Functional investigation of molecular crosstalk between PLCy1 and STAT3 in Cutaneous T-cell Lymphoma

皮肤T细胞淋巴瘤(CTCL)是一种罕见的非霍奇金T细胞淋巴瘤，具有高度的临床、病理和基因组异质性。由于这种异质性，通常很难诊断和治疗；目前的治疗选择通常是姑息性的。基因组测序研究，包括我们实验室的工作(1)，已经在CTCL的T细胞受体(TCR)信号通路中发现了频繁的异常，特别是在编码磷脂酶C-伽马1(Plcy1)的基因中。PLCG1基因是CTCL中第二个最常见的突变基因，在其他T细胞恶性肿瘤中也很常见。我们以前的工作表明，大多数发生在CTCL肿瘤细胞中的PLCG1突变是功能突变的骨性突变，这些突变赋予Plcy1结构性激活并驱动关键转录因子NFkB、AP-1和NFAT(2)的表达。我们还表明，信号转导和转录激活3(STAT3)的过度表达和结构性激活也是CTCL(3)的共同特征。然而，CTCL中的STAT3突变很少见，导致STAT3异常激活的机制(S)尚不清楚。已知TCR和STAT3信号通路调节细胞的存活和凋亡，因此这些通路在CTCL的发病机制中起着关键作用。导致这一提议的工作表明，Plcy1和STAT3之间可能存在分子串扰。我们已经证明，在体外，Plcy1上调STAT3的表达，并有初步证据表明Plcy1与STAT3之间存在直接相互作用。本论文将探讨PLCG1的激活突变可以在CTCL中驱动STAT3激活的假说。利用慢病毒在PLCG1缺失的J.Gamma1 T细胞中的过表达，我们将研究Plcy1及其激活突变p.R48W和p.S345F对STAT3调控的影响。这将包括研究WT、R48W和S345F Plcy1表达对STAT3表达、磷酸化和激活的影响。我们将分别在Y705和S727通过磷酸化来研究plcy1驱动的规范和非规范STAT3信号通路的激活。此外，我们还将研究Plcy1-STAT3蛋白与蛋白之间潜在的直接相互作用。总体而言，这项工作将评估Plcy1和STAT3之间的分子串扰，可能证实TCR和JAK-STAT信号通路在CTCL中的收敛。这项研究还将证实Plcy1作为治疗靶点的有效性，并支持开发Plcy1抑制剂作为CTCL和其他携带PLCG1突变的成熟T细胞恶性肿瘤的治疗选择。我们推测，Plcy1的激活突变可能导致CTCL中STAT3的异常激活。在CTCL中，Plcy1和STAT3的突变是互斥的，越来越多的证据表明，STAT3的表达和激活可以受到PLCy1下游效应物的调控。将解决以下目标：1)研究Plcy1及其激活突变对STAT3表达、激活和亚细胞定位的影响。2)确定Plcy1及其激活突变对规范和非规范STAT3信号通路的影响。3)在原代T细胞和CTCL肿瘤细胞中验证研究结果。

Cutaneous T-cell lymphoma (CTCL) is a rare non-Hodgkin's T-cell lymphoma which shows a high degree of clinical, pathological, and genomic heterogeneity. Due to this heterogeneity it is often difficult to diagnose and treat; current therapeutic optionsare generally palliative. Genomic sequencing studies including work in our laboratory (1) have identified frequent aberrations inthe T-Cell Receptor (TCR) signalling pathway in CTCL, particularly in the gene encoding phospholipase C gamma 1 (PLCy1). PLCG1 is the second most frequently mutated gene in CTCL and is commonly mutated in other T-cell malignancies. Our previous work has shown that the majority of PLCG1 mutations occurring in CTCL tumour cells are bone fidegain of function mutations that confer constitutive activation of PLCy1 and drive expression of key transcription factors NFkB, AP-1 and NFAT (2). We have also shown that overexpression and constitutive activation of signal transducer and activation of transcription 3 (STAT3) is also a common feature of CTCL (3). However, STAT3 mutations in CTCL are rare and the mechanism(s) driving aberrant STAT3 activation are unknown. TCR and STAT3 signalling are known to regulate cell survival and apoptosis and therefore these pathways are key players in the pathogenesis of CTCL. Work leading up to this proposal suggests that there may be molecular crosstalk between PLCy1 and STAT3. We have shown that in vitro, PLCy1 upregulates STAT3 expression and have preliminary evidence for a direct interaction between PLCy1 and STAT3. This thesis will investigate the hypothesis that activating mutations of PLCG1can drive STAT3 activation in CTCL. Using lentiviral overexpression in PLCG1-null J.gamma1 T-cells, we will investigate the effect of PLCy1 and its activating mutations p.R48W and p.S345F on the regulation of STAT3. This will include investigation of changes to STAT3 expression, phosphorylation and activation in response to WT, R48W, and S345F PLCy1 expression. We will investigate PLCy1-driven activation of both canonical and non-canonical STAT3 signalling pathways, via phosphorylation at Y705 and S727 respectively. Additionally, we will investigate a potential direct PLCy1-STAT3 protein-protein interaction. Overall, this work will evaluate molecular crosstalk between PLCy1 and STAT3, potentially confirming the convergence of the TCR and JAK-STAT signalling pathways in CTCL. This investigation will also confirm the validity of PLCy1 as a therapeutic target and support the development of PLCy1 inhibitors as a treatment option for CTCL and other mature T-cell malignancies harbouring PLCG1mutations. We hypothesise that activating mutations in PLCy1 may drive the aberrant activation of STAT3 in CTCL. Mutations in PLCy1 and STAT3 are mutually exclusive in CTCL, and accumulating evidence suggests that STAT3 expression and activation can be regulated by downstream effectors ofPLCy1. The following aims will be addressed1)Investigate the effect of PLCy1 and its activating mutations on STAT3 expression, activation, and subcellular localisation.2)Determine the effect of PLCy1 and its activating mutations on the canonical and non-canonical STAT3 signalling pathways.3)Validate findings in primary T-cells and CTCL tumour cells.