BCL6 Corepressor Research Articles

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

Role of the Polycomb Gene Bcor in Hematopoiesis

Polycomb group (PcG) proteins are epigenetic regulators crucial for the maintenance and differentiation of stem cells. PcG proteins form in the nucleus two kinds of complexes, PRC (polycomb repressive complex) 1 containing Pcgf family proteins and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Bcor (BCL6 corepressor) together with Pcgf1 has recently been demonstrated to form a non-canonical PRC1 distinct from canonical PRC1 containing Cbx and Bmi1/Pcgf4. Of note, recurrent somatic mutations have been identified in BCOR in myeloid malignancies such as acute myeloid leukemia and myelodysplastic syndrome. However, little is known about its role in hematopoiesis and hematological malignancies. In this study, we investigated the role of Bcor in hematopoiesis using Bcor conditional knockout mice. We first examined basal levels of Bcor expression in BM hematopoietic cell populations by real-time qPCR and found that Bcor is ubiquitously expressed with the highest expression in Flk2-CD34+c-Kit+Sca-1+Lineage- ST-HSCs. We transplanted total bone marrow (BM) cells (CD45.2+) from Bcor floxed male mice (Bcorf/Y ; Bcor is located at X chromosome) crossed with Rosa::Cre-ERT mice into lethally irradiated recipient mice with the equal numbers of BM cells from CD45.1+ congenic WT mice and deleted Bcor by inducing nuclear translocation of Cre by tamoxifen injection at 4 weeks post-transplantation. Chimerism of Bcor-/Y cells in the peripheral blood gradually decreased in all lineages by 6 months after tamxifen injection. BM analysis also showed decreased chimerism of Bcor-/Y cells in Lineage-Sca-1+c-Kit+ (LSK) HSCs/MPPs and myeloid progenitors at 7 months after tamoxifen injection. In non-competitive repopulating assays, recipient mice repopulated with Bcor-/Y BM cells showed leukopenia mainly due to impaired B lymphopoiesis. BM analysis showed CD34-LSK HSC number was significantly decreased in recipients repopulated with Bcor-/Y BM cells compared to the controls. However, no mice showed any signs of myeloid malignancies. These data suggested that Bcor is essential for the maintenance of repopulating capacity of HSCs. We then examined the effects of deletion of Bcor on HSC growth in vitro. Bcor-/Y CD34-LSK cells were cultured for 14 days. Deletion of Bcor profoundly impaired the growth of CD34-LSK cells in the presence of SCF, TPO and IL3 but not significantly in the presence of SCF and TPO only. This finding suggests that Bcor is critical for IL3-responsive proliferation and differentiation.To understand molecular mechanisms underlying defective hematopoiesis in the absence of Bcor, we performed western blot analysis in LK cells and gene expression analysis in LSK cells. Western blot analysis revealed no significant difference in the levels of global H3K27me3 and H3K36me2 between Bcor-/Y LK cells and control. Unexpectedly, however, the level of global H2A119ub1 was not reduced at all in Bcor-/Y LK cells compared to control. Gene expression analysis revealed a trend of de-repression of PRC2 genes, but major target genes of canonical PRC1, such as cyclin-dependent kinase inhibitor genes were not altered in expression in Bcor-/Y LSK cells. These results indicate that Bcor regulates hematopoietic cells in a way different from canonical PRC1.Taken together, Bcor plays an important role in normal hematopoiesis, but its simple loss is not sufficient to induce myeloid malignancies within a short latency. To identify the target genes of Bcor in more detail, we are currently working on RNA sequencing and ChIP-sequencing of histone modifications in Bcor-/Y stem/progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract 449: Exome sequencing reveals Bcl6 co-repressor (Bcor) as a frequently mutated tumor suppressor gene in Eμ-Myc lymphoma

