PAX5 Research Articles

The Function and Developmental Expression of Alternatively Spliced Isoforms of Amphioxus and Xenopus laevis Pax2/5/8 Genes: Revealing Divergence at the Invertebrate to Vertebrate Transition

Pax genes encode highly conserved transcription factors vital for metazoan development. Pax transcripts, particularly those in Group II (Pax2/5/8), are extensively alternatively spliced. This study compares the transcriptional activation capacity and developmental stage-specific expression of major isoforms of Group II Pax proteins in amphioxus (Branchiostoma floridae) and in Xenopus laevis. The comparison reveals considerable divergence of splice forms between the lineages, with the X. laevis Group II Pax genes (Pax2, Pax5, and Pax8) possessing a greater repertoire of regulated and functionally distinct splice forms than the single amphioxus gene (Pax2/5/8). Surprisingly, some apparently conserved splice forms are expressed at quite different levels during development in the two organisms and present different capacities to activate transcription. However, despite this divergence, the combinatorial transcriptional activation capacity of the isoforms present in early X. laevis and amphioxus development are broadly similar. This suggests that the some of the conserved functional roles, implied by the expression of Group II Pax genes in homologous tissues of amphioxus and X. laevis embryos, may depend upon the combination of isoforms expressed in a particular tissue at a particular time in development. Thus, during early development, the evolutionary constraint on the net effect of several isoforms co-expressed in a given tissue may be more strict than that on specific isoforms. This flexibility may facilitate the appearance of new exons and splicing patterns in the vertebrate duplicates, leading to isoforms with subtly distinct functions critical to the subsequent development of vertebrate-specific cell types and structures.

Promoter hypermethylation in male breast cancer: analysis by multiplex ligation-dependent probe amplification

IntroductionEpigenetic events are, along with genetic alteration, important in the development and progression of cancer. Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. The role of promoter hypermethylation in male breast cancer has not yet been studied.MethodsIn a group of 108 male breast cancers, the methylation status of 25 genes was studied using methylation-specific multiplex ligation-dependent probe amplification. Methylation of more than 15% was regarded indicative for promoter hypermethylation. Methylation status was correlated with clinicopathological features, with patients' outcome and with 28 female breast cancer cases.ResultsPromoter hypermethylation of the genes MSH6, WT1, PAX5, CDH13, GATA5 and PAX6 was seen in more than 50% of the cases, but was uncommon or absent in normal male breast tissue. High overall methylation status was correlated with high grade (P = 0.003) and was an independent predictor of poor survival (P = 0.048; hazard ratio 2.5). ESR1 and GSTP1 hypermethylation were associated with high mitotic count (P = 0.037 and P = 0.002, respectively) and high grade (both P = 0.001). No correlation with survival was seen for individual genes. Compared with female breast cancers (logistic regression), promoter hypermethylation was less common in a variety of genes, particularly ESR1 (P = 0.005), BRCA1 (P = 0.010) and BRCA2 (P < 0.001). The most frequently hypermethylated genes (MSH6, CDH13, PAX5, PAX6 and WT1) were similar for male and female breast cancer.ConclusionPromoter hypermethylation is common in male breast cancer and high methylation status correlates with aggressive phenotype and poor survival. ESR1 and GSTP1 promoter hypermethylation seem to be involved in development and/or progression of high-grade male breast cancer. Although female and male breast cancer share a set of commonly methylated genes, many of the studied genes are less frequently methylated in male breast cancer, pointing towards possible differences between male and female breast carcinogenesis.

Evolutional principles of homology in regulatory genes of myogenesis

Analysis of early steps in muscular system development of invertebrates and vertebrates shows that early steps of myogenesis are regulated by genes-orthologs mainly belonging to two families, Pax and bHLH. In the majority of the following organisms, muscles formation (steps of determination and the earliest steps of myogenesis) is regulated by genes orthologs Pax3 which belong to the family Pax: nematodes (Caenorhabditis elegans, Pristionchus pacificus), insects (Drosophila melanogaster), echinoderms (Strongylocentrotus purpuratus), sea squirts (Ciona intestinalis, Holocynthia roretzi), fishes (Danio rerio), amphibians (Xenopus laevis), birds, and mammals (mouse, rat). The nematode C. elegans is an exception since formation of its muscles in this period is regulated by homeobox gene Pal-1 belonging to the family Caudal. The sea squirt C. intestinalis is also an exception because the earliest steps of development involved in further muscle formation are accompanied by activation of the gene CiSna (snail) (gene family basic Zinc finger). The next steps of myogenesis in all analyzed species are regulated by genes orthologs belonging to the family of transcriptional factors bHLH. They along with genes Pax3 are characterized by a high extent of homology in all studied groups of animals.

