BCL6 Corepressor Research Articles

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.

BCOR modulates transcriptional activity of a subset of glucocorticoid receptor target genes involved in cell growth and mobility

Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.

Lenz microphthalmia syndrome in neurosurgical practice: a case report and review of the literature

Lenz microphthalmia syndrome (LMS) is an allelic X-linked syndrome correlated to a null mutation of B cell lymphoma (BCL-6) corepressor (BCOR) gene, which is essential in the early embryonic development. Phenotypically, this rare hereditary syndrome is characterized by microphthalmia/anophthalmia and other eye disorders; mental disability; dental, ear, and digital abnormalities; and variable malformations affecting the heart, skeleton (limbs and/or spine), and genitourinary tract. In this paper, a case of a young adult with LMS affected additionally by immuno-hematological disturbances was treated with decompressive craniectomy after domestic accidental fall. Case description and a brief review of the current literature about this rare condition are presented here.

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.

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor−/− knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor−/−/Dnmt3a−/− knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.

Developmental Programming: Sheep Granulosa and Theca Cell-Specific Transcriptional Regulation by Prenatal Testosterone.

Prenatal testosterone (T)-treated sheep, similar to polycystic ovarian syndrome women, manifest reduced cyclicity, functional hyperandrogenism, and polycystic ovary (PCO) morphology. The PCO morphology results from increased follicular recruitment and persistence of antral follicles, a consequence of reduced follicular growth and atresia, and is driven by cell-specific gene expression changes that are poorly understood. Therefore, using RNA sequencing, cell-specific transcriptional changes were assessed in laser capture microdissection isolated antral follicular granulosa and theca cells from age 21 months control and prenatal T-treated (100 mg intramuscular twice weekly from gestational day 30 to 90; term: 147 days) sheep. In controls, 3494 genes were differentially expressed between cell types with cell signaling, proliferation, extracellular matrix, immune, and tissue development genes enriched in theca; and mitochondrial, chromosomal, RNA, fatty acid, and cell cycle process genes enriched in granulosa cells. Prenatal T treatment 1) increased gene expression of transforming growth factor β receptor 1 and exosome component 9, and decreased BCL6 corepressor like 1, BCL9 like, and MAPK interacting serine/threonine kinase 2 in both cells, 2) induced differential expression of 92 genes that included increased mitochondrial, ribosome biogenesis, ribonucleoprotein, and ubiquitin, and decreased cell development and extracellular matrix-related pathways in granulosa cells, and 3) induced differential expression of 56 genes that included increased noncoding RNA processing, ribosome biogenesis, and mitochondrial matrix, and decreased transcription factor pathways in theca cells. These data indicate that follicular function is affected by genes involved in transforming growth factor signaling, extracellular matrix, mitochondria, epigenetics, and apoptosis both in a common as well as a cell-specific manner and suggest possible mechanistic pathways for prenatal T treatment-induced PCO morphology in sheep.

BCOR-coupled H2A monoubiquitination represses a subset of androgen receptor target genes regulating prostate cancer proliferation.

We have identified BCL6 corepressor (BCOR) as a hormone-dependent interaction partner of androgen receptor (AR), a key transcription factor in the development of normal and cancerous prostate. BCOR is often mutated in cancers and hematological diseases and as a component of a non-canonical polycomb repressive complex 1 (ncPRC1.1) required for arranging many facets of cellular differentiation. However, its role in androgen signaling or prostate cancer cells remains unknown. Here, our genome-wide analyses reveal that BCOR is recruited in an androgen-dependent fashion to majority of AR-binding chromatin sites in castration-resistant prostate cancer (CRPC) cells. Interestingly, depletion of BCOR has a significant effect on the expression of androgen-repressed genes linked to regulation of cell proliferation, differentiation and development. At many of these genes, such as HOX genes, the depletion leads to a decrease in H2A K119 monoubiquitination and an increase in mRNA expression. Consistently, BCOR depletion impairs the proliferation and viability of CRPC cells, inducing their apoptosis. Collectively, our data indicate a key role for the BCOR-ncPRC1.1 complex in the corepression of an important subset of AR target genes and the regulation of prostate cancer cell proliferation.

