Aberrant Cathepsin S (CTSS) Activity in Follicular Lymphoma

肿瘤细胞协调并依赖于与肿瘤微环境(TME)的免疫细胞的相互作用。滤泡性淋巴瘤(FL)是一种典型的、仍然无法治愈的血液系统癌症，它例证了这一不断演变的范例的临床意义。我们之前已经发现了一种新的、临床相关的淋巴瘤和免疫细胞相互作用的机制，并对其进行了功能表征。具体地说，组织蛋白酶S(CTs)的反复变化，通过体细胞功能获得点突变或扩增/过表达，导致这种溶酶体半胱氨酸蛋白酶异常高活性，并诱导促进肿瘤的CD4T细胞丰富的免疫微环境。我们现在的目标是从功能上定义和靶向CTSS高活性淋巴瘤细胞与TME体内和体外免疫细胞之间的促肿瘤相互作用(目标1)。为此，我们建立了原代FL样生发中心(GC)B细胞(有无CTSS高活性)和自体免疫细胞(包括抗原特异性CD4T细胞)的完全人类体外共培养模型。此外，我们还从Bcl2转基因小鼠中培养出具有和不具有B系限制性CTSS高活性的造血干细胞和祖细胞(HSPC)，用于体内移植到免疫活性动物的实验中。在目标2中，我们想要研究CTSS高活性在非溶酶体细胞过程中的作用。为此，我们通过蛋白质组邻近标记(BioID2)以及免疫共沉淀(Co-IP)和定量质谱仪(LC-MS/MS)研究了CTSS在CTSS高度活跃的淋巴瘤细胞中的相互作用，证实了CTSS在抗原处理和递呈、Toll样受体(TLR)和核因子-kB信号转导等通路中的作用。值得注意的是，我们的结果也表明了以前未被识别的CTSS在细胞核内的活动，例如转录调控，我们将从功能上对其进行表征。在目标3中，我们希望定义相关的调控网络，通过考虑其他蛋白酶和抑制剂的存在和丰度来确定CTSS的净酶活性。最后，在目标4中，我们将利用CTSS过度活跃肿瘤的治疗弱点，特别是通过溶酶体膜通透性(LMP)将CTSS释放到胞浆中。事实上，我们的初步数据表明，在CTSS过度活跃的肿瘤中，对LMP诱导化合物的敏感性增加，并与标准免疫化疗具有协同作用。值得注意的是，这种现象不仅限于淋巴瘤，而且也可以在其他CTSS过度活跃的肿瘤细胞中得到证实。所有目标都得到了有希望的初步数据的支持。我们将陆续将我们的研究扩展到其他CTSS高活动性肿瘤，这些肿瘤的临床需求最高。总之，这些研究将揭开CTSS过度活跃肿瘤的独特病理生物学，并有望为其转化为临床提供强有力的理论基础。

Tumor cells orchestrate and depend on interactions with immune cells of the tumour microenvironment (TME). Follicular lymphoma (FL), a prototypical and still incurable cancer of the blood system, exemplifies the clinical implications of this evolving paradigm. We have previously discovered and functionally characterized a novel, clinically relevant mechanism of lymphoma and immune cell interaction. Specifically, recurrent cathepsin S (CTSS) alterations, through somatic gain-of-function point mutations or amplification/overexpression, result in aberrant hyperactivity of this lysosomal cysteine protease and induce a tumor-promoting CD4 T cell enriched immune microenvironment. We now aim to functionally define and target the tumor-promoting interactions between CTSS-hyperactive lymphoma cells and immune cells of the TME ex vivo and in vivo (Aim 1). Towards this end, we have established a fully human ex vivo co-culture model of primary FL-like germinal center (GC) B cells (with and without CTSS hyperactivity) and autologous immune cells, including antigen-specific CD4 T cells. Furthermore, we have engineered haematopoietic stem and progenitor cells (HSPCs) with and without B-lineage restricted CTSS hyperactivity from Bcl2-transgenic mice for in vivo transplant experiments into immunocompetent animals. In Aim 2, we want to investigate the role of CTSS hyperactivity in non-lysosomal cellular processes. For this, we have explored the CTSS interactome in CTSS hyperactive lymphoma cells by proteomic proximity-based labelling methods (BioID2) as well as co-immunoprecipitation (Co-IP) followed by quantitative mass spectrometry (LC-MS/MS), confirming its recognized role in pathways such as antigen-processing and -presentation, Toll-like receptor (TLR) and NF-kB signalling. Remarkably, our results also suggest previously unrecognized activities of CTSS within the nucleus, such as transcriptional regulation, which we will functionally characterize. In Aim 3, we want to define the relevant regulatory networks that determine the net CTSS enzymatic activity by accounting for the presence and abundance of other proteases and inhibitors. Finally, in Aim 4, we will exploit the therapeutic vulnerabilities in CTSS hyperactive tumors, specifically the release of CTSS into the cytosol by lysosomal membrane permeabilization (LMP). Indeed, our preliminary data indicate increased sensitivity to LMP-inducing compounds in CTSS hyperactive tumors and synergy with standard immunochemotherapies. Of note, this phenomenon is not restricted to lymphoma, but could also be demonstrated for other CTSS hyperactive tumor cells. All aims are supported by promising preliminary data. We will successively expand our studies to other CTSS-hyperactive tumors with highest unmet clinical need. Together, these studies will unravel the unique pathobiology of CTSS hyperactive tumors and are expected to provide a strong rationale for its translation into the clinic.