Dissecting the interplay between deregulated signaling networks and aberrant YAP/TAZ activity in myxoid liposarcoma

黏液样脂肪肉瘤(MLS)是由编码异常转录因子的FUS-DDIT3融合基因驱动的恶性脂肪细胞肿瘤。FUS-DDIT3介导肉瘤发生的机制尚不完全清楚，选择性靶向MLS细胞的策略仍然难以捉摸。我们先前已经证明，MLS细胞依赖于河马途径的中心效应器YAP，FUS-DDIT3促进YAP的表达和转录活性，并且这两种蛋白质物理上相互作用。此外，我们通过PIK3CA突变、PTEN缺失或FUS-DDIT3依赖的转录诱导细胞自主的IGF-II/IGF-IR前馈机制，提供了MLS中PI3K/AKT信号通路反复激活的证据。最近来自其他人的数据记录了河马效应器YAP/TAZ与表观遗传调控SWI/SNF复合体成员之间的物理相互作用，这调节了YAP/TAZ-TEAD复合体的形成。此外，FUS-DDIT3蛋白本身也与SWI/SNF亚基有关。FUS-DDIT3、激活的激酶信号、Hippo-YAP/TAZ级联和SWI/SNF复合体之间的功能相互作用在MLS中尚未被研究。该项目的第一部分将包括免疫组织化学调查和分析整个外显子/基因组和大量MLS组织的RNA测序数据，以确定YAP/TAZ、PI3K/AKT和MEK/ERK激活的信息模式。第二部分将解决FUS-DDIT3、特定的激酶途径、第三部分将致力于通过系统的免疫共沉淀实验来阐明FUS-DDIT3、YAP/TAZ、TEAD和选定的SWI/SNF复合蛋白之间的物理联系，重点研究在MLS中调节YAP/TAZ信号的相互作用。采用抗体引导的染色质标签测序和RNA测序相结合的方法，第四部分将探讨YAP/TAZ、FUS-DDIT3、FUS-DDIT3、以及SWI/SNF复合体，通过鉴定以特定/重叠方式调节的基因组位点/染色质位置。在此背景下，本研究将对YAP/TAZ、FUS-DDIT3和SWI/SNF复合体成员调控后的染色质功能标记分布和基因表达谱进行比较分析，有助于对MLS生物学的理解，并为未来分子靶向治疗的发展奠定基础。

Dissecting the interplay between deregulated signaling networks and aberrant YAP/TAZ activity in myxoid liposarcomaMyxoid liposarcoma (MLS) are malignant adipocytic tumors driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. We have previously shown that MLS cells are dependent on YAP, the central effector of the Hippo pathway, that FUS-DDIT3 promotes YAP expression and transcriptional activity, and that both proteins physically interact. Furthermore, we provided evidence of recurrent PI3K/AKT signaling pathway activation in MLS through mutations in PIK3CA, PTEN loss, or FUS-DDIT3-dependent transcriptional induction of a cell-autonomous IGF-II/IGF-IR feed-forward mechanism. Recent data from others documented physical interactions between the Hippo effectors YAP/TAZ and members of the epigenetic regulatory SWI/SNF complex, which modulate the formation YAP/TAZ-TEAD complexes. In addition, the FUS-DDIT3 protein itself was shown to associate with SWI/SNF subunits. The functional interplay between FUS-DDIT3, activated kinase signaling, the Hippo-YAP/TAZ cascade, and the SWI/SNF complex has not been investigated in MLS.The first part of the project will include immunohistochemical investigations and analyses of whole-exome/genome and RNA sequencing data in a large cohort of MLS tissues to identify informative patterns of YAP/TAZ, PI3K/AKT, and MEK/ERK activation.The second part will address regulatory dependencies between FUS-DDIT3, specific kinase pathways, and YAP/TAZ activation in vitro through (i) interrogation of kinase pathways known to be of relevance in MLS and (ii) an unbiased CRISPR interference screen to identify signaling intermediates specifically acting between kinases and phosphatases and the Hippo pathway.The third part will be dedicated to elucidating physical associations between FUS-DDIT3, YAP/TAZ, TEAD, and selected SWI/SNF complex proteins by systematic co-immunoprecipitation experiments, focusing on interactions that modulate YAP/TAZ signals in MLS.Employing a combined antibody-guided chromatin tagmentation sequencing and RNA sequencing approach, the fourth part will approach the transcriptional interplay between YAP/TAZ, FUS-DDIT3, and the SWI/SNF complex through identification of genomic loci/chromatin sites regulated in a specific/overlapping manner. Against this background, it will comparatively analyze the distribution of functional chromatin marks and gene expression profiles upon modulation of YAP/TAZ, FUS-DDIT3, and selected SWI/SNF complex members.The results of this project will contribute to the understanding of MLS biology and will lay the groundwork for the development of future molecularly targeted therapies.