Abstract Genetically-engineered mouse models offer controlled and reproducible systems in which to study the genetics of cancer development. We have used massively-parallel sequencing to provide a comprehensive analysis of the step-wise progression of cancer in the highly tractable Eμ-Myc mouse model. The Eμ-Myc mouse has been engineered to harbor a t(8;14) translocation juxtaposing the IgH enhancer to the proto-oncogene Myc, a lesion frequently observed in mature B-cell malignancies. Eμ-Myc mice develop spontaneous Burkitt-like B-cell leukemia/lymphoma with 100% penetrance through predicable premalignant to malignant progression phases. Malignant progression ostensibly occurs as a result of secondary somatic mutations that co-operate with Myc. Despite common perception that the majority of tumors can be explained by disruption of the archetypal Cdkn2a and Trp53 tumor suppressor axis, in more than half of spontaneous Eμ-Myc tumors harbor secondary mutations that remain unknown. We hypothesize that the identification of genes that cooperate with Myc could therefore yield not only new biological insight into oncogenic pathways involved in B-cell lymphomagenesis but also improve therapy for patients in the future. We applied exome-sequencing to a cohort of 18 spontaneous murine Eμ-Myc lymphomas to determine novel somatic driver mutations capable of cooperating with Myc. In addition to exome-sequencing, we harvested blood samples at 2-week intervals throughout the premalignant to malignant phases to allow a temporal analysis of mutations that were detected at end-stage. End-stage tumors were found to be heterogeneous in nature, ranging from 11 to 36 single-base mutations, deletions or insertions. The screen identified genes previously described to be mutated in Eμ-Myc lymphoma (Trp53, Cdkn2a, Kras and Nras). Interestingly, concurrent mutations in known cancer genes were evident in clonal tumors from the sequenced cohort with, for example, a Cdkn2a deletion co-occurring with an activating Kras mutation. In addition to these mutations, we identified novel protein-truncating mutations in Bcl6 co-repressor (Bcor), which has not previously been described in Eμ-Myc lymphoma. Bcor truncating mutations were present at a high frequency, occurring in 3 out of 18 samples. RNAi mediated knockdown of Bcor in Eμ-Myc fetal liver hematopoietic stem cells reconstituted into lethally irradiated congenic recipient mice accelerated lymphomagenesis, validating Bcor as a tumor suppressor gene in this model. BCOR loss-of-function mutations have recently been identified in human cancers suggesting that BCOR acts as a tumor-suppressor in a wide range of malignancies. Given that Bcor mutations frequently occur in the Eμ-Myc lymphoma, we hypothesize that Bcor is critically important for B-cell development and that its disruption can induce malignancy in co-operation with other potent oncogenes such as Myc. Citation Format: Marcus P. Lefebure, Richard Tothill, Jason Li, Geoff Matthews, Jake Shortt, Edwin Hawkins, Elizabeth Kruse, Maria Doyle, Gretchen Poortinga, Ross Hannan, Vivian Bardwell, Micah Gearhart, Ricky W. Johnstone. Exome sequencing reveals Bcl6 co-repressor (Bcor) as a frequently mutated tumor suppressor gene in Eμ-Myc lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 449. doi:10.1158/1538-7445.AM2014-449

Clarifying the Impact of Polycomb Complex Component Disruption in Human Cancers

The dysregulation of proper transcriptional control is a major cause of developmental diseases and cancers. Polycomb proteins form chromatin-modifying complexes that transcriptionally silence genome regions in higher eukaryotes. The BCL6 corepressor (BCOR) complex comprises ring finger protein 1B (RNF2/RING1B), polycomb group ring finger 1 (PCGF1), and lysine-specific demethylase 2B (KDM2B) and is uniquely recruited to nonmethylated CpG islands, where it removes histone H3K36me2 and induces repressive histone H2A monoubiquitylation. Germline BCOR mutations have been detected in patients with oculofaciocardiodental and Lenz microphthalmia syndromes, which are inherited conditions. Recently, several variants of BCOR and BCOR-like 1 (BCORL1) chimeric fusion transcripts were reported in human cancers, including acute promyelocytic leukemia, bone sarcoma, and hepatocellular carcinoma. In addition, massively parallel sequencing has identified inactivating somatic BCOR and BCORL1 mutations in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia, medulloblastoma, and retinoblastoma. More importantly, patients with AML and MDS with BCOR mutations exhibit poor prognosis. This perspective highlights the detection of BCOR mutations and fusion transcripts of BCOR and BCORL1 and discusses their importance for diagnosing cancer subtypes and estimating the treatment responses of patients. Furthermore, this perspective proposes the need for additional functional studies to clarify the oncogenic mechanism by which BCOR and BCORL1 are disrupted in cancers, and how this may lead to the development of novel therapeutics.