Expression of pax5 gene in wildtype zebrafish embryos

Objective To study the role and related mechanisms of pax5 gene during the development of the embryos,especially in the early development of B cell and midbrain,and to observe the expression of pax5 gene in wildtype zebrafish embryos.Methods Total RNA of Tuebingen wildtype zebrafish embryos was extracted to obtain cDNA of pax5 gene by RT-PCR with specific primers.The cDNA of pax5 gene and pCS2+ vector were double digested with EcoRⅠ and XbaⅠ,and then ligated by T4 DNA ligase.The recombinant vector was verified by double digestion,colony PCR screening and sequencing.The verified recombinant vector was then used to synthesize digoxin labeled anti-sense mRNA probe of pax5 gene using T3 RNA polymerase in vitro transcription system.The generated probes were used to detect pax5 gene expression in zebrafish embryos by whole-mount in situ hybridization.Results The pCS2+-pax5 recombinant plasmid and the probe of digoxin-labeled anti-sense mRNA of pax5 gene was successfully constructed.Whole-mount in situ hybridization showed that pax5 gene was expressed in the cerebellum and midbrain hindbrain boundary from 18-72 h post-fertilization(hpf).In the cochlea pax5 gene was expressed from 24-72 hpf and the expression was increased as time went by.From 18-48 hpf pax5 gene is found in the notochord.Conclusion Pax5 gene is highly expressed in the brain and notochord of zebrafish embryos.Pax5 gene expression is found in the cochlea of the Tuebingen wildtype zebrafish's embryos for the first time.It is suggested that pax5 may play an important role in the development of the nervous system in the early zebrafish embryos.

Characterization and Manipulation of NZB miR15a/16-1 loci for CLL Therapy (46.50)

Abstract Chronic Lymphocytic Leukemia (CLL) is a malignancy characterized by accumulation of mature CD5+ B-cells in peripheral lymphoid organs, bone marrow and blood and is incurable with conventional therapy. We have previously shown that similar to human B-CLL cells, NZB mice (de novo mouse model of CLL) have 50% less level of miR15a/16-1 that is associated with a mutation and deletion in the miR15a/16-1 region. The NZB loci lead to a processing defect inhibiting the conversion of pri-miR15a/16-1 to pre-miR15a/16-1. We hypothesized that exogenously increasing its expression would lead to increased apoptosis in tumor cells. Using a lentiviral delivery system, we were able to stably increase the level of these two microRNAs in LNC (NZB derived B-CLL cell line). The apoptosis observed in miR-GFP transduced LNC was twice as much as in LNC transduced with GFP lentivirus (control). In addition to the miR defect, B-CLL cells have a very high level of B-cell specific activator protein (BSAP) and this prevents their terminal differentiation. Recent reports have suggested that BSAP and miR15a/16-1 form an autoregulatory loop. Alternatively, we found that the miR defect can also be corrected by knocking down BSAP (using RNAi) leading to a cell cycle arrest. The second strategy is clinically relevant, since developing a BSAP inhibitor is more feasible than systemic miR delivery. Based on our findings we propose that targeting BSAP could be used as a novel therapeutic strategy for B-CLL.

Marking the tempo for myogenesis: Pax7 and the regulation of muscle stem cell fate decisions

Post-natal growth and regeneration of skeletal muscle is highly dependent on a population of resident myogenic precursors known as satellite cells. Transcription factors from the Pax gene family, Pax3 and Pax7, are critical for satellite cell biogenesis, survival and potentially self-renewal; however, the underlying molecular mechanisms remain unsolved. This is particularly true in the case of Pax7, which appears to regulate myogenesis at multiple levels. Accordingly, recent data have highlighted the importance of a functional relationship between Pax7 and the MyoD family of muscle regulatory transcription factors during normal muscle formation and disease. Here we will critically review key findings suggesting that Pax7 may play a dual role by promoting resident muscle progenitors to commit to the skeletal muscle lineage while preventing terminal differentiation, thus keeping muscle progenitors poised to differentiate upon environmental cues. In addition, potential regulatory mechanisms for the control of Pax7 activity will be proposed.

