Selective targeting of sumoylation disrupts mitochondrial homeostasis via TRAP1 to suppress growth of non-Hodgkin lymphoma (NHL) B-cells

Abstract Introduction: Sumoylation involves the attachment of small ubiquitin-like modifiers (SUMO) proteins to target proteins, thereby modulating their function and affecting cellular and mitochondrial processes. Aberrant sumoylation pathways are frequently observed in tumors, highlighting their potential significance in cancer biology. Here we studied the mechanistic effects of targeting sumoylation in B-cell NHL using TAK-981 (subasumstat). Methods: B-NHL cell lines (OCI-LY3, VAL, U-2932, SU-DHL10, Raji) were treated with TAK-981 (10-10,000nM) for up to 72 h and assessed for cell viability (MTS and apoptosis), mitochondrial function and metabolism changes (Seahorse and metabolomics). For in vivo studies, 3×106 OCI-LY3 and Raji cells were injected subcutaneously into NSG mice; once tumors reached 100 mm³, mice were treated with 15 mg/kg TAK-981 IV twice weekly for five weeks. Proteomic analysis was performed by LC-MS/MS. HEK293T and Raji cells were transfected with 3xFLAG-TRAP1 for mitochondrial isolation, Co-IP, and SUMO2/3 western blot (WB). IP-MS was done on TAK-981–treated HEK293T TRAP1-FLAG-expressing cells. TAK-981 was provided by Takeda Development Center Americas Inc. (Cambridge, MA). Results: We observed high levels of sumoylation in B-NHL cell lines. Sumo pathway components were highly expressed in lymph nodes from NHL patients vs. reactive lymph nodes. TAK-981 inhibited growth of NHL cell lines (IC₅₀ ~10 nM) as demonstrated by apoptosis and MTS assays over 24-72 hours, accompanied by rapid, dose-dependent global protein desumoylation. Treatment with TAK-981 significantly prolonged survival of OCI-LY3 and Raji xenograft-bearing mice (by ~5.5 weeks) vs. control mice (log-rank test, p<.01). To identify SUMO–regulated pathways, we performed RNA-Seq of four TAK-981-treated B-NHL cell lines. GSEA revealed significant downregulation of MYC targets and OxPhos-related genes. Given the latter, subsequent Gene Ontology analysis using the MitoMiner dataset revealed strong downregulation of pathways involved in mitochondrial membrane organization and depolarization following TAK-981 treatment. Subcellular fractionation of Raji and U2932 cells after 8 hours of TAK-981 treatment revealed a dramatic desumoylation of proteins in the mitochondrial fraction, compared with cytosolic fraction. LC-MS/MS profiling of TAK-981-treated mitochondrial extracts identified 40 (U2932) and 44 (Raji) proteins with >4-fold reduction in SUMO2/3-ylation, including 7 proteins shared by both cell lines. Among these, AP1G2 and PKM2 completely lost SUMO2/3, while TNF receptor-associated protein 1 (TRAP1; hsp75) showed a ~5-fold reduction of SUMO2/3 binding. Given TRAP1's pro-oncogenic role involving regulation of mitochondrial integrity, glycolysis and ROS production, we next investigated how sumoylation modulates TRAP1. We first confirmed SUMO2/3–TRAP1 association in the mitochondria, which was then disrupted by TAK-981, using FLAG-TRAP1–expressing HEK293T cells. We then evaluated whether SUMO altered TRAP1 stability and localization using Cycloheximide chase assays. Our results revealed that desumoylation did not alter TRAP1 stability, however, it caused a shift in TRAP1 localization from the mitochondria (in untreated cells) to the cytosol, suggesting that sumoylation guides TRAP1 to the mitochondria. To assess how sumoylation influences TRAP1 protein interactome, we performed IP-MS in HEK293T cells stably expressing TRAP1-FLAG, with or without TAK-981 treatment. TAK-981 significantly reduced the number of proteins interacting with TRAP1 (206 vs. 130), confirming that sumoylation indeed governs TRAP1 interactome. Given that our finding that SUMOylation diverges proteins to mitochondria, we next examined the effects of desumoylation on global mitochondrial function in NHL cell lines. Treatment with TAK-981 induced mitochondrial depolarization, disrupted mitochondrial ultrastructure, and reduced OxPhos (Seahorse). Metabolomic profiling confirmed a decline in TCA cycle intermediates. Upon overexpression of TRAP1 in Raji cells, the mitochondrial dysregulation was partially rescued with protection from TAK 981–induced apoptosis, highlighting that sumoylation alters mitochondrial metabolism, in part via TRAP1. Conclusion: TAK981 disrupts mitochondrial sumoylation, resulting in metabolic dysfunction and apoptosis in B-NHL cells. These findings identify sumoylation and TRAP1 regulation as a potential therapeutic vulnerability in lymphoma.