Single cell sequencing data for patient AZ01

Flow cytometry analysis for Ki67, MitoBright Green, and JC-1 in Mino and JeKo-1 cells.

The Bridging the Gaps (BTG) in Leukemia, Lymphoma and Multiple Myeloma Consensus Conference 2025 brought together a multidisciplinary group of oncology experts to address the complexities of lymphoma management, focusing on mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and diffuse large B-cell lymphoma (DLBCL). This article presents consensus recommendations developed through a modified Delphi process, which emphasize the need for tailored therapeutic strategies in light of recent advancements in treatment options. Key recommendations include the screening for high-risk features in MCL, use of the BOVen regimen (zanubrutinib, obinutuzumab, and venetoclax) for TP53-aberrant cases, and integration of chimeric antigen receptor T-cell therapy for patients with mantle cell lymphoma that is refractory to covalent Bruton tyrosine kinase inhibitors. For CLL, recommendations include consideration of time-limited therapies for younger patients and a "watch and wait" strategy for asymptomatic patients despite the improved activity and safety of current treatment regimens. For DLBCL, this article highlights the challenges in treatment sequencing and the role of circulating tumor DNA and minimal residual disease testing in monitoring disease progression. Overall, the conference describes the importance of ongoing research to refine management strategies and improve patient outcomes in lymphoma care, addressing the gaps in clinical practice where high-level evidence is lacking.

Cell proliferation assay for MCL cell lines treated with IACS-010759

Single-cell RNA-seq analysis of PBMCs from a patient with MCL responsive to AZD4573 treatment on a clinical trial (AZ01). A, Uniform Manifold Approximation and Projection (UMAP) plot representation of integrated total PBMCs collected from the patient across four designated time points (n = 9,499 in total; baseline 0 hours, 4,663 cells; 4 hours, 859 cells; 24 hours, 2,666 cells; and C1D8, 1,311 cells) revealed four main cell populations. Population identities were determined based on marker gene expression. B, Normalized genes expression level heatmap of top marker genes per cluster. C and D, Dot plots showing the pathway enrichment on hallmark gene sets against differential expressed genes between time points, in B_cell_1 and B_cell_2 clusters, respectively. The ranks of genes per comparison group were calculated based on log FC and P value.

Single cell sequencing data for patient AZ04

The mechanisms of resistance to Bruton tyrosine kinase inhibitors (BTKi) in MCL are not well understood. We found that cyclin-dependent kinase 5 (CDK5), a serine/threonine kinase, was upregulated in ibrutinib-resistant MCL cell lines and in primary MCL cells obtained from patients who progressed on BTKi. Furthermore, primary MCL cells upregulated CDK5 in BAFF/CD40L stromal conditions, and CDK5 mRNA overexpression in primary MCL tumors was associated with inferior outcomes. Genetic and pharmacologic (GFB-12811) manipulation of CDK5 in MCL cell lines revealed that CDK5 contributed to proliferation and ibrutinib resistance in vitro , while engineered expression of CDK5 in JeKo-1 cells shortened survival in a murine xenograft model. CDK5 was upregulated by B-cell receptor and BAFF signaling and complexed with BTK. Mass spectrometry analysis of CDK5-manipulated cells revealed that cell cycle and metabolism-related pathways were most affected by CDK5; 29 kinases were differentially active, and Src kinase activity correlated with CDK5 expression. CDK5 complexed with IRE1α and Xbp1, promoted IRE1α phosphorylation and increased Xbp1 levels thus facilitating unfolded protein response. Meanwhile, Xbp1 knockdown resensitized MCL cells to ibrutinib. Collectively, CDK5 promotes BTKi resistance via modulation of the XBP1/IRE1α axis of the UPR pathway and represents a potential therapeutic target in MCL.

To understand the recent, evolving treatment landscape for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), we retrospectively analyzed US claims data from MarketScan Commercial Claims and Encounters and Medicare databases and Symphony Health Solutions database for adult patients who initiated first-line DLBCL therapy from January 2017 through June 2023 (MarketScan) or May 2024 (Symphony). The MarketScan analysis included 3552 patients (median follow-up: 15 months). The Symphony analysis included 49,370 patients (median follow-up: 35 months). Results from this contemporary real-world analysis showed heterogeneity in DLBCL treatments administered, with a lack of standard of care in the third-line/fourth-line setting. In both analyses, most patients (>50% per line) received rituximab-based therapy as second-line or later-line therapy. These results highlight a need for new regimens that demonstrate clinical benefit and improve overall survival in patients with R/R DLBCL, while also being feasible and widely applicable in routine practice.

