ITK/SYK融合基因在外周T细胞淋巴瘤发生发展中的作用及机制研究

Disease rarity and histologic subtype heterogeneity of peripheral T-cell lymphomas (PTCL) lead to the missing of rational treatment strategies and cause the poor prognosis. Recently, a recurrent translocation was identified in a subset of PTCL that fuses the inducible T cell kinase (ITK) gene on the chromosome 5 with the spleen tyrosine kinase (SYK) gene on the chromosome 9 to form the t (5; 9) (q33; q22) fusion gene. ITK/SYK is a functional tyrosine kinase and can transform NIH3T3 cells in vitro, leading to loss of contact inhibition and formation of anchorage-independent colonies in soft agar. Expression of ITK/SYK fusion resulted in the formation of highly malignant PTCL in mice, with a phenotype resembling that described in human patients in mice. The identification of the t (5; 9) (q33; q22) is an important step in understanding the pathogenesis of a subset of PTCL, but the mechanisms by which this translocation contributes to lymphoma development are unknown. In present proposal, it is about to detect the expression of fusion gene in clinical PTCL patients and assess its prognostic significance, and to establish a malignant transformation model of normal human T lymphocytes mediating by potent oncogene ITK/SYK. Stable isotope labeling with amino acid in cell culture (SILAC) combined with mass spectrometry will be used to study the proteomic changes caused by ITK/SYK fusion gene. Interaction networks and pathway analysis of the proteomic data will be highlighted using a bioinformatics analysis tool. Expressions of the key proteins in networks or pathway will be confirmed in clinical tissues, transformed primary T lymphocytes and xenograft tumor mouse. An unbiased approach will be believed to comprehensively assess the proteomic consequences of ITK/SYK fusion expression would lead to novel insights with respect to the signaling pathways and functional alterations induced by the oncogenic fusion and hope to provide better understanding of PTCL pathobiology and enable the identification of novel biomarkers and rational therapeutic targets.

外周T细胞淋巴瘤(PTCL)是一种侵蚀性很高的恶性淋巴瘤，存在高度异质性，缺乏分子治疗靶点，预后极差。ITK/SYK融合基因是PTCL病例中新进发现的一种遗传学异常，与PTCL发病密切相关，但具体发生机制尚不明确。本项目通过荧光实时定量PCR和FISH方法检测该融合基因在PTCL病例中的表达并分析临床意义；体外建立融合基因转化人外周血原代T淋巴细胞的模型，阐明转化细胞的生物学特性，将转化细胞接种免疫缺陷小鼠，分析其体内成瘤能力；采用定量蛋白质组方法，结合SILAC体内标记技术，对过表达融合基因的原代T细胞和对照细胞的蛋白质进行分离、比较、鉴定及生物信息学分析，构建信号传导通路，筛选关键蛋白质，在临床病例、转化细胞及致瘤小鼠模型中从mRNA及蛋白质水平验证关键蛋白质的表达变化，从整体细胞蛋白质组改变的角度探索融合基因与PTCL早期发生的分子机制，为PTCL的分子靶向治疗提供实验依据。

外周T细胞淋巴瘤是非霍奇金淋巴瘤中异质性颇高的一种恶性肿瘤，预后不良。可再现的染色体易位形成的ITK-SYK融合基因首次在非特指外周T细胞淋巴瘤中被报道，能够诱导NIH3T3细胞转化和移植小鼠模型中T细胞淋巴瘤的发展。然而，ITK-SYK融合基因导致肿瘤发生的下游信号通路仍然特征不明。我们构建了过表达ITK-SYK的慢病毒载体，转染人类急性T淋巴细胞白血病Jurkat细胞株。通过全基因表达谱方法检测ITK-SYK引发的基因改变，对芯片数据进行生物信息学分析，寻求差异基因以及特定的信号通路改变。研究发现ITK-SYK+Jurkat细胞高达CXCR5、PD-1、ICOS、BCL-6等基因，异常分泌IL-21，具有辅助B淋巴细胞产生抗体的能力，提示ITK-SYK+Jurkat具备T滤泡辅助细胞的特征。将其接种NOD/SCID小鼠，生成的肿瘤组织高表达Tfh特异性标志。进一步分析ITK-SYK致瘤的信号通路，首次发现融合基因激活JAK3/STAT5 信号通路。选择性抑制JAK3的活性可以抑制ITK-SYK+Jurkat细胞生长，诱导细胞凋亡，阻遏细胞周期停滞在G1/S期。在小鼠移植模型中，使用JAK3抑制剂可明显延缓肿瘤细胞生长速度。总而言之，研究结果第一次展示了ITK-SYK导致的差异基因的表达，并具有转化T细胞向Tfh分化的潜能。第一次阐述ITK-SYK调节下游的信号通路致瘤的详细机制，支持JAK3特异性的抑制剂用于带有ITK-SYK的外周外周T细胞淋巴瘤治疗的进一步研究。