Aberrant Cathepsin S (CTSS) Activity in Follicular Lymphoma

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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.