SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming.

Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A02.