Functional characterization of potentially oncogenic somatic mutations of cardiac sarcomas

心脏肉瘤，主要是血管肉瘤和肉瘤一氧化氮合酶，预后不良，发病机制尚不清楚。利用有针对性的下一代测序和全基因组SNP阵列分析，我们确定了心脏血管肉瘤中反复出现的基因组异常。在大多数情况下，除了KDR等受体酪氨酸激酶和受体酪氨酸激酶激活的信号分子外，染色质修饰物也受到基因组异常的影响。对于复发的PLCG1突变，我们可以证明该突变导致PLCG1永久激活，随后激活转录因子NFAT，并增加对凋亡素的抗性。在染色质修饰物中，MLL2最常受到失活突变的影响。因此，我们旨在分析PLCG1反复突变和MLL2失活如何在心脏血管肉瘤的发病机制中起作用。染色质修饰剂如MLL2和PLCG1通过激活转录因子NFAT影响基因表达。因此，我们将使用RNA下一代测序进行全基因组基因表达分析，以单独和联合使用CRISPR-Cas9系统在原代内皮细胞中分析通过异位表达突变体激活PLCG1和通过基因组编辑失活MLL2的影响。作为补充，我们将通过与原代心脏内皮细胞的比较来鉴定心脏血管肉瘤中差异表达的基因。通过这些方法，我们可以鉴定由于PLCG1激活和MLL2失活而异常表达的基因，从而识别PLCG1和MLL2下游的潜在治疗靶点。在基因表达分析中，还将包括miRNAs。心脏血管肉瘤和肉瘤一氧化氮合酶的miRNA表达谱已经产生。为了鉴定差异表达的miRNAs，必须建立作为参考的原代心脏内皮细胞的表达谱。进一步的目标是建立心脏血管肉瘤的模型。我们将利用原代人内皮细胞的基因组编辑，建立稳定表达PLCG1突变和MLL2失活的细胞系。利用这个模型，可以评估KDR/PLCG1途径的抑制剂以及PLCG1和MLL2下游的靶向干预措施，如对异常表达蛋白的失活或抑制。最后，我们将继续分析心脏肉瘤NOS中的基因组异常，以识别和功能分析，如心脏血管肉瘤中的潜在致病突变。

Cardiac sarcomas, mainly angiosarcomas and sarcomas NOS, have an unfavorable prognosis and the pathogenic mechanisms are largely unknown. Using targeted next generation sequencing and genome-wide SNP array analysis we identified recurrent genomic aberrations in cardiac angiosarcomas. In most cases in addition to receptor tyrosine kinases like KDR and signalling molecules activated by receptor tyrosine kinases also chromatin modifiers were affected by genomic aberrations. For a recurrent PLCG1 mutation we could show that the mutation causes permanent PLCG1 activation with subsequent activation of the transcription factor NFAT and increased apoptosuis resistance. Among the chromatin modifiers MLL2 was most frequently affected by inactivating mutations.Therefore we aim to analyse how the recurrent PLCG1 mutation and MLL2 inactivation contribute to the pathogenesis of cardiac angiosarcomas. Chromatin modifiers like MLL2 as well as PLCG1 via activation of the transcription factor NFAT influence gene expression. Therfore we will perform genome-wide gene expression analysis using RNA next generation sequencing to analyse the effects of PLCG1 activation by ectopic expression of the mutant and MLL2 inactivation by genome editing using the CRISPR-Cas9 system, individually and in combination, in primary endothelial cells. Complementary we will identifiy differentially expressed genes in cardiac angiosarcomas by comparison with primary cardiac endothelial cells. By these approaches we should identify genes aberrantly expressed due to PLCG1 activation and MLL2 inactivation and thereby also identify potential therapeutic targets downstream of PLCG1 and MLL2.In the gene expression analysis also miRNAs will be included. miRNA expression profiles of cardiac angiosarcomas and sarcomas NOS have already been generated. For the identification of differentially expressed miRNAs expression profiles of primary cardiac endothelial cells as reference have to be generated.A further goal is the establishment of a model for cardiac angiosarcomas. We shall establish cell lines with stable expression of the PLCG1 mutation and MLL2 inactivation using genome editing of primary human endothelial cells. With this model inhibitors of the KDR/PLCG1 pathway as well as targetd interventions downstream of PLCG1 and MLL2 like inactivation or inhibition of aberrantly expressed proteins could be evaluated.Finally, we will continue our analysis of genomic aberrations in cardiac sarcomas NOS to identify and functionally analyse, like in cardiac angiosarcomas, potentially pathogenic mutations.