Abstract 5618: Tumor suppressor genes? The status of genes PAX 5&amp;6 in primary and secondary gliomas and glioblastoma multiforme tumors

Abstract Background: Gliomas are the most common brain tumors, and glioblastoma multiforme (GBM) tumors have the poorest prognosis, and due to their molecular heterogeneity, these tumors remain incurable and highly resistant to treatments, in most cases. The PAX (paired box) family of genes is comprised of are nine members that are subdivided based on the products of their protein domain structures. PAX5 and PAX6 are developmental genes that are vital during embryonic stages of development, and their transcription factors have supportive, if not, key functions in tumorigenesis. Interestingly, gene PAX6 has been reported to be over and under expressed in high grade gliomas, GBMs, and glioma cell lines. However, these results could be an effect of upstream or downstream manipulation of other transcription factor(s), the origin of the tissue (PAX6 expression is restricted to the forebrain, optic cup, hindbrain, and spinal cord, lens placode, and nasal epithelium), and the epigenetic regulation of some other gene. In addition, there are no reported mutations in either gene PAX5 or PAX6 in the cells from GBMs. Therefore, confusion exists as to role these genes have in tumorigenesis, and whether either gene has the properties or characteristics of a tumor suppressor gene. Study Objective: The overall study objective was to determine if either PAX5 or PAX6 are tumor suppressor genes, and can be silenced or over-expressed to inhibit the progression of cultured human adult primary glioma tissue samples, by employing small interfering RNA (siRNA) methodology. Methods: Primary cell cultures were incubated in reduced serum conditions for at least 1 month prior to transfections and cells were passed every 7 to 10 days or when flask was 90% confluent. To investigate and measure true inhibition of cell proliferation or progression of the cells, several techniques were used, such as, real-time quantitative reverse transcription PCR, enzyme-linked immunosorbent assays, and immunohistochemistry. Results: The high grade (IV) primary glioma tissue samples and their respective cell cultures showed relatively high expression levels for the gene PAX6 and lower levels of expression for gene PAX5 when compared to GAPDH gene and standard controls. Our initial transfections for knocking down of PAX6 using siRNA (TriFECTA, IDT) showed 90% of control samples were transfected, and our primary cells were knockdown by 82%, 70%, and 65% for each of PAX6 isoforms (N000280.21.1-S, 2-S, and 3-S) respectively. We are currently analyzing the rest of the PAX6 data and all of the PAX5 data. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5618. doi:1538-7445.AM2012-5618

Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

BackgroundRetinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers) during treatment of human retinal pigment epithelium (RPE) cells with amniotic fluid (AF), RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age.ResultsCompared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1) confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation.ConclusionTaken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

Expression of essential B cell development genes in horses with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p<0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p<0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients.

Parallel reduction in expression of the eye development gene hedgehog in separately derived cave populations of the amphipod Gammarus minus

Caves provide excellent settings to examine evolutionary questions. Subterranean environments are characterized by similar and consistent conditions. Cave-adapted species often share characteristics such as diminished pigmentation, elongated limbs and reduced or absent eyes. Relatively little is known about the evolution and development of troglomorphic traits in invertebrates. In this study, we compare expression of the eye development genes hedgehog, pax6, sine oculis and dachshund in individuals from multiple independently derived cave populations of the amphipod Gammarus minus. hedgehog expression was significantly reduced in cave populations, compared to genetically related surface populations. Interestingly, no differences were found in pax6, sine oculis or dachshund expression. Because hedgehog-related genes are also involved in eye reduced in Astyanax mexicanus, these genes may be consistent targets of evolution during cave adaptation. These results provide support for the hypothesis of genomic 'hotspots' of evolution and allow comparison of adaptive mechanisms among diverse animals in subterranean environments.

The Temporal Sequence of the Mammalian Neocortical Neurogenetic Program Drives Mediolateral Pattern in the Chick Pallium

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.