A potential molecular pathogenesis of cardiac/laterality defects in Oculo-Facio-Cardio-Dental syndrome

Pitx2 is the last effector of the left–right (LR) cascade known to date and plays a crucial role in the patterning of LR asymmetry. In Xenopus embryos, the expression of Pitx2 gene in the left lateral plate mesoderm (LPM) is directly regulated by Xnr1 signaling, which is mediated by Smads and FoxH1. Previous studies suggest that the suppression of Pitx2 gene in the left LPM is a potential cause of cardiac/laterality defects in Oculo-Facio-Cardio-Dental (OFCD) syndrome, which is known to be caused by mutations in BCL6 co-repressor (BCOR) gene. Recently, our work has revealed that the BCL6/BCOR complex blocks Notch-dependent transcriptional activity to protect the expression of Pitx2 in the left LPM from the inhibitory activity of Notch signaling. These studies indicated that uncontrolled Notch activity in the left LPM caused by dysfunction of BCOR may result in cardiac/laterality defects of OFCD syndrome. However, this Notch-dependent inhibitory mechanism of Pitx2 gene transcription still remains unknown. Here we report that transcriptional repressor ESR1, which acts downstream of Notch signaling, inhibits the expression of Pitx2 gene by binding to a left side-specific enhancer (ASE) region in Pitx2 gene and recruiting histone deacetylase 1 (HDAC1) to this region. Once HDAC1 is tethered, histone acetyltransferase p300 is no longer recruited to the Xnr1-dependent transcriptional complex on the ASE region, leading to the suppression of Pitx2 gene in the left LPM. The study presented here uncovers the regulatory mechanism of Pitx2 gene transcription which may contribute to an understanding of pathogenesis of OFCD syndrome.

Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia

Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis.We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis.1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). Disclosures:No relevant conflicts of interest to declare.

The Histone Demethylase LSD1 Acts As a BCL6 Corepressor In Germinal Center B Cells

The BCL6 transcription repressor (B-Cell Lymphoma 6) is required for B cells to form germinal centers (GC) and undergo antibody affinity maturation. BCL6 mediates its functions mainly through the recruitment of corepressor complexes with distinct enzymatic activities including the SMRT/NCOR, BCOR and MTA3 corepressors. Preliminary chromatin immunoprecipitation (ChIP) studies designed to dissect the biochemical actions of BCL6 on chromatin, showed that BCL6 inhibition results in considerable induction of H3K4me2 levels at selected BCL6 target loci. Consistent with these observations a BCL6 expressing lymphoma cell line had lower levels of H3K4me2 compared to BCL6 negative lymphoma cells. Hence, we hypothesized that in addition to HDAC and PRC1-like complexes BCL6 associates with distinct complexes bearing H3K4me2 demethylase activity. We observed that one of the few H3K4 demethylases known to target H3K4me1/2 residues, Lysine Specific Demethylase-1 (LSD1) is highly expressed in GC B cells. More specifically both LSD1 transcript and protein levels were induced in the transition from naïve to GC B cell state, similar to BCL6 levels. Collectively these results indicate that BCL6 might functionally interact with LSD1 in GC B cells to mediate transcriptional repression of key gene networks. We initially asked if BCL6 and LSD1 proteins interact in GC-derived B cells using coimmunoprecipitation and GST pull down experiments. We found that BCL6 and LSD1 proteins endogenously interact in these cells. Additional GST pull-down experiments using full length GST-LSD1 and in-vitro translated S35 BCL6 confirmed this interaction suggesting it is possibly direct. Moreover, immunoprecitated BCL6 complexes could demethylate H3K4me1 but not H3K4me3 in in vitro assays using histone substrates. To further evaluate the role of LSD1 in the physiological GC response we challenged C57BL/6 mice with T-cell dependent antigen (sheep red blood cells) and we examined GC formation in mice treated with a specific LSD1 inhibitor compared to untreated control mice. LSD1 inhibition resulted in GC formation deficiency indicating that LSD1 enzymatic activity plays key roles in normal immune response. To study the functional role of BCL6-LSD1 complexes we performed Q-ChIP assays for LSD1 at BCL6 target loci and showed that several BCL6 target loci, including MLL2, CDKN1A, BRAF and CCR6 are also bound by LSD1. To extend these findings we performed LSD1 ChIP-seq in a GC-derived lymphoma cell line. These genomic studies revealed an extensive overlap of BCL6 and LSD1 binding sites (1509 common loci). Roughly 560 gene promoters were bound by both BCL6 and LSD1 at the same site. Moreover, about 1,000 BCL6-LSD1 binding sites were outside promoters and might play a role in the regulation of inter/intragenic elements similar to the role of LSD1 in enhancer regulation in embryonic stem cells. Loss of BCL6 at several targeted loci resulted in LSD1 release from the chromatin suggesting that BCL6 mediates tethering of LSD1 at these loci. Genes bound or associated with BCL6-LSD1 complexes were significantly enriched in pathways such as B cell activation, chromatin organization and cell cycle control. Taken together these results suggest that LSD1 acts as a BCL6 corepressor in GC B cells and is an essential transcriptional regulator for the formation of normal GC response. BCL6 and LSD1 possibly cooperate to establish a transcriptional program crucial for the GC stage of B cell differentiation. Because LSD1 is expressed at high levels in lymphoma cells, targeting the LSD1 enzymatic activity could potentially be exploited as a lymphoma targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Prenatal diagnosis of X‐linked recessive Lenz microphthalmia syndrome

