetic abnormalities, accounting for ~5% of ALL cases. The 1;19 rearrangement results in a fusion between the E2A and PBX1 genes, and pre B-ALL cells carrying the E2A-PBX1 fusion are consistently arrested at an intermediate stage of B cell lineage development in which the cells express a functional pre B-cell receptor (pre-BCR). We have previously shown that receptor orphan tyrosine kinase receptor 1 (ROR1) is highly expressed on t(1;19) pre B-ALL blasts and required for their viability, even though ROR1 is not directly regulated by either the E2A-PBX1 fusion or the pre-BCR. Moreover, ROR1 exhibits compensatory signaling cross-talk with the pre-BCR such that maximal killing of t(1;19) pre B-ALL cells is achieved by combining silencing of ROR1 with siRNA and inhibition of the pre-BCR with kinase inhibitors such as dasatinib. However, clinical employment of this strategy is hampered as ROR1 is a pseudokinase whose biology is poorly understood and no ROR1 small-molecule inhibitors currently exist. Therefore, it is critical to advance our understanding of the upstream regulatory mechanisms responsible for ROR1 expression to uncover new therapeutic strategies for t(1;19) pre B-cell ALL.Methods: To identify potential regulators of ROR1 expression, we designed two siRNA panels targeting transcription factors predicted to bind the ROR1 locus or an array of chromatin remodeling factors and epigenetic regulators. Pre B-ALL cell lines that are positive or negative for the 1;19 rearrangement were screened with these siRNA panels and any gene targets required for the viability of t(1;19) pre B-cell ALL cells but not control cells lines were considered candidate regulators of ROR1 expression. Candidates were validated for the capacity to regulate ROR1 expression by qRT-PCR and immunoblot.Results: Screening of the t(1;19) pre B-cell ALL cell line, RCH-ACV, with our siRNA panels targeting transcription factors and chromatin remodeling/epigenetic regulators identified four potential regulators of ROR1 that were required for viability of RCH-ACV cells but not our negative control cell line, REH (t(12;21)-positive). Among these four candidates was UHRF1, which interacts with histone and DNA methyltransferases. Interestingly, silencing of UHRF1 by siRNA led to reduced ROR1 protein levels, but had no significant impact on ROR1 mRNA expression. Since one primary function of UHRF1 is to regulate DNA methylation, we hypothesized that UHRF1 regulates ROR1 protein expression through an intermediate gene whose transcription is impacted by UHRF1-mediated DNA methylation. To test this hypothesis, RCH-ACV cells were treated with the RNA synthesis inhibitor actinomycin D 48 hours after silencing UHRF1. This resulted in a transient rescue of ROR1 protein levels, indicating that UHRF1 regulation of ROR1 protein depends on the transcription of an intermediate gene whose gene product subsequently regulates ROR1 protein levels.Conclusions: Data from siRNA screens and subsequent validation experiments suggest that UHRF1 functions to regulate ROR1 protein levels, but not mRNA expression. This is intriguing due to the primary role of UHRF1 as a modulator of DNA and histone methylation. The observed rescue of ROR1 protein with actinomycin D suggests that UHRF1 regulation of ROR1 protein occurs through an undefined intermediary whose locus is methylated in a UHRF1-dependent manner and whose gene product modulates ROR1 protein levels. Inhibitors of additional stages of gene transcription, translation, and stabilization are currently being tested to continue characterizing UHRF1-dependent regulation of ROR1. Cumulatively, this work has identified a novel regulatory mechanism of ROR1 protein expression by UHRF1 in t(1;19) pre B-cell ALL. In addition to advancing our understanding of ROR1 biology, this regulatory mechanism suggests a new therapeutic strategy to target ROR1-expressing cancers. DisclosuresTyner:Incyte: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding.

Associations of Polymorphisms in WNT9B and PBX1 with Mayer-Rokitansky-Küster-Hauser Syndrome in Chinese Han.

BackgroundMayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare syndrome that is characterized by congenital aplasia of the uterus and the upper portion (2/3) of the vagina. Previous attempts to identify causal mutations of MRKH syndrome have primarily resulted in negative outcomes. We investigated whether these reported variants are associated with MRKH syndrome (types I and II) in a relatively large sample size of Chinese Han patients, and whether any gene-gene epistatic interactions exist among these variants.MethodsThis study included 182 unrelated Chinese women with MRKH syndrome (155 with type I and 27 with type II) and 228 randomized female controls. Seventeen candidate loci in the AMH, PBX1, WNT4, WNT7A, WNT9B, HOXA10, HOXA11, LHXA1 and GALT genes were genotyped using the Sequenom MassARRAY iPLEX platform. Single-marker association, additive effects and multifactor interactions were investigated.ResultsThe gene frequency distributions of MRKH type 1 and type 2 were similar. Rs34072914 in WNT9B was found to be associated with MRKH syndrome (P = 0.024, OR = 2.65, 95%CI = 1.14–6.17). The dominant models of rs34072914 and rs2275558 in WNT9B and PBX1, respectively, were significantly associated with MRKH syndrome risk in the Chinese Han patients. Additive gene-gene interaction analyses indicated a significant synergetic interaction between WNT9B and PBX1 (RERI = 1.397, AP = 0.493, SI = 4.204). Multifactor dimensionality reduction (MDR) analysis revealed novel dimensional epistatic four-gene effects (AMH, PBX1, WNT7A and WNT9B) in MRKH syndrome.ConclusionsThis association study successfully identified two susceptibility SNPs (WNT9B and PBX1) associated with MRKH syndrome risk, both separately and interactively. The discovery of a four-gene epistatic effect (AMH, PBX1, WNT7A and WNT9B) in MRKH syndrome provides novel information for the elucidation of the genetic mechanism underlying the etiology of MRKH syndrome.

EWSR1‐PBX3: A novel gene fusion in myoepithelial tumors

The genetics of myoepithelial tumors (ME) of soft tissue and bone have recently been investigated, with EWSR1-related gene fusions being seen in approximately half of the tumors. The fusion partners of EWSR1 so far described include POU5F1, PBX1, ZNF444 and, in a rare case, ATF1. We investigated by RNA sequencing an index case of EWSR1-rearranged ME of the tibia, lacking a known fusion partner, and identified a novel EWSR1-PBX3 fusion. The fusion was further validated by reverse transcriptase polymerase chain reaction and fluorescence in situ hybridization (FISH). To evaluate if this is a recurrent event, an additional cohort of 22 EWSR1-rearranged ME cases lacking a fusion partner were screened by FISH for abnormalities in PBX3 gene. Thus, two additional cases were identified showing an EWSR1-PBX3 gene fusion. One of them was also intraosseous involving the ankle, while the other occurred in the soft tissue of the index finger. The morphology of the EWSR1-PBX3 fusion positive cases showed similar findings, with nests or sheets of epithelioid to spindle cells in a partially myxoid to collagenous matrix. All three cases showed expression of S100 and EMA by immunohistochemistry. In summary, we report a novel EWSR1-PBX3 gene fusion in a small subset of ME, thereby expanding the spectrum of EWSR1-related gene fusions seen in these tumors. This gene fusion seems to occur preferentially in skeletal ME, with two of the three study cases occurring in intraosseous locations.

Overexpression of Pbx1-d, a novel splice isoform of Pbx1, changes the portion of follicular CD4 T cell populations and impairs regulatory T cell homeostasis (BA14P.201)

Abstract We have previously shown that the differential expression of a novel splice isoform, Pbx1-d, of the Pbx1 gene was associated with the production of autoreactive CD4+ T cells in the Sle1a1 lupus susceptibility locus and lupus susceptibility in the B6.Sle1.Sle2.Sle3 mouse model. Pbx1 regulates the transcription of a large number of genes by controlling chromatin accessibility. Pbx1-d is the only known Pbx1 isoform lacking the DNA binding domain, which suggests a dominant negative function, and its expression is more frequently in the CD4+ T cells from lupus patients than in healthy controls. With the hypothesis that Pbx1-d contributes to lupus development by regulating CD4+ T cell effector functions through novel mechanisms, we have generated B6.CD4-Pbx1-d Tg (Pbx1-d Tg) mice, which specifically express Pbx1-d in CD4+ T cells. These Tg mice reproduce the phenotypes of B6.Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired Treg cell homeostasis. Interestingly, the composition of follicular helper and regulatory T cell (Tfh and Tfr) populations, is different in the Pbx1-d Tg mice as well as the B6.Sle1.Sle2.Sle3 mice as compared with wild-type mice. Currently, we are investigating the mechanisms by which Pbx1 regulates Treg homeostasis and follicular T cell differentiation in the Pbx1-d Tg mice, as well as its role in autoimmune pathology in the B6.Sle1.Sle2.Sle3 mice.