The Pax-5 Gene: A Pluripotent Regulator of B-cell Differentiation and Cancer Disease

The Pax-5 oncogene encodes a potent transcription factor that plays a key role in B-cell development and cancerous processes. In normal B-lymphopoiesis, Pax-5 accomplishes a dual function by activating B-cell commitment genes while concomitantly repressing non-B-lineage genes. Given the pivotal importance of Pax-5-mediated processes in B-cell development, an aberrant regulation of Pax5 expression has consistently been associated with B-cell cancers, namely, lymphoma and lymphocytic leukemias. More recently, Pax-5 gene expression has been proposed to influence carcinogenic events in tissues of nonlymphoid origin by promoting cell growth and survival. However, in other cases, Pax-5 products have opposing effects on proliferative activity, thus redefining its generally accepted role as an oncogene in cancer. In this review, we attempt to summarize recent findings about the function and regulation of Pax-5 gene products in B-cell development and related cancers. In addition, we present new findings that highlight the pleiotropic effects of Pax-5 activity in a number of other cancer types.

Absence of mutations in four genes encoding for congenital cataract and expressed in the human brain in Tunisian families with cataract and mental retardation

BackgroundTo identify the genetic defect associated with autosomal recessive congenital cataract (ARCC), mental retardation (MR) and ARCC, MR and microcephaly present in most patients in four Tunisian consanguineous families.MethodsWe screened four genes implicated in congenital cataract by direct sequencing in two groups of patients; those affected by ARCC associated to MR and those who presented also microcephaly. Among its three genes PAX6, PITX3 and HSF4 are expressed in human brain and one gene LIM2 encodes for the protein MP20 that interact with the protein galectin-3 expressed in human brain and plays a crucial role in its development. All genes were screened by direct sequencing in two groups of patients; those affected by ARCC associated to MR and those who presented also microcephaly.ResultsWe report no mutation in the four genes of congenital cataract and its flanking regions. Only variations that did not segregate with the studied phenotypes (ARCC associated to MR, ARCC associated with MR and microcephaly) are reported. We detected three intronic variations in PAX6 gene: IVS4 -274insG (intron 4), IVS12 -174G>A (intron12) in the four studied families and IVS4 -195G>A (intron 4) in two families. Two substitutions polymorphisms in PITX3 gene: c.439 C>T (exon 3) and c.930 C>A (exon4) in one family. One intronic variation in HSF4 gene: IVS7 +93C>T (intron 7) identified in one family. And three intronic substitutions in LIM2 gene identified in all four studied families: IVS2 -24A>G (intron 2), IVS4 +32C>T (intron 4) and c.*15A>C (3'-downstream sequence).ConclusionAlthough the role of the four studied genes: PAX6, PITX3, HSF4 and LIM2 in both ocular and central nervous system development, we report the absence of mutations in all studied genes in four families with phenotypes associating cataract, MR and microcephaly.

Successive Development of T-Cell Acute Lymphoblastic Leukemia, Non-Langerhans-Cell Histiocytose and Histiocytic Sarcoma with a Common Origin: Evidence for Common Oncogenetic Transformation Before Full Lineage Commitment,

Abstract 3470Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into lineage committed cells. Recent data are suggestive for some degree of lineage flexibility in both normal as malignant cells. We here present a boy that presented with T-cell acute lymphoblastic leukemia (T-ALL). During T-ALL treatment, the patient developed in short time a non-Langerhans-cell histiocytose in the ileum, and subsequently a disseminated form of histiocytic sarcoma (a.o. also in the liver). Interestingly, the same clonal T cell receptor (TCR) gene rearrangements were found in all three malignancies (TCRB-VJ, TCRG-VJ and TCRB-DJ) indicating they were related. There were no immunoglobulin rearrangements. To understand the evolution from T-ALL to non-Langerhans-cell histiocytose and to histiocytic sarcoma, an extensive genetic analysis was performed. Using a SNP6.0 array platform we analyzed DNA isolated from T-ALL cells, a bone marrow sample obtained during complete remission from theT-ALL, a biopsy from the ileum tumor, the liver tumor, and DNA isolated from skin fibroblasts.The three tumor samples were not fully concordant in the SNP analysis, with lesions acquired but also absent from subsequent samples. All three tumor samples showed identical TCR gene rearrangements as well as a loss of the CDKN2A/B region, the only gene-containing copy number aberration (CNA) present in the T-ALL. The T-ALL sample showed a homozygous loss of the CDKN2A/B region, whereas both the ileum and liver samples showed a heterozygous loss. In addition, the three tumor samples shared two 1Mb regions of loss of heterozygosity (LOH) on chromosomes 6p and 11p, containing DDB2, MADD and RUNX2, amongst others. A gain on chromosome 19q containing 12 genes was shared between the ileum and liver sample only. In addition, the ileum tumor showed 14 non-shared CNAs of which 11 losses, 2 homozygous losses, and 1 gain, affecting 11 genes including ZFAT, PTPRK and beta-catenin (CTNNB1). The liver tumor acquired gains of a large part of chromosome 5, the p-arm of chromosome 11 and the q-arm of chromosome 22 reminiscent of a chromothrypsis event. The PAX5 gene indicated in the progression of an ALL to histiocytic sarcoma was not affected in either of the samples. These results indicate that the three tumors are related but did not evolve from each other in a linear manner. Apparently, the common oncogenetic transformation occurred at an early differentiation stage with lineage plasticity, i.e. with some lineage specification but without full lineage commitment. Disclosures:No relevant conflicts of interest to declare.