Lenz microphthalmia syndrome comprises microphthalmia-anophthalmia with mental retardation, malformed ears and skeletal anomalies, and is inherited in an X-linked recessive pattern. In 2004, it was reported that the missense mutation (BCL-6 co-repressor gene [BCOR] c.254C>T, p.P85L) in a single family with Lenz microphthalmia syndrome co-segregated with the disease phenotype. We report a case of prenatal diagnosis for X-linked recessive Lenz microphthalmia syndrome with the mutation. A 32-year-old gravida 5, para 2 Japanese woman was referred to Nagoya City University Hospital at 15 weeks of gestation. After genetic counseling and informed consent, amniocentesis was performed for fetal karyotyping, which was 46,XY. Using the extracted DNA from cultured amniotic cells, fetal search for BCOR c.254C>T mutation was undertaken. The couple requested medical termination of pregnancy, and the postabortion examination confirmed the diagnosis. This is the third report of a BCOR mutation, associated with X-linked syndromic microphthalmia, and most importantly, it is always the same mutation. The prenatal genetic diagnosis of the Lenz microphthalmia syndrome allowed time for parental counseling and delivery planning.

Bcl6a function is required during optic cup formation to prevent p53-dependent apoptosis and colobomata

Mutations in BCOR (Bcl6 corepressor) are found in patients with oculo-facio-cardio-dental (OFCD) syndrome, a congenital disorder affecting visual system development, and loss-of-function studies in zebrafish and Xenopus demonstrate a role for Bcor during normal optic cup development in preventing colobomata. The mechanism whereby BCOR functions during eye development to prevent colobomata is not known, but in other contexts it serves as a transcriptional corepressor that potentiates transcriptional repression by B cell leukemia/lymphoma 6 (BCL6). Here, we have explored the function of the zebrafish ortholog of Bcl6, Bcl6a, during eye development, and our results demonstrate that Bcl6a, like Bcor, is required to prevent colobomata during optic cup formation. Our data demonstrate that Bcl6a acts downstream of Vax1 and Vax2, known regulators of ventral optic cup formation and choroid fissure closure, and that bcl6a is a direct target of Vax2. Together, this regulatory network functions to repress p53 expression and thereby suppress apoptosis in the developing optic cup. Furthermore, our data demonstrate that Bcl6a functions cooperatively with Bcor, Rnf2 and Hdac1 in a common gene regulatory network that acts to repress p53 and prevent colobomata. Together, these data support a model in which p53-dependent apoptosis needs to be tightly regulated for normal optic cup formation and that Bcl6a, Bcor, Rnf2 and Hdac1 activities mediate this regulation.

The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG–binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex– and the NuRD complex–associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

Structure of the Polycomb Group Protein PCGF1 in Complex with BCOR Reveals Basis for Binding Selectivity of PCGF Homologs

Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) arecritical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.