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.

Undifferentiated round cell sarcomas with CIC-DUX4 gene fusion: expanding the clinical spectrum

Round cell sarcomas are a heterogeneous group of mesenchymal neoplasms with overlapping morphology and immunohistochemical profile. Ewing sarcoma is the most well-known tumour in this group characterised by EWSR1/FUS rearrangements with members of the ETS family of transcription factors. Undifferentiated round cell sarcomas lacking these rearrangements, known as 'Ewing-like' sarcomas, usually show atypical clinical presentation and focal CD99 positivity. This group of tumours can be subdivided into: capicua transcriptional repressor (CIC)-rearranged sarcomas, Bcl6 corepressor (BCOR)-rearranged sarcomas, sarcomas with EWSR1 fusion to non-ETS family members and unclassified round cell sarcomas. We describe seven new cases of CIC-DUX4 rearranged sarcomas with their clinicopathological features, two of which presented in unusual locations (skin and lymph node). Patient age ranged between 23 and 54 years, three of whom were female. In five cases, aggressive behaviour was observed with rapid disease progression and lethal outcome within 15 months. One patient achieved a complete response after chemotherapy. The last patient whose tumour was located purely in the dermis demonstrated no residual tumour in the re-resection specimen, was not given any further treatment and showed no sign of disease after 24 months. Immunohistochemically, tumour cells in all cases showed focal membranous CD99 positivity, while WT1 N-/C-terminus were positive in all 5/5 cases (nuclear and/or cytoplasmic). NGS analysis revealed a CIC-DUX4 fusion in all cases. This study expands the spectrum of anatomical locations of CIC-DUX4 rearranged sarcomas, highlighting the inclusion of this rare entity in the differential diagnosis of undifferentiated tumours in various anatomical locations outside of soft tissues.

Expression of Normal or Mutated X-Linked BCOR Transcripts in OFCD iPSCs

Reprogramming diseased cells with mutated genes into induced pluripotent stem cells (iPSCs) can allow studies of disease mechanism and correct the mutation. Oculofaciocardiodental (OFCD) syndrome is a developmental disorder caused by heterozygous mutations in the X-linked BCL-6 corepressor (BCOR) gene. In this present study, we aimed to reprogram stem cells from a tooth apical papilla (SCAP) of a patient with OFCD, termed SCAP-O, into iPSCs. The SCAP-O carry a copy of the BCOR gene having 1 nucleotide deletion in 1 of the alleles, therefore harboring a mixture of cells expressing either normal (SCAP-OBCOR-WT) or mutated (SCAP-OBCOR-mut) BCOR transcripts. We subcloned SCAP-O and separated SCAP-OBCOR-WT and SCAP-OBCOR-mut as verified by sequencing. The selected subclone SCAP-OBCOR-mut expressed only the mutated BCOR transcripts and remained in such condition after multiple passages. We reprogrammed SCAP-O and subclone SCAP-OBCOR-mut into transgene-free iPSCs using an excisable lentiviral vector system (hSTEMCCA-loxP) carrying 4 reprogramming factors in a single cassette, followed by removal of transgenes via Cre-mediated excision. We found that after reprogramming SCAP-O or subclone SCAP-OBCOR-mut into iPSCs, some of the iPSC clones expressed either solely the normal BCOR-WT or BCOR-mut transcripts, while other clones expressed both BCOR-WT and BCOR-mut transcripts. This is our first step toward establishing OFCD study models by generating isogenic control BCOR-WT iPSCs versus BCOR-mut iPSCs.

Concurrent mutations in other epigenetic modulators portend better prognosis in BCOR-mutated myelodysplastic syndrome

IntroductionThe role of single mutations has been extensively studied myelodysplastic syndromes (MDS), but the impact of genetic aberrations in the context of other mutations is less well understood. BCOR is...