PAX5 Is a Frequently Altered Transcription Factor In Genetically Unclassified Childhood Common/B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract 1458 Introduction:Genetic lesions define treatment and prognosis in childhood common/B cell-precursor Acute Lymphoblastic Leukemia (c/pre-B ALL) (Pui CH et al., J Clin Oncol, 2011). However, up to 40% of childhood pre-B ALL cases, lack the presence of prognostically significant abnormalities; and relapses are common in this group (Mejerink J et al., Semin Hem, 2009). Molecular genetic characterization of genetically unclassified ALL is critical for a better understanding of the biology of this disease, to determine new prognostic markers and to optimize treatment for this group of patients. Several transcription factors, including EBF1, IKZF1 and PAX5, which regulate B-cell development, have recently been shown often to be altered in B-ALL (Mullighan CG et al., Nature, 2007; den Boer ML et al., Lancet, 2009). We decided to investigate the incidence of abnormalities involving B-cell transcription factors, in a cohort of patients with childhood c/pre-B ALL lacking significant prognostic genetic aberrations. Materials and methods:60 patients diagnosed with c/pre-B ALL in Great Ormond Street Hospital (London, UK) from 1994 to 2011 were included in the study. The median age was 3.43 (range: 1.14–12.24), with a median white blood cell count of 14.0 (range: 1.36–213.2). All patients showed an absence of high-hyperploidy/hypodiploidy, BCR-ABL1, MLL, ETV6-RUNX1 and E2A rearrangements, and also i(amp)21 by karyotype, fluorescence in-situ hybridization (FISH) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) investigations. We performed FISH using commercially available probes and BAC clones, in parallel with RT-PCR, to detect deletions/rearrangements of the EBF1, IKZF1 and PAX5 gene regions. Direct sequence analysis was performed for all exons of the PAX5 gene. Real-time PCR was used to determine the level of expression of PAX5 and its transcriptional targets, mostly components of the pre-B cell receptor (pre-BCR) complex including CD19, CD79α, VpreB, BLNK and BLK genes. Western blotting was further performed for the identification of an abnormal PAX5 protein. Results:25/60 patients (42%) showed abnormalities involving PAX5, 2/60 patients (3%) showed deletions of IKZF1. No deletions/rearrangements of EBF1 were detected. Several forms of PAX5 deletions were observed: the entire loss of one PAX5 allele (32%), loss of 5’PAX5 (20%), loss of 3’PAX5 (12%) and loss of 3’PAX5 due to unbalanced rearrangements (8%). Balanced PAX5 rearrangements comprise 12% of the PAX5 alterations. Sequence analysis revealed 9 somatic mutations of PAX5 in 8/60 patients (13%): 6 frameshifts, 2 splice-site and 1 non-stop mutations. The mutations were located in functional exons of PAX5 and resulted in the initiation of premature stop-codons, exon skips, and the formation of abnormal transcripts. Interestingly, 2 patients with an identical splice site mutation, showed the same karyotypic abnormality of an isochromosome of the long arm of chromosome 9 leading to loss of the second PAX5 allele. Moreover, further investigation DNA and mRNA from remission samples demonstrated that in one case the mutation was acquired, and that in the second case the mutation was a germ-line variant. In total, 20% of patients with PAX5 abnormalities had bi-allelic alterations of the PAX 5 gene. PAX5, BLNK and BLK gene-expression levels were lower (p<0.05) in the group of patients with loss of one entire PAX5 allele. There were no significant changes in expression levels of the tested genes in patients with partial PAX5 deletions, rearrangements or mutations. Expression of an abnormal PAX5 protein was confirmed by western blot analysis. Conclusion:The high incidence of PAX5 alterations in childhood c/pre-B ALL, without prognostically significant genetic abnormalities, suggests a pathogenetic role for PAX5 in these acute lymphoblastic leukemia cases. Down-regulation of expression of the BLNK and BLK genes could be one of the mechanisms by which abnormal PAX5 blocks B-cell differentiation and contributes to the initiation of leukemia. Disclosures:No relevant conflicts of interest to declare.