Demethylation of Epiregulin Gene by Histone Demethylase FBXL11 and BCL6 Corepressor Inhibits Osteo/dentinogenic Differentiation

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. Histone methylation, controlled by histone methyltransferases and demethylases, may play a key role in MSC differentiation. Here, we investigated FBXL11, a histone demethylase, lysine (K)-specific demethylase 2A, which is evolutionarily conserved, ubiquitously expressed, and a member of the JmjC-domain-containing histone demethylase family. We tested whether FBXL11 could inhibit the osteo/dentinogenic differentiation potential in MSC cells with gain- and loss-of-function assays. We found that FBXL11 regulated osteo/dentinogenic differentiation in MSC cells. Furthermore, we found that the gene encoding the epidermal growth factor, Epiregulin (EREG), was a downstream target of FBXL11, and that EREG mediated FBXL11 regulation of MSC differentiation. Moreover, we found that the FBXL11 histone demethylase function was activated by associating with BCL6 corepressor, and this complex could repress EREG transcription by increasing histone K4/36 methylation in the EREG promoter. In conclusion, our results elucidated a new function for FBXL11 and EREG, explored the molecular mechanism underlying directed differentiation in MSC cells, and identified potential target genes for improving tissue regeneration techniques.

Comprehensive Cytogenetic and Molecular Genetic Characterization of T-PLL Identifies for the First Time BCOR Mutations in a Lymphatic Disease.

Abstract 2539 Introduction:T-cell prolymphocytic leukemia (T-PLL) is a rare mature post-thymic T-cell neoplasm with aggressive clinical course and a median overall survival of less than one year. Due to the rareness of this disease (∼2% of cases of mature lymphocytic leukemias in adults) only few cases with cytogenetic and molecular genetic aberrations have been reported so far. However, almost 75% of T-PLL cases are reported to harbor chromosome 14 abnormalities involving the TCRA/D locus resulting in the aberrant activation of the proto-oncogenes TCL1A or MTCP1. Aim:Perform a comprehensive cytogenetic and molecular characterization of T-PLL. Patient and Methods:The cohort comprised 36 T-PLL cases diagnosed between 10/2005 and 07/2012 (23 male, 13 female patients). Median age was 71.0 yrs (range: 26.8–82.8 yrs). According to the WHO classification all T-PLL cases were characterized using immunophenotyping and cytomorphology. Patients were further investigated using chromosome banding analysis (CBA) (n=30), FISH for deletions of ATM (n=30), TP53 (n=29), and 13q (n=26), and CGH arrays (n=3, Human CGH 6×630K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation analyses for BCOR and TP53 were performed using amplicon sequencing (Roche 454, Branford, CT). Results:Aberrant karyotypes were observed by CBA in 25/30 cases (83.3%). However, the 5 remaining cases with normal karyotype were due to insufficient proliferation of the T-PLL clone as gains, losses and rearrangements were detected in these 5 cases using FISH analyses. In more detail, combined CBA and FISH data revealed in 20/30 (66.7%) cases an inv(14)(q11q32)/t(14;14)(q11;q32)/TCRA/D-TCL1A (n=18) or t(X;14)(q27;q11)/TCRA/D-MTCP1 (n=2). Further, a gain of chromosome 8q and concomitant loss of 8p was observed in 13/30 (43.3%) cases. In addition, in 10/25 (40.0%) cases a 6q deletion and in 7/25 (28.0%) an 12p deletion were observed. Based on FISH data, deletions were detected of ATM in 19/30 (63.3%) cases, TP53 in 7/29 (24.1%), and 13q in 9/26 (34.6%) cases.In addition, 3 cases were studied using array CGH. Hereby, an intragenic deletion in the BCOR gene was observed in one patient. BCOR is a BCL6 corepressor and located on chromosome Xp11.4. BCOR mutations were recently described in cases with AML. BCOR mutation frequency was determined at 3.8% in AML with normal karyotype and mutations were associated with shorter overall and event-free survival. The deletion in BCOR identified in one of our cases and the TP53 deletions in 7 T-PLL cases prompted us to screen 35 cases for molecular mutations in these two genes. Overall, BCOR mutations were detected in 5/35 (14.3%) patients and TP53 mutations in 4/35 (11.4%) cases. In total, 7 missense, one frame-shift and one nonsense mutations were found. Median mutation load was 90.0% for BCOR (range: 38–100%) and 80.0% (59–87%) for TP53.Next, we performed correlation analyses between mutations in BCOR and TP53, rearrangements involving chromosome 14, deletions of ATM, TP53, 6q, 12p, and 13q and gain of 8q. Here, BCOR was not associated with any of these parameters. In contrast, TP53 mutations were accompanied in all 4 cases by TP53 deletions, while only 3/24 TP53 wild-type cases harbored a TP53 deletion (P=0.002). In addition, only one of 4 TP53 mutated cases harbored a chromosome 14 rearrangement while 18/25 (72%) TP53 wild-type cases did (P=0.105). In line with this result, TP53 deletions were also negatively associated with chromosome 14 rearrangements (2/7 vs 17/22, P=0.030). Further, all cases with a gain of 8q harbored a 14q rearrangement (13/13 vs 8/18 without gain of 8q, P=0.001). Conclusions:1. CBA, FISH and mutation analysis of TP53 and BCOR revealed genetic abnormalities in all 36 analyzed cases. 2. The most frequent abnormality involved the TCRA/D locus (14q11) (20/30; 66.7%) activating the proto-oncogenes TCL1A on chromosome 14q32 (90.0%) or MTCP1 on chromosome Xq28 (10.0%). 3. Deletions were detected for ATM (63.3%), TP53 (24.1%), 6q (40.0%), 13q (34.6%), 12p (28.0%), and a gain was detected for the long arm of chromosome 8 (43.3%). 3. In addition to the detection of TP53 mutations in 11.4%, BCOR mutations were observed for the first time in a lymphatic malignancy with a mutation frequency of 14.3%. 4. The prognostic relevance of such cytogenetic and molecular genetic aberrations has to be determined in T-PLL, given that in myeloid malignancies both BCOR and TP53 are associated with shorter OS. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Oculo-facio-cardio-dental syndrome in three succeeding generations: genotypic data and phenotypic features

Oculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked disorder mainly manifesting in females. Patients show ocular, facial, cardiac, and dental abnormalities. OFCD syndrome is caused by heterozygous mutations in the BCOR gene, located in Xp11.4, encoding the BCL6 co-repressor. We report a Croatian family with four female members (grandmother, mother and monozygotic female twins) diagnosed with OFCD syndrome who carry the novel BCOR mutation c.4438C>T (p.R1480*). They present high intrafamilial phenotypic variability with special regard to cardiac defect and cataract that showed more severe disease expression in successive generations. Clinical and radiographic examination of the mother of the twins revealed a talon cusp involving the permanent maxillary right central incisor. This is the first known report of a talon cusp in OFCD syndrome with a novel mutation in the BCOR gene.

IRF8 Interacts with the BCL6 Co-repressor, BCOR (109.5)

Abstract BCOR has been discovered as a BCL-6 interacting co-repressor, but little is known about its biological activities in normal B cell development and function. Previously, we found that the interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, ICSBP, directly targets a large number of genes in germinal center (GC) B cells including BCL6. In this study, we screened potential binding partners of IRF8 using a retrovirus-based protein complementation assay in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and the alpha helical region of IRF8 is required for this interaction. Using a siRNA-mediated IRF8 knockdown mouse B cell lymphoma cell line, we showed that IRF8 represses BCOR and enhances BCL6 transcription. Taken together, these data suggest that a complex comprised of BCOR-IRF8 modulates BCL6 associated transcriptional regulation of B cell function. Additional studies to evaluate the possible role of BCOR-IRF8 complex are in progress.

A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion

The identification of subtype-specific translocations has revolutionized the diagnostics of sarcoma and has provided new insight into oncogenesis. We used RNA-seq to investigate samples from individuals diagnosed with small round cell tumors of bone, possibly Ewing sarcoma, but which lacked the canonical EWSR1-ETS translocation. A new fusion was observed between BCOR (encoding the BCL6 co-repressor) and CCNB3 (encoding the testis-specific cyclin B3) on the X chromosome. RNA-seq results were confirmed by RT-PCR and through cloning of the tumor-specific genomic translocation breakpoints. In total, 24 BCOR-CCNB3-positive tumors were identified among a series of 594 sarcoma cases. Gene profiling experiments indicated that BCOR-CCNB3-positive cases are biologically distinct from other sarcomas, particularly Ewing sarcoma. Finally, we show that CCNB3 immunohistochemistry is a powerful diagnostic marker for this subgroup of sarcoma and that overexpression of BCOR-CCNB3 or of truncated CCNB3 activates S phase in NIH3T3 cells. Thus, the intrachromosomal X-chromosome fusion described here represents a new subtype of bone sarcoma caused by a newly identified gene fusion mechanism.

