Yan-Yi Jiang

Genomic and molecular characterization of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.

BCL6 promotes glioma and serves as a therapeutic target

Significance Glioblastoma (GBM) is the most lethal brain malignancy lacking effective treatment. In this study, we demonstrate that BCL6 is a prognostic marker and a targetable GBM-promoting factor. Silencing of BCL6 inhibits the malignant phenotype of GBM cells and triggers cellular senescence. We also identify AXL as an important BCL6 transcriptional target, the expression of which is also regulated positively by NCoR, a BCL6 cofactor. Either silencing of BCL6 or targeted disruption of the BCL6/NCoR complex diminishes AXL expression and inhibits GBM growth. This study elucidates a crucial BCL6-mediated signaling pathway in GBM biology. More importantly, our results highlight the promise and merit of targeting BCL6 for treating this deadly disease. ZBTB transcription factors orchestrate gene transcription during tissue development. However, their roles in glioblastoma (GBM) remain unexplored. Here, through a functional screening of ZBTB genes, we identify that BCL6 is required for GBM cell viability and that BCL6 overexpression is associated with worse prognosis. In a somatic transgenic mouse model, depletion of Bcl6 inhibits the progression of KrasG12V-driven high-grade glioma. Transcriptome analysis demonstrates the involvement of BCL6 in tumor protein p53 (TP53), erythroblastic leukemia viral oncogene homolog (ErbB), and MAPK signaling pathways. Indeed, BCL6 represses the expression of wild-type p53 and its target genes in GBM cells. Knockdown of BCL6 augments the activation of TP53 pathway in response to radiation. Importantly, we discover that receptor tyrosine kinase AXL is a transcriptional target of BCL6 in GBM and mediates partially the regulatory effects of BCL6 on both MEK-ERK (mitogen-activated protein/extracellular signal-regulated kinase kinase–extracellular signal-regulated kinase) and S6K-RPS6 (ribosomal protein S6 kinase–ribosomal protein S6) axes. Similar to BCL6 silencing, depletion of AXL profoundly attenuates GBM proliferation both in vitro and in vivo. Moreover, targeted inhibition of BCL6/nuclear receptor corepressor 1 (NCoR) complex by peptidomimetic inhibitor not only significantly decreases AXL expression and the activity of MEK-ERK and S6K-RPS6 cascades but also displays a potent antiproliferative effect against GBM cells. Together, these findings uncover a glioma-promoting role of BCL6 and provide the rationale of targeting BCL6 as a potential therapeutic approach.

Identification of distinct mutational patterns and new driver genes in oesophageal squamous cell carcinomas and adenocarcinomas

Objectives Oesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance. Design Previously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations. Results OSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, CUL3 and ZFP36L2, were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. CUL3 was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, ZFP36L2 is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor. Conclusions These data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.

Mutational profiling of acute lymphoblastic leukemia with testicular relapse

Relapsed acute lymphoblastic leukemia (ALL) is the leading cause of deaths of childhood cancer. Although relapse usually happens in the bone marrow, extramedullary relapse occasionally occurs including either the central nervous system or testis (<1–2%). We selected two pediatric ALL patients who experienced testicular relapse and interrogated their leukemic cells with exome sequencing. The sequencing results and clonality analyses suggest that relapse of patient D483 directly evolved from the leukemic clone at diagnosis which survived chemotherapy. In contrast, relapse leukemia cells (both bone marrow and testis) of patient D727 were likely derived from a common ancestral clone, and testicular relapse likely arose independently from the bone marrow relapsed leukemia. Our findings decipher the mutational spectra and shed light on the clonal evolution of two cases of pediatric ALL with testicular relapse. Presence of CREBBP/NT5C2 mutations suggests that a personalized therapeutic approach should be applied to these two patients.

ROCK‐dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type‐specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation‐inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine–glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.

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.

Abstract 891: Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription

Purpose: We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription. Experimental Design: Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2. Results: Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha. Conclusions: These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration. Citation Format: Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription [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 891. doi:10.1158/1538-7445.AM2017-891

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