Mesenchymal chondrosarcoma is a rare, high‐grade, primitive mesenchymal tumor. It accounts for around 2–10% of all chondrosarcomas and mainly affects adolescents and young adults. We previously described the HEY1–NCOA2 as a recurrent gene fusion in mesenchymal chondrosarcoma, an important breakthrough for characterizing this disease; however, little study had been done to characterize the fusion protein functionally, in large part due to a lack of suitable models for evaluating the impact of HEY1–NCOA2 expression in the appropriate cellular context. We used iPSC‐derived mesenchymal stem cells (iPSC‐MSCs), which can differentiate into chondrocytes, and generated stable transduced iPSC‐MSCs with inducible expression of HEY1–NCOA2 fusion protein, wildtype HEY1 or wildtype NCOA2. We next comprehensively analyzed both the DNA binding properties and transcriptional impact of HEY1–NCOA2 expression by integrating genome‐wide chromatin immunoprecipitation sequencing (ChIP‐seq) and expression profiling (RNA‐seq). We demonstrated that HEY1–NCOA2 fusion protein preferentially binds to promoter regions of canonical HEY1 targets, resulting in transactivation of HEY1 targets, and significantly enhances cell proliferation. Intriguingly, we identified that both PDGFB and PDGFRA were directly targeted and upregulated by HEY1‐NCOA2; and the fusion protein, but not wildtype HEY1 or NCOA2, dramatically increased the level of phospho‐AKT (Ser473). Our findings provide a rationale for exploring PDGF/PI3K/AKT inhibition in treating mesenchymal chondrosarcoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
: c-Jun N-terminal kinases (JNKs), members of the Mitogen-activated protein kinase (MAPK) signaling pathway, play a key role in the pathogenesis of many diseases including cancer, inflammation, Parkinson’s disease, Alzheimer’s disease, cardiovascular disease, obesity, and diabetes. Therefore, JNKs represent new and excellent target by therapeutic agents. Many JNK inhibitors based on different molecular scaffolds have been discovered in the past decade. However, only a few of them have advanced to clinical trials. The major obstacle for the development of JNK inhibitors as therapeutic agents is the JNK-isoform selectivity. In this review, we describe the recent development of JNK inhibitors including ATP competitive and ATP non-competitive (allosteric) inhibitors, bidentate-binding inhibitors and dual inhibitors, the challenges, and future direction of JNK inhibitors as potential therapeutic agents.
Mesenchymal chondrosarcoma is a rare and often aggressive cancer mostly affecting children and young adults. Genetically, this tumor is mainly characterized by the recurrent HEY1-NCOA2 fusion. Localized tumor is managed surgically; traditional chemotherapy and radiation therapy, however, did not appear to substantially improve the event-free survival rate of mesenchymal chondrosarcoma patients. It is clear that better therapeutic options are needed for the treatment of this tumor. In our previous study, an in vitro model for studying mesenchymal chondrosarcoma tumorigenesis was developed using stably transduced iPSC-derived mesenchymal stem cells (iPSC-MSCs) with inducible-expression of HEY1-NCOA2. With the in vitro model, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq to identify HEY1-NCOA2-dependent transcriptional regulation and demonstrated the dramatic activation of PDGF-PDGFR and PI3K-AKT signaling pathways (manuscript in review). Our data also suggested that PDGFB is one of the direct targets of HEY1-NCOA2 fusion protein. In the present study, with the in vitro iPSC-MSC model, we verified that HEY1-NCOA2 fusion protein, but not wild-type HEY1 or NCOA2, dramatically increased phospho-AKT (Ser473, a readout of PI3K) expression by western blotting. Further on, we tested the effect of two kinase inhibitors CP-673451 and PF-04691502, a potent and selective PDGFR inhibitor and the mTOR/PI3K inhibitor, respectively. Stably transfused iPSC-MSC were treated with the kinase inhibitors, respectively, at different dosages (0.1 µM, 0.5 µM and 1.0 µM) for one week. At the concentration of 1.0 µM, CP-673451 efficiently suppressed AKT phosphorylation by ~23% as well as suppressed cell growth by ~67% which is significantly higher than that observed in the controls (e.g. ~20% suppression in HEY1- and NCOA2- expression iPSC-MSCs). Similarly, at the concentration of 0.5 µM, PF-04691502 suppressed the HEY1-NCOA2-expression iPSC-MSCs growth by ~24% more than that in the control (p=0.02). In summary, both CP-673451 and PF-04691502 were active against the in vitro mesenchymal chondrosarcoma model tested. Based on these data, the potential benefit of targeting PDGF/PI3K/AKT signaling pathways in the treatment of mesenchymal chondrosarcoma was suggested. Citation Format: Wenqing Qi, Lu Wang. Pdgf/Pi3k/akt axis activation by hey1-ncoa2 fusion and potential target in the treatment of mesenchymal chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB200.