Clear cell sarcoma of the kidney in a 62-year-old patient presenting with generalized pruritus

BackgroundClear cell sarcoma of the kidney (CCSK) is the second most common renal tumor in children following Wilms’ tumor. CCSK is extremely rare in adults, with only 25 adult cases reported in the medical literature.Case presentationWe reported a 62-year-old man with a right renal mass presenting only with generalized pruritus who underwent radical right nephrectomy. With immunostaining, tumor cells were positive for expressed vimentin, neural cell adhesion molecule (NCAM, CD56), and Ki-67 and focally positive for p53, CD10 and Bcl-2. The histopathological diagnosis was CCSK. Two weeks after the operation, the generalized pruritus ended. One month after the operation, the patient started treatment with a regimen combining doxorubicin, vincristine, cyclophosphamide, and etoposide. At the 20-month follow-up visit, there was no evidence of local recurrence or metastases.ConclusionsIn a patient presenting with generalized pruritus, further evaluation for an underlying malignancy should be considered. It is difficult to distinguish CCSK from undifferentiated renal neoplasms. Immunohistochemistry could help to make exact histopathological diagnoses. The BCL-6 corepressor (BCOR) gene could play a significant role in CCSK tumorigenesis and be a good marker for CCSK diagnosis. Surgery with combination chemotherapy and radiation therapy could be used to treat CCSK in older patients.

Genetic alterations in 47 patients with a novel myelodysplastic syndrome diagnosis at a single center.

At least one mutation is present in 70–80% of patients with myelodysplastic syndrome (MDS). Genetic alterations and other molecular biological markers have been included in the diagnostic and treatment guidelines for MDS. The aim of the present study was to analyze the association between genetic alterations and clinicopathological features among 47 Chinese patients with a novel diagnosis of MDS using a next-generation sequencing approach. The results indicated that from the 47 patients, 66.0% had genetic alterations. Furthermore, seven genes, U2 small nuclear RNA auxiliary factor 1 (23.4%), splicing factor 3b subunit (12.8%), ASXL transcriptional regulator 1 (10.6%), tet methylcytosine dioxygenase 2 (8.5%), BCL6 corepressor (8.5%), TP53 (8.5%) and DNA methyltransferase 3α (6.4%), indicated a higher prevalence of alterations in >5% of patients. Among the 16 (51.6%) patients with ≥2 mutations, 12 (75%) had mutations in different genetic functional groups. Variant allele frequencies in signaling pathways were generally low, suggesting that mutations in the corresponding genes were acquired relatively late during the evolution of the leukemic clones. The mutation prevalence rates of Janus kinase 2 and SH2B adaptor protein 3 were significantly higher in the MDS unclassified group and in the very high-risk groups with a karyotype as a prognostic indicator, respectively (both P<0.05). The mutation prevalence rates of SET binding protein 1 and enhancer of zeste 2 polycomb repressive complex 2 subunit were significantly higher in the high-risk group (both P<0.05). In summary, 66.0% of the 47 patients with a novel MDS diagnosis had a genetic mutation as detected by 127-target gene next-generation sequencing. The results for the genetic alterations in the present study will supplement the database of patients with MDS in China.

BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors.

CD4+ T helper 17 (Th17) cells protect vertebrate hosts from extracellular pathogens at mucosal surfaces. Th17 cells form from naive precursors when signals from the T cell antigen receptor (TCR) and certain cytokine receptors induce the expression of the RORγt transcription factor, which activates a set of Th17-specific genes. Using T cell-specific loss-of-function experiments, we find that two components of the Polycomb repressive complex 1.1 (PRC1.1), BCL6 corepressor (BCOR) and KDM2B, which helps target the complex to unmethylated CpG DNA islands, are required for optimal Th17 cell formation in mice after Streptococcus pyogenes infection. Genome-wide expression and BCOR chromatin immunoprecipitation studies revealed that BCOR directly represses Lef1, Runx2, and Dusp4, whose products inhibit Th17 differentiation. Together, the results suggest that the PRC1.1 components BCOR and KDM2B work together to enhance Th17 cell formation by repressing Th17 fate suppressors.

Bcor loss perturbs myeloid differentiation and promotes leukaemogenesis

The BCL6 Corepressor (BCOR) is a component of a variant Polycomb repressive complex 1 (PRC1) that is essential for normal development. Recurrent mutations in the BCOR gene have been identified in acute myeloid leukaemia and myelodysplastic syndrome among other cancers; however, its function remains poorly understood. Here we examine the role of BCOR in haematopoiesis in vivo using a conditional mouse model that mimics the mutations observed in haematological malignancies. Inactivation of Bcor in haematopoietic stem cells (HSCs) results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Gene expression analysis and chromatin immunoprecipitation sequencing reveals differential regulation of a subset of PRC1-target genes including HSC-associated transcription factors such as Hoxa7/9. This study provides mechanistic understanding of how BCOR regulates cell fate decisions and how loss of function contributes to the development of leukaemia.

Clear Cell Sarcoma of the Kidney.

Clear cell sarcoma of the kidney is the second most common primary renal malignancy in childhood. It is histologically diverse, making accurate diagnosis challenging in some cases. Recent molecular studies have uncovered BCOR exon 15 internal tandem duplications in most cases, and YWHAE-NUTM2 fusion in a few cases, with the remaining cases having other genetic mutations, including BCOR-CCNB3 fusion and EGFR mutations. Although clear cell sarcoma of the kidney has no specific immunophenotype, several markers including cyclin D1, nerve growth factor receptor, and BCOR (BCL6 corepressor) have emerged as potential diagnostic aides. This review provides a concise account of recent advances in our understanding of clear cell sarcoma of the kidney to serve as a practical update for the practicing pathologist.

Bcor insufficiency promotes initiation and progression of myelodysplastic syndrome

AbstractBCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (BcorΔE4/y) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (BcorΔE9-10/y), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. BcorΔE9-10/y mice developed lethal T-ALL in a similar manner to BcorΔE4/y mice, whereas BcorΔE9-10/y hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2. Tet2Δ/ΔBcorΔE9-10/y mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2Δ/ΔBcorΔE9-10/y MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in BcorΔE9-10/y progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

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.

BCOR Regulates Cell Fate Transition, Myeloid Differentiation and Leukaemogenesis

Stem cell self-renewal and lineage specification are highly dynamic and tightly controlled processes that are essential for normal haematopoiesis and are dysregulated in cancer. The X-linked BCL6 Corepressor (BCOR) gene encodes a protein that is widely expressed across adult human tissues and is a component of a non-canonical Polycomb repressive complex 1 (PRC1). The BCOR gene is recurrently mutated in various malignant and non-malignant blood disorders, and we and others have recently provided experimental evidence that BCOR has cell-context dependent functions in regulating the proliferation, differentiation and survival of haematopoietic cells. To comprehensively examine the role of BCOR in haematopoiesis in vivo we used a conditional mouse model that mimics the truncating mutations observed in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Using stem and progenitor populations isolated ex vivo we comprehensively analysed the role of BCOR in regulating gene expression, modifying chromatin and altering genome architecture. We demonstrate that BCOR has a pivotal role in down-regulating haematopoietic stem cell (HSC) associated transcriptional networks during the transition from multi-potent stem cells to lineage-committed myeloid progenitors. Inactivation of Bcor in HSCs results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Mechanistically, Bcor regulates a subset of PRC1-target genes including key HSC super-enhancer-linked transcription factors that are normally down-regulated during myeloid differentiation. We used CRISPR/Cas9 to explore the function of Bcor target genes and identified those that are necessary for the proliferation of Bcor mutant leukaemic cells. This study provides a comprehensive mechanistic understanding of how BCOR regulates cell fate decisions and contributes to the development of leukaemia. DisclosuresNo relevant conflicts of interest to declare.