Ectopic Expression of the Leukemogenic Protein E2A-PBX1 in Early Hematopoietic Progenitors Blocks B-Lymphoid Commitment

Abstract 1393The oncoprotein E2A-PBX1 is produced by t(1;19)(q23;p13) in pre-B acute lymphoblastic leukemia (ALL). Perplexingly, in vitro and in vivo models of E2A-PBX1 leukemogenesis to date generate either myeloid or T-lymphoblasts, neither of which are representative of the human disease. Furthermore, we have observed recently that lineage-depleted (lin-) murine bone marrow cells infected with an E2A-PBX1 retrovirus selectively fail to repopulate the B-lymphoid compartment on transplantation into irradiated recipients. Therefore, we have now begun to investigate the mechanism by which enforced expression of E2A-PBX1 antagonizes B-lymphopoiesis.We show that E2A-PBX1-transduced lin- fetal liver progenitor cells (FLPs) fail to differentiate to committed CD45R+ pro-B-cells when cultured with IL-7 in the presence of OP9 stromal cells. Instead these cells manifest a relatively immature immunophenotype (CD45R-, CD11b-, CD117+), are murine stem cell factor (SCF) dependent, and undergo myeloid differentiation upon stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF) or transplantation into irradiated mice. Amino acid substitutions in E2A-PBX1 that impair co-activator recruitment or DNA binding completely rescue the differentiation block, implying a critical role for target-gene regulation by E2A-PBX1. E2A-PBX1-transduced cells fail to induce the B-lymphoid commitment genes Ebf1 and Pax5, and chromatin immunoprecipitation (ChIP) analysis shows aberrant persistence of the Polycomb silencing mark H3K27me3 at these promoters. Previous studies have identified the Polycomb gene Bmi1 as a candidate E2A-PBX1 target gene. However, enforced expression of Bmi1 neither blocked induction of Ebf1 or Pax5 nor impaired B-lymphopoiesis, indicating that Bmi1 is not sufficient to mediate blocked differentiation by E2A-PBX1. Gene expression microarray analysis identified cytokines (ex., Csf2, IL1a, Il6) and transcription factors (HoxA9, Tal1) up-regulated in E2A-PBX1-transduced FLPs that are known to antagonize B-lymphoid development.In summary, our results support the notion that enforced expression of E2A-PBX1 in early hematopoietic progenitors blocks B-lymphopoiesis, to an important degree, by inducing the transcription of genes whose products antagonize early steps of B-lymphoid commitment, including induction of Ebf1 and Pax5. HoxA9 is particularly interesting as a mediator of E2A-PBX1 effects in view of its extreme induction ratio in our system (at least 1000-fold relative to controls), its propensity to promote myeloid over lymphoid differentiation, and its well-established role in leukemogenesis. Studies to investigate a potential requirement for HoxA9 and other candidates in mediating the E2A-PBX1-driven B-lymphoid differentiation block are ongoing. Disclosures:No relevant conflicts of interest to declare.

The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Abstract 397Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER (Ung−/−) and MMR (Msh2−/−). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung−/−Msh2−/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung−/−Msh2−/−mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p<0.0001). All 5 of the Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung−/−Msh2−/−mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung−/−Msh2−/−mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung−/−Msh2−/−tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung−/−Msh2−/−mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung−/−Msh2−/−tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes (cMyc, Pim1, RhoH, Pax5, Cd79a, Fas, H2ax and OcaB) in tumors from 3 Iμ-Bcl6 Ung−/−Msh2−/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10−4, suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung−/− and Iμ-Bcl6 Msh2−/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures:No relevant conflicts of interest to declare.