RNA-seq and chIP-seq profiling identifies genes and pathways dysregulated by hey1-ncoa2 fusion and shed a light on mesenchymal chondrosarcoma tumorigenesis Wenqing Qi1, Wojciech Rosikiewicz2, Zhaohong Yin1, Beisi Xu2, Shibiao Wan2, Yiping Fan2, Gang Wu1,2 and Lu Wang1 1. Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 2. Center for Applied Bioinformatics, St. Jude Children's Research Hospital, Memphis, TN Correspondence and requests for materials should be addressed to Lu Wang (email: lu.wang2@stjude.org) Mesenchymal chondrosarcoma is a rare and often aggressive cancer that accounts for 2-10% of all chondrosarcomas. Genetically, this tumor is characterized by the recurrent HEY1-NCOA2 fusion. However, the function of HEY1-NCOA2 fusion in mesenchymal chondrosarcoma remains to be elucidated. In this study, we transduced iPSC-derived mesenchymal stem cells (MSCs) with inducible expression HEY1, NCOA2 and HEY1-NCOA2 (HN) construct, respectively. The iPSC-MSCs expressing HEY1-NCOA2 (HN_MSCs) showed significantly accelerated growth rates and higher percentage of cells in S phase compared with cells expressing wild-type HEY1 or NCOA2 (HEY1_MSCs or NCOA2_MSCs). Intriguingly, in 3-D spheroidal growth model, we observed both accelerated cell proliferation and distinct morphological features of the HN_MSCs in contrast to the control cells. To identify direct target genes and the pathways modulated by HEY1-NCOA2, stably transduced iPSC-MSCs with and without the expression of the exogenous HN, HEY1 and NCOA2, were subjected to Chromatin Immunoprecipitation Sequencing and RNA-seq analyses. ChIP-seq data showed a largely overlap in DNA-binding peaks observed in HN_MSCs and HEY1_MSCs (~90% of HN binding peaks). In contrast, very few (0.1%) overlaps observed between HN_MSCs and NCOA2_MSCs. In addition, ~25% of HN binding peaks contain the canonical HEY1 binding E-box motif (CACGTG). Through RNA-seq analysis, HN_MSC and HEY1-MSC can be robustly separated by gene expression profiling, and GSEA pathway enrichment analysis revealed that genes encoding cell cycle related targets of E2F transcription factors and genes involved in the G2/M checkpoint as in progression through the cell division cycle are significantly enriched in genes highly expressed in HN_MSC. Differential expression analysis identified 316 genes that were significantly up-regulated by HN but down-regulated by HEY1, of which CCND1 is one of the most differentially expressed genes. When correlating with ChIP-seq analysis, 59 of the 316 genes showed DNA-binding peaks with the canonical HEY1-binding motif, including HES1 and SOX4 which were reported to be involved in chondrogenesis. We further verified the significant upregulation of CCND1, HES1 and SOX4 in HN_MSCs by Q-RT-PCR. Taken together, our study demonstrated that HN is oncogenic by significantly accelerating cell proliferation. RNA-seq and ChIP-seq profiling revealed that HEY1-NCOA2 directly binds to HEY1 target genes through HEY1-binding motif to dysregulate their expression and consequently rewire the downstream pathways. The combined RNA-seq and ChIP-seq analysis suggested that HES1, SOX4 and CCND1 are critical targets of the transcriptional dysregulation mediated by HN and may play key roles in mesenchymal chondrosarcoma tumorigenesis. Citation Format: Wenqing Qi. RNA-seq and chIP-seq profiling identifies genes and pathways dysregulated by hey1-ncoa2 fusion and shed a light on mesenchymal chondrosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB188.
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