Whole-exome sequencing identifies somatic mutations of BCOR in acute myeloid leukemia with normal karyotype

AbstractAmong acute myeloid leukemia (AML) patients with a normal karyotype (CN-AML), NPM1 and CEBPA mutations define World Health Organization 2008 provisional entities accounting for approximately 60% of patients, but the remaining 40% are molecularly poorly characterized. Using whole-exome sequencing of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3-ITD, IDH1, and MLL-PTD, we newly identified a clonal somatic mutation in BCOR (BCL6 corepressor), a gene located on chromosome Xp11.4. Further analyses of 553 AML patients showed that BCOR mutations occurred in 3.8% of unselected CN-AML patients and represented a substantial fraction (17.1%) of CN-AML patients showing the same genotype as the AML index patient subjected to whole-exome sequencing. BCOR somatic mutations were: (1) disruptive events similar to the germline BCOR mutations causing the oculo-facio-cardio-dental genetic syndrome; (2) associated with decreased BCOR mRNA levels, absence of full-length BCOR, and absent or low expression of a truncated BCOR protein; (3) virtually mutually exclusive with NPM1 mutations; and (4) frequently associated with DNMT3A mutations, suggesting cooperativity among these genetic alterations. Finally, BCOR mutations tended to be associated with an inferior outcome in a cohort of 422 CN-AML patients (25.6% vs 56.7% overall survival at 2 years; P = .032). Our results for the first time implicate BCOR in CN-AML pathogenesis.

Whole-Exome Sequencing Identifies Recurrent Mutations of BCOR in Acute Myeloid Leukemia with Normal Karyotype

Abstract 71Acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) represents about half of all adult AML. NPM1 and CEBPA mutations define WHO provisional entities accounting for ∼60% of CN-AML, but the remaining cases (∼40%) remain poorly characterized. To address this issue, we carried out whole-exome-sequencing (WES) of leukemic and normal cells from one patient with CN-AML that lacked mutations in NPM1, CEBPA, FLT3-ITD, and MLL-PTD. Using this approach, we identified a clonal somatic mutation of BCOR, a gene located on chromosome Xp11.4, that was present in the leukemic but not normal cells of the index AML case. The BCOR (BCL6 co-repressor) gene encodes for an ubiquitously expressed nuclear protein that is involved in repressing the activity of BCL6 and other transcriptional factors. BCOR is a key transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis. Germline mutations of BCOR are responsible for the oculo-facio-cardio-dental (OFCD) genetic syndrome that is inherited in an X-linked pattern and comprises microphtalmia, dysmorphic appearance, dental abnormalities (radiculomegaly), hammer-toe deformity and cardiac defects. WES findings in the index case were subsequently validated and further studied in a total cohort of 514 AML patients. We first performed deep-sequencing analyses of all exons of the BCOR gene in an initial set of 82 AML cases that were selected because they showed the same genetic characteristics of our index patient (i.e. normal karyotype without NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Disruptive BCOR mutations (i.e., nonsense mutations, out-of-frame small indels, and consensus splice-site mutations) were detected in 14/82 (17.1%) of these cases. We next assessed the frequency of BCOR mutations in a series of unselected CN-AML patients (n=262) and found that they occurred in 4.2% of cases, mostly showing the typical features of BCOR-mutated cases (absence of NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Almost mutual exclusion of BCOR and NPM1 mutations was further confirmed in a separate series of 71 NPM1-mutated only AML patients. No BCOR mutations were observed in the 89 AML cases with recurrent cytogenetic abnormalities investigated, including t(8;21)(q22;q22) (n= 29), inv(16)(p13q22) (n=40), t(15;17)(q22;q12) (n=10), and t(11q23)/MLL (n=10), and in the 10 patients with double CEBPA-mutated AML studied. BCOR mutations were: i) scattered across the whole length of the coding sequence with no hotspots identified; ii) somatic in origin and disruptive molecular events similar to germline BCOR mutations causing the OFCD genetic syndrome; iii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iv) almost mutually exclusive with NPM1 (only 1.5% of the 197 NPM1-mutated AML investigated carried BCOR mutations); v) rarely associated with FLT3-ITD; and vi) frequently associated with DNMT3A and RUNX1 mutations, suggesting cooperation with the respective mutated pathways. Clinically, BCOR mutations correlated with poor outcome among the cohort of 160 CN-AML patients evaluated (28.0% versus 66.3% overall survival at 2 years, P=0.024). We also searched for BCOR mutations in the human AML cell lines OCI-AML2, OCI-AML3, KG1a, U937, HL-60, HL-60R, HB4, AML193, and MVP-11. Only HL-60 and HL-60R (a ATRA-resistant derivative of HL-60) carried a BCOR mutation that consisted of a hemizygous G to T transition at position 4616 in exon 10, leading to the Glu1442X nonsense mutation. Western blot analysis of HL-60 cells resulted in the absence of the full-length BCOR protein (predicted MW: 192 kDa) and presence of a low intensity 156 kDa band likely corresponding to a truncated BCOR protein. In conclusion, our results implicate for the first time BCOR in the pathogenesis of CN-AML and suggest it may act as tumor suppressor gene. Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Role of the Intrinsically Disordered and Multifunctional AF9 C-Terminal Domain in MLL-AF9 Leukemia,

