BCL6 Corepressor Mutations Research Articles

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1

ObjectivesOculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. MethodsPeriodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. ResultsRNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. ConclusionsOur findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

BCOR Loss Confers Increased Stemness and Partially Rescues RUNX1-Deficient Phenotypes in Human Hematopoietic Stem and Progenitor Cells

Introduction: Loss of function mutations in BCL-6 corepressor (BCOR) commonly occur in de novo acute myeloid leukemia (AML) and germline hematopoietic malignancies, most notably in those arising from RUNX1 familial platelet disorder (RUNX1-FPD). Second only to loss of heterozygosity of RUNX1, mutations in BCOR are the most commonly acquired mutations upon progression to myeloid malignancy in RUNX1-FPD. Additionally, RUNX1 and BCOR mutations commonly co-occur in de novo AML, and both are classified as “adverse risk” by the European LeukemiaNet if found at AML diagnosis. However, the role of BCOR in leukemogenesis is poorly understood, and there are limited studies investigating co-loss of BCOR and RUNX1 in hematopoietic stem and progenitor cells (HSPCs). Methods: We developed a model of BCOR loss in primary human CD34+ HSPCs using CRISPR/Cas9 and AAV6-mediated homology directed repair to knock-in a fluorescent reporter expression cassette into the BCOR locus. We used this system to investigate how BCOR knockout (KO) perturbs normal HSPC function. We combined this approach with a previously developed analogous system to disrupt the RUNX1 locus to investigatehow BCOR loss in the setting of RUNX1 KO modulates the effects of RUNX1 loss on HSPC function. Results: Here, we found that BCOR loss increased capacity for serial replating and conferred a competitive advantage over control HSPCs. In serial replating assays, BCOR-deficient HSPCs had a 5-fold increase in colony formation after one passage with a striking majority of colonies being erythroid. In liquid differentiation assays, BCOR-deficient HSPCs consistently had a 2-fold increase in CD34+ cells over a 21 day time course. When co-cultured with control HSPCs in a 1:1 ratio in stem expansion media (base media + SCF, FLT3-L, TPO, IL-6, UM-171), BCOR-deficient HSPCs expanded to comprise 70% of the culture at 14 days. This competitive advantage was recapitulated in vivo. At the 12-week timepoint of a competitive transplantation assay in NSG mice using HSPCs from 3 different donors, BCOR KO cells (marked by BFP) on average made up 75% of the engrafted human CD45+ population, while control cells (expressing GFP from the AAVS1 safe harbor locus) made up only 21%. Additionally, BCOR loss partially rescued RUNX1-induced stem cell dysfunction in vitro. Compared to RUNX1 deficiency alone, HSPCs deficient in both RUNX1 and BCOR had a 4-fold increase in colony formation after two passages in serial replating assays. Our lab has previously shown that RUNX1 loss ablates erythroid colony formation and skews towards monocytic colonies. Despite BCOR loss alone having a strong skew towards erythroid colonies, BCOR loss in RUNX1-deficient HSPCs did not induce erythroid colony formation. This suggests that in double mutant HSPCs, RUNX1 loss drives lineage decisions while BCOR loss drives the increase in serial replating potential. Ongoing efforts are aimed at characterizing combinatorial effects of BCOR and RUNX1 loss on hematopoiesis and stem cell function through in vitro liquid differentiation assays and in vivo transplantations, as well as investigating the molecular underpinnings of these phenotypes through RNA-seq. Conclusion: In summary, we have established a model of BCOR loss in primary human HSPCs to evaluate not only the role of BCOR in hematopoiesis, but also to characterize how BCOR is involved in leukemic transformation when combined with RUNX1. We show that BCOR deficiency enhances several attributes associated with stem cell function, including increased potential for serial replating, increased CD34 positive population, and increased engraftment compared to control HSPCs. When combined with RUNX1 loss, BCOR loss increases the serial replating potential of RUNX1-deficient cells, suggesting some rescue of RUNX1 KO-induced stem cell dysfunction. Ultimately, this model will enable us to elucidate the molecular mechanisms underlying increased stemness and competitive advantage in BCOR-deficient HSPCs, interrogate RUNX1-FPD disease progression associated with BCOR mutation, and validate potential therapeutics.

Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma

Abstract Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye. Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2557.

CSIG-33. BCOR LOSS PROMOTES RETINOBLASTOMA GROWTH AND DISSEMINATION BY MODULATING INSULIN-LIKE GROWTH FACTOR 2

Abstract While loss of RB function is the most common genetic alteration in the pediatric embryonal eye tumor retinoblastoma, mutations leading to BCL-6 corepressor (BCOR) loss of function are the second most common alteration. BCOR mutations are found in more aggressive tumors, and are associated with metastasis and poor prognosis. We investigated the effects of BCOR loss or overexpression in retinoblastoma cells in order to better understand its functional role, and to determine if it represents a new therapeutic target in these tumors. Six established retinoblastoma cell lines were examined, and BCOR protein was most highly expressed in WERI-RB1, but absent in Y79 and two other lines. BCOR knock down in WERI-RB1 cells using shRNA caused markedly increased migration and proliferation. BCOR was also knocked out in these cells using CRSPR, resulting in more than three-fold increased size of intraocular murine xenografts and extensive extraocular extension of tumors. In terms of mechanism, BCOR loss of function induces IGF2 transcription, resulting in an activation of IGF1R and the ERK signaling pathway. In contrast, overexpressing BCOR in Y79 retinoblastoma cells with no baseline protein had opposite effects on IGF2. Targeting IGF2/IGF1R signaling pharmacologically using Lincitinib inhibited proliferation and migration in BCOR-negative retinoblastoma cells. Increased IGF2 transcription was regulated at least in part by hyper methylation of CCCTC-binding factor (CTCF)-binding regions in the IGF2/H19 imprinting control region (ICR), suggesting that BCOR knock down promotes increased methylation at this site. In summary, our studies revealed that BCOR loss promotes the growth and invasion of retinoblastoma cells in vitro and in vivo, at least in part via modulating IGF2 transcription, suggesting new potential therapeutic targets for these aggressive childhood tumors of the eye.

Mutations in BCOR, a co-repressor of CRX/OTX2, are associated with early-onset retinal degeneration.

Many transcription factors regulating the production, survival, and function of photoreceptor cells have been identified, but little is known about transcriptional co-regulators in retinal health and disease. Here, we show that BCL6 co-repressor (BCOR), a Polycomb repressive complex 1 factor mutated in various cancers, is involved in photoreceptor degenerative diseases. Using proteomics and transcription assays, we report that BCOR interacts with the transcription factors CRX and OTX2 and reduces their ability to activate the promoters of photoreceptor-specific genes. CUT&RUN sequencing further shows that BCOR shares genome-wide binding profiles with CRX/OTX2, consistent with a general co-repression activity. We also identify missense mutations in human BCOR in five families that have no evidence of cancer but present severe early-onset X-linked retinal degeneration. Last, we show that the human BCOR mutants cause degeneration when expressed in the mouse retina and have enhanced repressive activity on OTX2. These results uncover a role for BCOR in photoreceptors in both health and disease.

Loss-of-function mutations in BCOR contribute to chemotherapy resistance in acute myeloid leukemia

Primary refractory acute myeloid leukemia (AML) is unresponsive to conventional chemotherapy and has a poor prognosis. Despite the recent identification of novel driver mutations and advances in the understanding of the molecular pathogenesis, little is known about the relationship between genetic abnormalities and chemoresistance in AML. In this study, we subjected 39 samples from patients with primary refractory AML to whole-exome and targeted sequencing analyses to identify somatic mutations contributing to chemoresistance in AML. First, we identified 49 genes that might contribute to chemotherapy resistance through the whole-exome sequencing of samples from 6 patients with primary refractory AML. We then identified a significantly higher frequency of mutations in the gene encoding BCL-6 co-repressor (BCOR) in patients with primary refractory AML through the targeted sequencing of all coding sequence of 49 genes. Notably, the presence of BCOR mutations appeared to have a negative impact on prognosis in our cohort and previous larger studies. Subsequently, to investigate the biological effect of BCOR mutations on sensitivity to anticancer drugs, we established BCOR knockout human leukemic cell lines using the CRISPR/Cas9 system. Here, BCOR knockout cell lines exhibited statistically significant reductions in sensitivity to anticancer drugs, compared with the wild-type controls both in vitro and in vivo in xenograft mouse models. In conclusion, loss-of-function BCOR mutations appear to contribute to chemotherapy resistance and may be a promising therapeutic target in primary refractory AML.

Loss-of-Function Mutations of BCOR Are an Independent Marker of Adverse Outcomes in Intensively Treated Patients with Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is a genetically heterogeneous disease. Clinical phenotypes of frequent mutations and their impact on patient outcome are well established. However, the role of rare mutations often remains elusive. We retrospectively analyzed 1529 newly diagnosed and intensively treated AML patients for mutations of BCOR and BCORL1. We report a distinct co-mutational pattern that suggests a role in disease progression rather than initiation, especially affecting mechanisms of DNA-methylation. Further, we found loss-of-function mutations of BCOR to be independent markers of poor outcomes in multivariable analysis. Therefore, loss-of-function mutations of BCOR need to be considered for AML management, as they may influence risk stratification and subsequent treatment allocation.Acute myeloid leukemia (AML) is characterized by recurrent genetic events. The BCL6 corepressor (BCOR) and its homolog, the BCL6 corepressor-like 1 (BCORL1), have been reported to be rare but recurrent mutations in AML. Previously, smaller studies have reported conflicting results regarding impacts on outcomes. Here, we retrospectively analyzed a large cohort of 1529 patients with newly diagnosed and intensively treated AML. BCOR and BCORL1 mutations were found in 71 (4.6%) and 53 patients (3.5%), respectively. Frequently co-mutated genes were DNTM3A, TET2 and RUNX1. Mutated BCORL1 and loss-of-function mutations of BCOR were significantly more common in the ELN2017 intermediate-risk group. Patients harboring loss-of-function mutations of BCOR had a significantly reduced median event-free survival (HR = 1.464 (95%-Confidence Interval (CI): 1.005–2.134), p = 0.047), relapse-free survival (HR = 1.904 (95%-CI: 1.163–3.117), p = 0.01), and trend for reduced overall survival (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056) in multivariable analysis. Our study establishes a novel role for loss-of-function mutations of BCOR regarding risk stratification in AML, which may influence treatment allocation.

PATH-03. HIGH-GRADE NEUROEPITHELIAL TUMOR SHOWING BCOR IMMUNOPOSITIVITY WITHOUT EXON 15 INTERNAL TANDEM DUPLICATIONS IN A FIVE-YEAR-OLD BOY: A CASE REPORT

Recent DNA methylation profiling clarified several rare entities of pediatric CNS tumors from institutionally-diagnosed primitive neuroectodermal tumors (PNETs). One of which is CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR), and it carries in-frame internal tandem duplications (ITD) of the BCL6 corepressor (BCOR) in exon 15. In the report, we describe a case of immunohistologically-diagnosed CNS HGNET-BCOR, which lacks exon 15 ITD of BCOR. A five-year-old boy visited a local hospital complaining uncontrolled vomiting for two months, and magnetic resonance imaging (MRI) showed a large well-circumscribed mass in his left cerebellum with ventricular dilatation. He referred to our hospital, and an additional MRI revealed diffuse and weak enhancement of gadolinium and low ADC values in mass. Immediately, he underwent total removal of the tumor and ventricular drainage, and his consciousness recovered soon after surgery. The tumor presented high BCOR expression by IHC, but target PCR did not identify exon 15 ITD of BCOR. As the previously-reported clinical and imaging features of CNS HGNET-BCOR resembled our case, we clinically diagnosed it as a similar phenotype of CNS HGNET-BCOR without exon 15 ITD. He received 60 Gy of extended-local irradiation with concomitant temozolomide and discharged without any neurological deficits. Since BCOR alterations, including ITD, gene fusions, and mutations, play an oncogenic role in several cancers, the present case might harbor another gene aberration of BCOR.

Age-Related Co-Expression of BCOR and BCORL1 mRNA in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy caused by a variety of genetic abnormalities and epigenetic dysregulation. The incidence of AML is strongly related to age, with the highest incidence rates being in older adults. The loss of function mutations in BCOR and BCORL1 genes have been identified in AML. BCL6 corepressor (BCOR) and BCL6 corepressor like 1 (BCORL1) are important epigenetic regu-lators as a member of Polycomb repressive complex 1 (PRC1.1), involved in histone modification processes. We analyzed the BCOR and BCORL1 mRNA expression in 74 adult and 22 pediatric patients with AML by Real-Time quantitative PCR in this study. Our results indicated that both BCOR and BCORL1 mRNA expressions decrease with age (p = 0.009 and p = 0.008, respectively) and there is a positive correlation between BCOR and BCORL1 mRNA expression (p < 0.001). BCOR and BCORL1 mRNA expressions were not significantly different in both adult and pediatric patients with AML compared to control (p > 0.05). Our findings indicate that expression of BCOR and BCORL1 mRNA are down-regulated with age. The increase in AML incidence with age suggests that age-associated BCOR and BCORL1 down-regulation might potentially contribute to age-related epigenetic alterations and form a predisposing condition for the development of elderly AML.

Direct Binding of BCOR, but Not CBX8, to MLL-AF9 Is Essential for MLL-AF9 Leukemia Via Regulation of the EYA1/SIX1 Gene Network

The mixed lineage leukemia (MLL) protein is a histone methyltransferase that writes the histone H3 lysine 4 trimethyl (H3K4me3) mark at the promoters of target genes such as HOXA9 and MEIS1. MLL is the target of chromosomal translocations that fuse it in frame to one of over 90 partners, leading to acute myeloid and lymphoid leukemias (AML and ALL, respectively) characterized by poor prognoses1. MLL fusions activate transcription by recruiting the AF4 family/ENL family/P-TEFb (AEP) complex and the DOT1L-AF10 family-ENL family complex (DOT1L complex or DotCom). Transcriptional activation via AF4 recruitment and transcriptional maintenance via DOT1L recruitment are required for MLL leukemias.Despite the large number of fusion partners, members of the AEP complex account for nearly 70% of MLL rearrangements1. These fusions constitutively activate MLL targets by bypassing recruitment via ENL (MLLT1) and AF9 (MLLT3) YEATS domain binding to crotonylated or acetylated histone H3. The AF9 ANC1 homology domain (AHD), retained in MLL fusions, is intrinsically disordered, but undergoes coupled folding and binding upon interaction with its binding proteins2. The AHD recruits AF4 and DOT1L, which support transcriptional elongation, as well as the BCL6 corepressor (BCOR) and chromobox homolog 8 (CBX8), which are implicated in transcriptional repression.CBX8 (HPC3) is a mammalian ortholog of Drosophila polycomb that binds trimethylated histone H3 lysine 9 and 27 (H3K9me3 and H3K27me3) with variable affinity. Previous reports indicate CBX8 is required for MLL-AF9 and MLL-ENL. BCOR is a transcriptional corepressor that augments BCL6-mediated repression. The BCL6 POZ domain forms a ternary complex with BCOR and SMRT, repressing targets via recruitment of PRC1.1 and HDAC3. BCOR translocations and mutations have been found in a range of cancers. Although it is broadly expressed throughout the hematopoietic system (Bloodspot), little is known about BCOR function in hematopoiesis. Recently, BCOR was shown to have a role in maintenance of human embryonic stem cell pluripotency. BCOR has also been implicated in regulation of myeloid cell proliferation and differentiation and is necessary for MLL-AF9 leukemogenesis.While the roles of the direct MLL-AF9/AF4 and MLL-AF9/DOT1L interactions have been the subject of previous structural and functional studies2-4, the roles of the direct interactions of MLL-AF9 with CBX8 and BCOR remain relatively uncharacterized. We determined the structures of the AF9 AHD-CBX8 and AF9 AHD-BCOR complexes. Based on the structures, we developed point mutants to increase and decrease affinity of CBX8 for AF9. Increased affinity decreased colony forming ability and induced differentiation of MLL-AF9-transformed cells, while decreased affinity had no effect. An additional point mutant was developed to selectively disrupt BCOR binding to AF9. In the context of MLL-AF9, this mutant increases proliferative ability without an effect on colony formation and is unable to cause leukemia in vivo. RNAseq analysis reveals that this mutant affects a different set of genes than loss of DOT1L or AF4 binding or gain of CBX8 binding, leading to a phenotype distinct from that seen with perturbation of other AF9 interactions, functionally distinguishing proliferative capacity from in vivo leukemogenesis. In particular, substantial effects were observed on EYA1 expression, suggesting a critical role for the EYA1/SIX gene network in MLL-AF9 leukemia.1 Meyer, C. et al. The MLL recombinome of acute leukemias in 2017. Leukemia32, 273-284, doi:10.1038/leu.2017.213 (2018).2 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).3 Kuntimaddi, A. et al. Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 Di- and tri-methylation on target genes and transformation potential. Cell reports11, 808-820, doi:10.1016/j.celrep.2015.04.004 (2015).4 Lokken, A. A. et al. Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4. Leukemia research38, 1309-1315, doi:10.1016/j.leukres.2014.08.010 (2014). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament

Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

Loss of BCOR Protein in Development of Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with a dismal clinical outcome and represents an unmet medical need. However, therapeutic progress will not be made without a greater understanding of the varied molecular pathogenesis of this heterogeneous disease. Recently, the majority of cases of the largest subgroup of AML, AML with a normal karyotype (AML-NK), were found to harbour recurrent loss-of-function mutations in epigenetic regulators.Germline mutations in the BCL6 Co-Repressor (BCOR) gene, situated at Xq27-28, are associated with two overlapping X-linked dominant disorders, the Oculo-facio-cardio-dental (OFCD) syndrome and syndromic micropthalmia. In addition, somatic mutations in BCOR were described within the last few years in adults and children with AML, particularly in cases arising in the context of aplastic anaemia and in patients with antecedent myelodysplastic syndromes.BCOR encodes a ubiquitously expressed protein which is involved in BCL6-mediated transcriptional repression. In contrast to other BCL6 partners, NCOR and SMRT, it was recently demonstrated to occupy the majority of its DNA binding sites independently from BCL6, reflecting its multiple interaction partners. BCOR also forms a Polycomb repressor complex 1 (PRC1)-like complex with PCGF1, KDM2B, RING1, SKP1, RYBP, and RNF2, and can bind to both class I and II histone deacetylases. Its spectrum of protein-binding partners and studies of mutant forms suggest that BCOR is an epigenetic modulator. Therefore, we predict that loss of BCOR in AML results in loss of its complex repressive function and leads to an activation of an important leukaemogenic transcriptional programme.To test this hypothesis, we generated a mouse model of leukaemia using CRISPR-Cas9 genome editing. Using a common endonuclease assay, we validated 6 different gRNA constructs against Bcor targeting the region of known human mutations described in AML. The three most efficient constructs were subsequently used to create a pool of lentiviral vectors. In order to increase the efficiency of targeting the haematopoietic progenitor cells, we harvested lineage depleted bone marrow cells from mice expressing the Cas9 cDNA from the Rosa locus (Rosa-Cas9). In parallel, we performed the same experiments with Rosa-Cas9 mice harbouring a Flt3 internal tandem duplication (Flt3-ITD), a mutation that can co-occur with BCOR mutations in individual AML patients.First, we transduced bone marrow lineage negative cells with lentiviruses carrying the three BCOR sgRNAs at 60-70% efficiency and performed a serial re-plating assay to investigate the clonogenic potential of targeted cells. We observed a significant increase in the colony numbers in both Cas9 only and Cas9 Flt3-ITD targeted cells in comparison to the empty lentiviral vector controls from the second re-plating onwards. Using a standard endonuclease assay we have confirmed the presence of Cas9-induced mutations within the target Bcor exons, indicative of clonal selection for Bcor targeted cells and an increased self-renewal potential of hematopoietic precursors that lack BCOR. Transduced cells were also injected into lethally irradiated recipient mice in the presence of helper cells to investigate development of leukaemia in vivo. We have been able to confirm the presence of the Bcor mutated cells in the blood of tested mice 12 weeks post injection and these mice continue to be monitored for evidence of leukemia.We are currently conducting integrated genomic analysis with ChIP-Seq, RNA-Seq and methylation analysis to link the BCOR mutations and their altered cellular phenotype to aberrant epigenetic control and pre/leukaemic gene expression programmes. Finally, we are also going to assess co-operativity of Flt3-ITD and BCOR mutations in AML leukaemogenesis. We anticipate that our work will inform the biology of AML-NK associated with BCOR, identify potential therapeutic targets and provide platforms for the assessment of novel therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Identification of nuclear localization signals within the human BCOR protein

Mutations in the BCL-6 corepressor (BCOR) gene, which encodes a transcriptional corepressor, were described to cause oculofaciocardiodental syndrome (MIM 300166). The purpose of this study was to localize the classical nuclear localization signals (NLSs) of the BCOR using reported human BCOR mutations with comparable phenotypes. The genotype–phenotype correlation among the mutations could not be clearly explained; however, the classical NLSs were identified at two possible sites; RVDRKRKVSGD at aa1131–1141 (NLS1) and LKAKRRRVSK at aa1158–1167 (NLS2). In addition, according to our results, NLS2 displayed a more efficient nuclear import function than NLS1.

Massively Parallel Sequencing Identifies Recurrent Mutations in TP53 in Thymic Carcinoma Associated with Poor Prognosis

The characterization of the molecular alterations in thymic epithelial tumors may lead to a better understanding of tumorigenesis, new therapeutic targets, and biomarkers in these tumors. Paired tissue (tumor and matched normal) from 15 thymic carcinomas (TCA) and six B3 thymomas were evaluated by exon capture of 275 cancer-related genes, followed by deep coverage next-generation sequencing, which identifies somatic sequence variants, small insertions and deletions, and copy number alterations involving all exons of the captured genes. Non-silent somatic mutations were identified in 12 of 15 (80%) TCA with a median of one mutation per tumor (range 0-26). Recurrent mutations were identified in tumor suppressor genes TP53 (n = 4), SMAD4 (n = 2), and CYLD (n = 2); and chromatin remodeling genes KDM6A (n = 3), SETD2 (n = 2), MLL3 (n = 2), and MLL2 (n = 2). Tumors with TP53 mutation appeared to exhibit more aggressive behavior. Therefore, the role of P53 was evaluated by immunohistochemistry in an additional ten cases. P53 overexpression correlated with TP53 mutation. These tumors had a higher rate of recurrence and death of disease compared to carcinoma with normal p53 expression (p = 0.02 for disease-free survival and p = 0.05 for overall survival). Among the B3 thymomas, mutations were identified in four of six tumors. Mutations in BCOR (BCL6 co-repressor) were seen in three thymomas and MLL3 (involved in histone methylation) in one tumor. Next-generation sequencing of cancer genes in thymic epithelial tumors revealed a low frequency of mutation, with different patterns between TCA and B3 thymomas. TP53 and BCOR were the most frequently mutated genes in TCA and B3 thymomas, respectively. Alterations in p53 are associated with worse prognosis in TCA.

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

BCOR and BCORL1 mutations in pediatric acute myeloid leukemia

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.

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.

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.