Lentiviral Vector-MediatedPAX6Overexpression Promotes Growth and Inhibits Apoptosis of Human Retinoblastoma Cells

The cancer-associated gene PAX6 is a key regulator in the embryological development of the retina. The authors assessed whether PAX6 was associated with the development of retinoblastoma. Methods. Two human retinoblastoma cell lines (Y79 and SO-Rb50) were transfected with PAX6-GFP recombinant lentiviral vectors and were compared with cells undergoing transfection with GFP lentiviral vectors and cells without any intervention. Overexpression of PAX6 was assessed by quantitative real-time polymerase chain reaction analysis and Western blot analysis. Cell proliferation assays were evaluated by colorimetric cell counting kit-8. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Apoptosis rates were assessed by TUNEL assay followed by FACS analysis. Using Western blot analysis, the authors measured levels of proteins p53, p21, p27, cdc2, proliferating cell nuclear antigen, and cleaved caspase-3. Three days after transfection, both cell lines showed a statistically significant (P < 0.001) overexpression of PAX6, parallel to significantly (P < 0.001) increased cell proliferation. At 7 days after transfection, cell cycle analysis showed a significant (P < 0.001) reduction of G0/G1 arrest and a significant induction of G2/M arrest (P < 0.01). Parallel to a reduction in caspase-3 levels, the apoptosis rate significantly (P < 0.001) decreased. Levels of p53, p21, and p27 were reduced, and the levels of cdc2 were increased. Lentiviral vector-mediated overexpression of PAX6 in human retinoblastoma cells was associated with increased cell proliferation parallel to a reduced caspase-3-dependent apoptotic rate and a change in the p53 regulated cell cycle. PAX6 may be further explored for the diagnosis of and therapy for retinoblastomas.

Coordinated Expression of Pax-5 and FAK1 in Metastasis

The Pax-5 gene encodes a B-cell-specific activator protein (BSAP) that plays a key role in B lymphocyte differentiation and embryogenesis. The deregulation of this transcription factor is also linked to B cell malignancies and recently to other cancers. More specifically, the downstream effects of Pax-5 promote cell-cell interactions and mediate the activation of adhesion genes which result in an epithelial phenotypic behavior of human carcinoma cells. To gain a better understanding of Pax-5-mediated gene regulation, we studied available gene expression data in depth and identified several Pax-5 downstream targets. Among these, we found that Pax-5 activity is consistently inversely correlated with the expression of Focal Adhesion Kinase 1 (FAK1). FAK1 is known to enhance migration of cancer cells and promote metastatic dissemination to distant sites. Further analysis looking at genome wide profiling of Pax-5 DNAbinding points to both direct and indirect regulation of FAK1 expression by Pax-5 and its downstream targets. These findings suggest a key role for Pax-5 in phenotypic transitioning during metastasis through the regulation of FAK1 activity.

Evaluation of PAX5 gene in the early stages of leukemic B cells in the childhood B cell acute lymphoblastic leukemia

B-lineage acute lymphoblastic leukemia (B-ALL) is a common subtype of acute leukemia in children. PAX5 plays a central role in B-cell development and differentiation. In this study, we analyzed PAX5 expression levels, transactivation domain mutations/deletions in B-ALL patients ( n = 115) and healthy controls ( n = 10). Relative PAX5 mRNA levels were significantly increased in B-ALL patients ( p < 0.0001). PAX5 expression was also evaluated in three different B-ALL subgroups (pro B, Common B and Pre B ALL) and showed stage specific expression levels. Pro B ( p = 0.04) and pre B ( p = 0.04) patients showed significantly high PAX5 mRNA levels compared to stage specific controls. At least one deletion of exons 7–8 or 9 has been identified in the 41% of the patients. CD34 positivity in patients and presence of large deletions (Δ7/8/9) showed a significant correlation ( p = 0.05). None of our patients showed PAX5 point mutations, but two previously identified SNPs (rs3780135 and rs35469494) were detected. Our results support that PAX5 is a critical factor in B-ALL development and aberrant PAX5 expression especially at early stages may leads to leukemic transformation.