Clinical features and implications of childhood acute lymphoblastic leukemia with E2A/PBX1 fusion gene

Objective To explore the clinical features and prognosis in childhood acute lymphoblastic leukemia(ALL) with E2A/PBX1 fusion gene. Methods From Jan.2007 to Dec.2011, E2A/PBX1 fusion gene from bone marrow sample in 450 newly diagnosed childhood patients with ALL was evaluated by nested reverse transiption-polymerase chain reaction (RT-PCR). Cases with B-lineage ALL accounted for 4.76% (20/420 cases). The patients with E2A/PBX1 gene were classified to the intermediate-risk group, and 85 cases with negative E2A/PBX1 gene were regarded as control group at the same time.Clinical characteristics, 5-year event-free survivial (EFS) rate and overall survi-vial(OS) rate were compared between 2 groups. Results Statistic differences were not found in age, gender, initial white blood cell count, hemoglobin level between 2 groups(all P>0.05). Level of minimal residual disease(MRD)was lower in E2A/PBX1 fusion gene group than that in control group at day 33 of induction chemotherapy(χ2=15.78, P=0.001). The 5-year EFS rates were (84.4±15.6)% and (73.5±15.6)%, respectively in 2 groups.The OS rates were (86.0±17.6)% and (81.8±17.6)%, respectively.The relapse rates were 10.5% and 12.9% and the median times of relapse were(8.9±5.3)months and(26.2±8.0)months, respectively.There was no significant differences in EFS rates, OS rates, relapse rates and median times of relapse between 2 groups(all P>0.05). Conclusions There was no significant differences between the E2A/PBX1 gene group and control group.E2A/PBX1 fusion gene plays an intermediate-risk role in pediatric B-ALL. Key words: Acute lymphoblastic leukemia; Child; E2A/PBX1 fusion gene; Clinical characteristics; Prognosis

The atypical homeoprotein Pbx1a participates in the axonal pathfinding of mesencephalic dopaminergic neurons

BackgroundThe pre B-cell leukemia transcription factor 1 (Pbx1) genes belong to the three amino acid loop extension family of homeodomain proteins that form hetero-oligomeric complexes with other homeodomain transcription factors, thereby modulating target specificity, DNA binding affinity and transcriptional activity of their molecular associates.ResultsHere, we provide evidence that Pbx1 is expressed in mesencephalic dopaminergic neurons from embryonic day 11 into adulthood and determines some of the cellular properties of this neuronal population. In Pbx1-deficient mice, the mesencephalic dopaminergic axons stall during mid-gestation at the border between di- and telencephalon before entering the ganglionic eminence, leading to a loose organization of the axonal bundle and partial misrouting. In Pbx1-deficient dopaminergic neurons, the high affinity netrin-1 receptor, deleted in colon cancer (DCC), is down-regulated. Interestingly, we found several conserved Pbx1 binding sites in the first intron of DCC, suggesting a direct regulation of DCC transcription by Pbx1.ConclusionsThe expression of Pbx1 in dopaminergic neurons and its regulation of DCC expression make it an important player in defining the axonal guidance of the midbrain dopaminergic neurons, with possible implications for the normal physiology of the nigro-striatal system as well as processes related to the degeneration of neurons during the course of Parkinson’s disease.

DifferentialHoxExpression in Murine Embryonic Stem Cell Models of Normal and Malignant Hematopoiesis

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFRβ. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFRβ) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis.