Jinfen Xiao

SETDB1 accelerates tumourigenesis by regulating the WNT signalling pathway.

We investigated the oncogenic role of SETDB1, focusing on non‐small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT–β‐catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1. Copyright

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD)

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

LNK (SH2B3): paradoxical effects in ovarian cancer.

LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography–mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.

Clonality and clonal evolution analysis of paediatric ALL based on B-cell receptor/T-cell receptor rearrangement

The prognosis of paediatric acute lymphoblastic leukaemia (ALL) has greatly improved in recent decades, with currently 85–90% of the patients cured with routine chemotherapy. However, once the disease relapses, the leukaemic cells often became refractory to therapy, leading to cancer death. Malignant B/T lymphocytes of ALL cells have a unique variable, diversity and joining (VDJ) junction sequence of B-cell/T-cell receptor genes (BCR/TCR), which can be used as a ‘molecular fingerprint’ to trace the clonality. To gain a better insight into ALL clonal dynamic and evolution, we initially investigated the clonality of 34 diagnosis-relapse matched paediatric ALL samples by analysing the IGH rearrangement repertoire. Genomic DNA was extracted and the VDJ junction at the IGH loci was amplified from each leukaemic sample using primer pools designed based on the FR1 region (van Dongen et al, 2003) (Table S1). Polymerase chain reaction products were gel purified, ligated with adaptors and sequenced with Hiseq4000 (PE250, Illumina, San Diego, CA, USA). Sequencing reads were mapped to the IGH database using MiXCR (Bolotin et al, 2015) and IgBLAST (https://www.ncbi.nlm. nih.gov/igblast/). Several clonal evolutionary patterns were observed: Pattern I, the dominant clone (defined as the clone having the highest clonal fractions of BCR/TCR in each case) observed at diagnosis survived chemotherapy and persisted as a dominant clone at relapse (top, Fig 1A); Pattern II, multiple clones present at diagnosis and relapse (middle, Fige 1A. IIa, subdominant clones emerged or increased at relapse; IIb, subdominant clones eliminated or decreased at relapse; IIc, persistent subdominant clones at relapse); Pattern III, minor subclone at diagnosis became dominant clone at relapse or minor subclones at diagnosis emerged as oligoclones at relapse (bottom, Fig 1A). In line with a recent finding (Bashford-Rogers et al, 2016), a large number of potential minor subclonal rearrangements (<2% of the clonal fraction) was detected at diagnosis, most of which persisted at relapse. In total, 170 index rearrangements (defined as rearrangement with clonotype fraction >2%) were detected in these 34 ALL patients. Among these index rearrangements, IGHV4-34 was the most frequently used heavy chain variable gene segment, followed by IGHV4-39 and IGHV6-1 (Fig 1B); and a highly diversified complementarity-determining region 3 (CDR3) was observed (Fig 1C). Notably, more than 45% of these index rearrangements encoding nonproductive BCR, containing either out-of-frame CDR3 or CDR3 disrupted by inside stop-codon (Fig 1D, Figure S1). The prevalence of nonproductive IGH rearrangements support the recent hypothesis that BCR may act as a tumour suppressor in most cases of precursor ALL (Muschen, 2015). Alternatively, these cells may have bypassed the BCR checkpoint by gaining a functional oncogenic driver that mimics the BCR signalling. The study was subsequently extended to another cohort of 219 paediatric ALL patients at diagnosis (Qian et al, 2017). BCR/TCR reads were extracted from the RNA sequencing data and mapped to the BCR/TCR sequence database using MiXCR. A total of 201 samples had a dominant rearrangement of either IGH/IGK/IGL or TRA/TRB (supported by more than 30 reads, Fig 2A,B, Table S2). Eighteen patients lacked expression of clonal BCR/TCR. Forty-eight patients (21%) had multiple clonal rearrangements (either ≥ 3 IGH/ or ≥ 3 IGL/or ≥ 3 TRA/or >4 IGK/or > 4 TRB (Langerak & van Dongen, 2012)), indicating a potential bi/oligoclonality of these samples. Table S3). Remarkably, 91 ALL samples (41%) carried nonproductive BCR/TCR on both alleles or on the only expressed allele. The stop-codon interrupted/frameshift mRNA had often undergone nonsense-mediated mRNA decay and, the vast majority of the non-productive BCR/TCR transcripts were only barely expressed (Fig 2D). Potential VH replacement events were detected in 12 patients (Table S4), suggesting a potential ongoing clonal diversification in these patients. Notably, unusual VJ rearrangement of IGH was detected in the dominant clone of 5 samples (Table S5). The mechanism of this VJ rearrangement, which may be attributed to the aberrant expression of RAG1/2 and AID in ALL cells, requires further study. Patients were grouped based on the expression of their haematopoietic lineage marker genes and BCR/TCR rearrangements. Most of the patients showed a good correlation between their B/T cell lineage gene expressions and their BCR/TCR rearrangement pattern (Fig 2A). However, 24 cases of B-ALL (defined by expression of CD19/CD79 etc.) surprisingly expressed a dominant TCR rearrangement (Table S6); 12 were supported by more than 100 TCR reads and thus considered as high confident rearrangements. For example, one patient (Immunophenotype: HLA-DR+/CD34 + / CD38 + /CD10-/CD19 + /CD20-/CD22 + /cyCD79a+/cyIgM/cyCD3-/CD2-/CD5-/CD7-/CD13dim/CD15dim/CD33 + / MPO-) expressed a productive TCR rearrangement in the dominant clone (TRBV28-TRBD-TRBJ2-7, 2,939 reads). In Correspondence

Diagnosis and relapse: cytogenetically normal acute myelogenous leukemia without FLT3 -ITD or MLL -PTD

Diagnosis and relapse: cytogenetically normal acute myelogenous leukemia without FLT3 -ITD or MLL -PTD

SETDB1 accelerates tumorigenesis by regulating WNT signaling pathway

We investigated the oncogenic role of SETDB1, focusing on non‐small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT–β‐catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1. Copyright

The c-MYC-BMI1 axis is essential for SETDB1-mediated breast tumourigenesis: SETDB1 mediates breast tumourigenesis via c-MYC-BMI1

Characterising the activated oncogenic signalling that leads to advanced breast cancer is of clinical importance. Here, we showed that SET domain, bifurcated 1 (SETDB1), a histone H3 lysine 9 methyltransferase, is aberrantly expressed and behaves as an oncogenic driver in breast cancer. SETDB1 enhances c‐MYC and cyclin D1 expression by promoting the internal ribosome entry site (IRES)‐mediated translation of MYC/CCND1 mRNA, resulting in prominent signalling of c‐MYC to promote cell cycle progression, and provides a growth/self‐renewal advantage to breast cancer cells. The activated c‐MYC–BMI1 axis is essential for SETDB1‐mediated breast tumourigenesis, because silencing of either c‐MYC or BMI1 profoundly impairs the enhanced growth/colony formation conferred by SETDB1. Furthermore, c‐MYC directly binds to the SETDB1 promoter region and enhances its transcription, suggesting a positive regulatory interplay between SETDB1 and c‐MYC. In this study, we identified SETDB1 as a prominent oncogene and characterised the underlying mechanism whereby SETDB1 drives breast cancer, providing a therapeutic rationale for targeting SETDB1–BMI1 signalling in breast cancer. Copyright

Co-activation of super-enhancer-driven CCAT1 by TP63 and SOX2 promotes squamous cancer progression

Squamous cell carcinomas (SCCs) are aggressive malignancies. Previous report demonstrated that master transcription factors (TFs) TP63 and SOX2 exhibited overlapping genomic occupancy in SCCs. However, functional consequence of their frequent co-localization at super-enhancers remains incompletely understood. Here, epigenomic profilings of different types of SCCs reveal that TP63 and SOX2 cooperatively and lineage-specifically regulate long non-coding RNA (lncRNA) CCAT1 expression, through activation of its super-enhancers and promoter. Silencing of CCAT1 substantially reduces cellular growth both in vitro and in vivo, phenotyping the effect of inhibiting either TP63 or SOX2. ChIRP analysis shows that CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR expression by binding to the super-enhancers of EGFR, thereby activating both MEK/ERK1/2 and PI3K/AKT signaling pathways. These results together identify a SCC-specific DNA/RNA/protein complex which activates TP63/SOX2-CCAT1-EGFR cascade and promotes SCC tumorigenesis, advancing our understanding of transcription dysregulation in cancer biology mediated by master TFs and super-enhancers.Master regulator transcription factors TP63 and SOX2 have been reported to overlap in genomic occupancy in squamous cell carcinomas (SCCs). Here, the authors demonstrate that TP63 and SOX2 promote co-operatively long non-coding RNA CCAT1 expression through activating its super-enhancer, and CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR super-enhancers and enhances both the MEK/ERK1/2 and PI3K/AKT signaling pathways in SCC.

Profiling the B/T cell receptor repertoire of lymphocyte derived cell lines

BackgroundClonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations.MethodsWe systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data.ResultsRearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution.ConclusionsOur analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.

Abstract 806: MGA is a potential tumor suppressor in acute myeloid leukemia

MGA is an incompletely studied gene with a high mutation frequency in MLL-PTD AML (9%) and in core bind factor AML (8%). This gene encodes a MAX-interacting protein and is believed to act as a transcription factor that suppresses MYC binding to its target. By in silico analysis, we found that MGA is expressed in normal myeloid hematopoietic cells and AML, and the expression level is comparable with TET2 or DNMT3A. Further data mining of TCGA revealed a high frequency of inactivating mutations of the MGA gene in a variety of cancers such as various adenocarcinomas. To interrogate functionally its role in leukemogenesis, lentiviral constructs containing either shRNA or CRISPR-sgRNA targeted to different regions of the MGA gene were generated. MGA expressing AML cell line EOL-1 was silenced by shRNA or CRISPER system. Silencing was confirmed by western blot (shRNA) and Sanger Sequencing (sgRNA). An increase of methylcellulose colony number (~30%) was observed in MGA silenced cell lines. Control EOL-1 cells or EOL-1 cells silenced with MGA CRISPR sgRNAs were injected into both flanks of NSG mice, and tumor masses were harvested 21 days after injection. Silencing of MGA by CRISPR-sgRNA consistently enhanced in vivo xenograft cell growth. In addition, western blot analyses revealed silencing of MGA in EOL-1 cells increased protein levels of Cyclin E1 and phos-RB (S807 phosphorylation inhibits the ability of RB to target protein allowing cell cycle progression), indicative of a proliferative advantage conferred by the silencing of MGA. MGA may be a potential regulator of the MYC pathway. We, therefore, examined whether silencing of MGA alters MYC transcriptional activity. Luciferase reporter assay was carried out in 293FT cells stabilized with either scramble or shRNA- targeting MGA. Luciferase activities were measured 48 h after transfection of cells with MYC activity reporter pMyc4ElbLuc and normalized to the corresponding co-transfected Renilla luciferase activity. A fourfold increase in luciferase activity was observed in MGA silenced cells when compared with non- targeting shRNA controls. Furthermore, Kaplan–Meier survival analysis was performed in the TCGA-AML patients by comparison of cases with highest versus lowest expression of MGA. P-values were calculated by log-rank test. MGA expression data and patient survival data were retrieved from TCGA-AML patients RNA seq, or microarray (70 AML patients). The MGA expression ‘high’ and ‘low’ groups were defined by 15% higher than the median or 15% lower than the median, respectively. AML patients with lower levels of MGA in their leukemic samples had a worse outcome compared with those whose leukemic cells expressed higher levels of MGA. Collectively, our results suggest that MGA may function as a potential tumor-suppressor in AML. Citation Format: Qiaoyang Sun, Lingwen Ding, Kar-Tong Tan, Wenwen Chien, Xinyi Loh, Jinfen Xiao, Anand Mayakonda, Dechen Lin, Yanyi Jiang, Henry Yang, Sigal Gery, H. Phillip Koeffler. MGA is a potential tumor suppressor in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2017-806