WT1 and BCORL1 Identified By Computational Biology Modeling Analysis of Patient Genomics Are Novel Predictors of Response to Azacitidine (AZA) and Lenalidomide (LEN) Treatment in Acute Myeloid Leukemia (AML)

Introduction: AML patients with relapsed/refractory (R/R) disease have few effective treatment options. LEN+AZA may be an active and better tolerated regimen compared with conventional chemotherapy. This combination has been tested in a phase 2 pilot study of LEN+AZA in 37 R/R older patients with a 49% overall response rate (4 complete remission (CR) / CR with incomplete recovery (CRi) and 14 with morphologic leukemia free state (MLFS). The objective of this study was to utilize the genomics of these patients to retrospectively predict response to AZA+LEN treatment using CBM workflow and identify key genomic characteristics of the responder profiles.Methods: We analyzed the clinical and genomic (NGS WES, cytogenetics) data from pre-treatment sample for a cohort of R/R AML patients. (Blood 2017 130:1337) All available genomic data was input into the CBM predictive workflow (Cellworks Group). Customized, individualized AML patient models - patient specific disease network maps of activated and inactivated dysregulated pathways were created.Digital drug models of AZA and LEN were created for CBM by programming their mechanism of action and effects on specific protein targets and pathways determined from published literature. The digital drug models were simulated individually and in combination on every patient's disease at varying doses. Drug impact was assessed by quantitatively measuring a disease inhibition score (DIS), the degree to which cancer phenotypes (proliferation, viability, apoptosis) were repressed, along with impact on the identified patient specific biomarkers. CBM predictions were compared with clinical outcomes in a retrospective blinded manner. Clinical response was defined as partial response or better as per International Working Group 2006 criteria.Results: 32 of 37 patients were analyzed by CBM based on availability of genomic profiling. 24 of the 32 patients were clinically evaluated, of which 4 had CR or CRi and 9 had MLFS response. CBM predictions matched for 20 of 24 profiles. The statistics of prediction accuracy against clinical outcome are presented in Table 1.A common aberration associating with sensitivity discovered in 7 profiles was a synonymous WT1 SNP rs16754. It was found in 1 CR and 5 MLFS cases and in the 1 non-responder case that along with WT1 SNP also had mutations in EZH2 and NOTCH1 that caused drug resistance. This WT1 SNP rs16754 correlated with higher mRNA expression of Wt1 and was associated with significantly improved outcome in pAML (PMID: 21189390) WT1 directly transactivates DNMT3A expression, thus improving response to hypomethylating agent. (PMID: 23042785)Another surprising find was the LOF mutation in BCORL1 (BCL6 Corepressor Like 1) L622P, that was the only common aberration found in all the 4 CR profiles. BCORL1 is a transcriptional corepressor that can also bind and regulate class II HDACs. LOF mutations in BCORL1 have been found in AML patients with abnormal karyotypes (PMID:22210327) In aplastic anemia, clones with BCORL1 mutations correlated with better response to immunosuppressive therapy and higher OS (PMID: 26132940), but no such association has been established in AML and may be worth exploring further in larger datasets.Conclusion: The CBM prediction of AZA+LEN in R/R AML patients based on genomic abnormalities and clinical data showed high predictive accuracy of 83.33%. The study validates the approach to a priori predict response and identify the right therapy option for the patient and could be used to establish criteria for precision enrollment in drug development trials. In addition, analysis uncovered novel predictors of response to AZA+LEN therapy that include presence of WT1 SNP rs16754 and possibly BCORL1 LOF, that were found in the responder profiles and linked to enhancing the drug efficacy network. This clinical decision support technology and these biomarkers may improve patient selection for AZA+LEN treatment in R/R AML. [Display omitted] DisclosuresAbbasi:Cell Works Group Inc.: Employment. Singh:Cellworks Research India Private Limited: Employment. Ullal:Cellworks Research India Private Limited: Employment. Kumari:Cellworks Research India Private Limited: Employment. Tyagi:Cellworks Research India Private Limited: Employment. Alam:Cellworks Research India Private Limited: Employment. Lunkad:Cellworks Research India Private Limited: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry. Vali:Cell Works Group Inc.: Employment. Pollyea:AbbVie: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Curis: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.