KDR/VEGFR2 regulation of HTLV-1 oncogenesis and viral gene expression

人类T细胞白血病病毒1型逆转录病毒(HTLV-1)是成人T细胞白血病/淋巴瘤(ATLL)的病原体。成人T细胞白血病/淋巴瘤是一种侵袭性肿瘤，在长期潜伏期后约5%的感染者会发生这种肿瘤。HTLV-1感染还与许多炎症性疾病有关，包括HTLV-1相关性脊髓病/热带痉挛性瘫痪(HAM/TSP)。HTLV-1Tax是一种多功能反式激活蛋白，在其发病机制中起着关键作用。病毒基因的表达需要Tax来协调CREB转录因子和CBP/p300共激活子在5‘端病毒长末端重复序列(LTR)上的募集。Tax也是一种有效的癌基因，可诱导核因子-kB途径的持续激活，从而驱动HTLV-1感染的T细胞克隆的存活、扩增和持续。新出现的研究表明，HTLV-1基因的表达是高度动态的，具有较长的潜伏期来逃避抗病毒免疫反应。然而，调节Tax表达的宿主蛋白或蛋白稳定性可能影响病毒基因表达、宿主抗病毒免疫和HTLV-1感染的T细胞的生存，人们对此知之甚少。此外，对于ATLL这种高度侵袭性和致命性疾病，临床上迫切需要新的治疗方法，但尚未得到满足。我们已经进行了全基因组的shRNA筛选，以确定对HTLV-1转化的T细胞系的生存至关重要的宿主因素。屏幕上最热门的是酪氨酸激酶受体KDR/VEGFR2，我们已经证实它是HTLV-1转化的T细胞亚群中的一个关键生存因子。用shRNA或小分子抑制剂抑制KDR，选择性地诱导KDR+HTLV-1转化的T细胞死亡。此外，抑制KDR可钝化核因子-kB的慢性激活，并触发Tax蛋白的降解。在这一探索性应用中，我们将研究KDR信号调节税收稳定性的机制，以及KDR在HTLV-1转化的T细胞激活致癌信号通路、细胞存活和迁移中的作用。推动这些研究的中心假设是KDR激活了HTLV-1感染的T细胞中的致癌信号通路，促进了细胞存活和Tax蛋白的稳定性。我们将通过实验验证这一假设，具体目的如下：(1)确定VEGFR2/KDR在HTLV-1转化的T细胞中的致癌作用；(2)确定KDR信号如何调节Tax蛋白的稳定性和病毒基因的表达。拟议研究的完成将为HTLV-1 Tax癌蛋白和病毒基因表达的复杂调控提供新的见解，并可能导致减少HTLV-1感染者前病毒载量的新策略。

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm that occurs in ~5% of infected individuals after a long latent period. HTLV-1 infection is also associated with a host of inflammatory diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax is a multifunctional trans-activating protein and key player in pathogenesis. Tax is required for viral gene expression by coordinating the recruitment of the CREB transcription factor and CBP/p300 co-activators to the 5’ viral long terminal repeat (LTR). Tax is also a potent oncogene and induces the persistent activation of the NF- kB pathway to drive the survival, expansion and persistence of HTLV-1-infected T cell clones. Emerging studies have revealed that the expression of HTLV-1 genes is highly dynamic, with prolonged periods of latency to evade antiviral immune responses. However, little is known about host proteins that regulate Tax expression or protein stability which may impact on viral gene expression, host antiviral immunity and the survival of HTLV-1-infected T cells. Furthermore, there is an urgent and unmet clinical need for novel therapeutic approaches for ATLL, a highly aggressive and lethal disease. We have conducted a kinome-wide shRNA screen to identify host factors important for the survival of an HTLV-1- transformed T cell line. The top hit in the screen was the tyrosine kinase receptor KDR/VEGFR2, which we have validated as a critical survival factor in a subset of HTLV-1-transformed T cell lines. Inhibition of KDR with shRNA or a small molecule inhibitor induced cell death selectively in KDR+ HTLV-1-transformed T cells. Furthermore, inhibition of KDR blunted the chronic activation of NF-kB and triggered the degradation of the Tax protein. In this exploratory application, we will investigate the mechanisms by which KDR signaling regulates Tax stability, as well as the role of KDR in the activation of oncogenic signaling pathways, cell survival and migration of HTLV-1- transformed T cells. The central hypothesis driving these investigations is that KDR activates oncogenic signaling pathways in HTLV-1-infected T cells that promote cell survival and the stability of the Tax protein. We will test this hypothesis experimentally with the following Specific Aims: (1) determine the oncogenic role of VEGFR2/KDR in HTLV-1-transformed T cells, and (2) determine how KDR signaling regulates Tax protein stability and viral gene expression. Completion of the proposed studies will provide new insight into the complex regulation of the HTLV-1 Tax oncoprotein and viral gene expression and may lead to new strategies to curtail the proviral load in HTLV-1-infected individuals.