Abstract 3464Mixed Lineage Leukemia (MLL) fusion proteins disrupt HOX gene regulation through recruitment of transcriptional elongation factors, leading to acute leukemia. AF9 is one of the most common MLL fusion partners and has roles in normal regulation of HOX genes. AF9 has also been shown to interact with multiple transcriptional regulators, including AF4 family proteins which recruit and activate P-TEFb, suggesting a pivotal role in regulation of transcriptional elongation at HOX loci. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription in MLL-AF9 leukemia is poorly understood. We have recently shown that the AF9 ANC1 Homology Domain (AHD), which is retained in MLL fusion proteins, is intrinsically disordered, i.e. it is unstructured on its own, and recruits AF4 family members by coupled folding and binding. In order to further understand this behavior, we have determined the solution structure of the AF9 AHD in complex with AF4 and identified a point mutation which selectively disrupts the AF4-AF9 interaction. Introduction of this mutation into MLL-AF9 results in loss of HOXA9 upregulation and loss of transformation of hematopoietic progenitor cells, establishing the importance of this interaction for MLL-AF9 leukemia. We propose that AF9 functions as a signaling hub which regulates transcriptional elongation by alternative recruitment of transcriptional regulators. Indeed, the AF9 ANC1 homology domain (AHD) has been shown to also bind the H3K79 methyltransferase Dot1L, human polycomb 3 (hPC3), and the BCL6 corepressor, necessitating a carefully orchestrated choreography of binding partners to regulate transcription. Intrinsic disorder may facilitate dynamic exchange between binding partners to mediate rapid changes of HOX gene expression in both normal tissues, as required for specific differentiation programs, and in leukemia. Our results also validate the AF9-AF4 interaction as a potential target for therapeutic development. Disclosures:No relevant conflicts of interest to declare.

Abstract C28: Binding to BCOR defines a subfamily of Psc ortholog mediated polycomb group complexes

Abstract NSPC1 is a Polycomb group (PcG) protein that functions within a multiprotein complex containing BCOR (BCL6 Co-Repressor), a protein which has been shown to drive formation of diffuse large B cell leukemia. NSPC1 is one of six human homologs (BMI1, MEL18, NSPC1, MBLR, PCGF3 and PCGF5) of the Drosophila Polycomb group (PcG) protein Psc. While BMI1 and MEL18 can bind directly to another PcG protein called Polyhomeotic (PH), multi-protein complexes involving MBLR and NSPC1 are absent of PH and no function has yet to be assigned to PCGF3 or PCGF5. We show that the ubiquitin (Ub) fold of NSPC1 and PCGF3 does not bind PH, but instead directly interacts with a previously unrecognized 115 residue region within BCOR while the Ub folds of BMI1 and MEL18 bind only PH. Preliminary structural studies using nuclear magnetic resonance and analytical ultracentrifugation indicate that this region of BCOR can independently fold and possesses a novel structure thereby identifying this domain as a unique PcG recruiting module. Thus, we propose the presence of at least two distinct functional classes of Psc orthologs: Class I, (BMI1 and MEL18) that binds PH and Class II (PCGF3, NSPC1 and likely PCGF5) which bind BCOR. The existence of different classes of Psc homologs demonstrates the evolution of the PcG in order to diversify its silencing function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C28.