Abstract Aberrant sumoylation is frequently observed in human tumors, highlighting its significance in cancer biology and therapeutic targeting. We investigated the mechanistic effects of inhibiting SUMOylation in B-cell non-Hodgkin lymphoma (NHL) using the inhibitor TAK-981 (subasumstat). SUMO pathway components were highly expressed in lymph nodes from NHL patients compared with reactive lymph nodes. TAK-981 potently inhibited growth of NHL cell lines (IC50 ∼10 nM), inducing apoptosis and reducing cell viability over 24-72 hours, alongside rapid, dose-dependent global protein desumoylation. Consistent with these in-vitro effects, TAK-981 treatment significantly prolonged survival of OCI-LY3 and Raji xenograft-bearing mice by approximately 5.5 weeks relative to control. To uncover SUMO-regulated pathways relevant to the TAK-981 response, we performed RNA-Seq on B-NHL cell lines. Gene set enrichment analysis revealed significant downregulation of MYC targets and oxidative phosphorylation-related genes. MitoMiner gene-ontology analysis further indicated suppression of mitochondrial membrane organization and depolarization pathways. Subcellular fractionation showed that TAK-981 caused dramatic desumoylation of proteins in the mitochondrial fraction compared to the cytosol. LC-MS/MS profiling of mitochondrial extracts from U2932 and Raji cells identified 40 and 44 proteins, respectively, with >4-fold reductions in SUMO2/3 binding. The mitochondrial chaperone TRAP1 (Hsp75), which in Raji cells exhibited ∼5-fold decreased SUMO association. Given TRAP1’s pro-oncogenic role in maintaining mitochondrial integrity and metabolism, we investigated how SUMOylation affects TRAP1. We confirmed SUMO2/3-TRAP1 association in mitochondria, which was disrupted by TAK-981 in FLAG-TRAP1-expressing HEK293T cells. Cycloheximide chase assays demonstrated that desumoylation did not alter TRAP1 protein stability; however, it caused a redistribution of TRAP1 from mitochondria to the cytosol, indicating that SUMOylation guides TRAP1 mitochondrial localization. Consistent with widespread loss of mitochondrial SUMOylation, TAK-981 induced mitochondrial depolarization, ultrastructural defects, and reduced oxidative phosphorylation as measured by Seahorse analysis. Metabolomic profiling confirmed decreases in TCA cycle intermediates. Importantly, TRAP1 overexpression in Raji cells partially restored mitochondrial function and protected cells from TAK-981-induced apoptosis, supporting a functional requirement for SUMOylated TRAP1 in lymphoma cell survival. Collectively, our findings show that TAK-981 disrupts mitochondrial SUMOylation, leading to metabolic dysfunction and apoptosis in B-NHL cells. SUMO-dependent regulation of TRAP1 emerges as a mechanistic driver and a promising therapeutic vulnerability in lymphoma. Citation Format: Vi Lam, Tingting Liu, Heifeng Shen, Olga V. Danilova, Katarzyna Dabrowska, Sonia Rodriguez, Courtney Jones, Martina Cusan, Angelo D’Alessandro, Lapo Alinari, Lili Wang, Tycel Phillips, Patrick Pirrotte, Zheng Xia, Alexey Danilov. Selective targeting of sumoylation disrupts mitochondrial homeostasis via TRAP1 to suppress growth of non-Hodgkin lymphoma (NHL) B-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3057.

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.

Pathway analysis of murine MCL splenocytes. A, GSEA of spleen-resident tumor cells from MCL01 mice treated with high-dose AZD4573 (2× weekly at 15 mg/kg) and vehicle that were subjected to RNA-seq analysis. Graph shown represents the top 10 hallmark pathways that were positively enriched and top 10 hallmark pathways that were negatively enriched in the AZD4573- vs. vehicle-treated mice. (* indicates nominal P value ≤ 0.05). B, GSEA plots for select gene sets. C and D, Heatmap depicting the ranked list of genes that had core enrichment for the TNFα signaling via NF-κB pathway and OxPhos as seen in B. Top row indicates treatment. Gray, three control-treated mice. Orange, four AZD4573-treated mice The range of expression values are visualized as red (high) to blue (low).

MCL cell lines treated with AZD4573 prolifereation assay

AZD4573 induces apoptosis and thwarts survival of MCL cells. A, MCL cell lines were treated with the indicated doses of AZD4573 for 72 hours. Cell proliferation was assessed using a colorimetric tetrazolium-based assay. The mean ± SEM is shown. IC50 value was calculated using GraphPad Prism software set to variable slope. B, Cells were treated with indicated doses of AZD4573 for 8 hours. Apoptosis was determined using Annexin V–FITC staining. Data are the mean ± SEM. *, P < 0.05 and **, P < 0.01 vs. untreated control. C, Primary MCL cells (n = 5 individual samples tested in technical triplicates) were cocultured with stromal cells for 24 hours and then treated with indicated doses of AZD4573 for 24 hours. Apoptosis of the CD19+ B-cell population was quantified using Annexin V–FITC staining. *, P < 0.05 and **, P < 0.01 vs. untreated control. D, Parental and IR MCL cell lines were treated with 10 nmol/L of AZD4573 or vehicle control for the indicated time points. Cell lysates were subjected to immunoblotting. E, Mice were inoculated with MCL. Two distinct MCL PDX models were used. Once circulating CD5+/CD19+ MCL cells were detected in the peripheral blood, mice began treatment as indicated in the methods (n = 3–11 per group). Kaplan–Meier survival curves are shown, with significance determined by the log-rank test. *, P < 0.05; **, P < 0.01.

The treatment of relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains challenging, with inadequate responses to salvage chemoimmunotherapy limiting patients’ ability to receive potentially curative treatments like autologous stem cell transplantation (ASCT). Epcoritamab, a subcutaneous CD3×CD20 bispecific antibody, has demonstrated antitumor activity in R/R DLBCL as a monotherapy and in combination with chemotherapy. In Arm 4 of the EPCORE® NHL-2 phase 1b/2 trial (NCT04663347), transplant-eligible patients with CD20+ R/R DLBCL received epcoritamab plus rituximab, dexamethasone, cytarabine, oxaliplatin/carboplatin (R-DHAX/C). Patients could continue epcoritamab until ASCT or progression. Twenty-nine patients received epcoritamab plus R-DHAX/C; 72% had stage IV disease; 66% had primary refractory disease. As of January 15, 2025 (median follow-up 40.4 months), overall response rate (primary endpoint) was 79%, and complete response rate was 69%. Sixteen patients (55%) proceeded to ASCT and five remained on epcoritamab monotherapy. At 36 months, an estimated 70% of responses were ongoing, 59% of patients were progression-free, and 76% were alive. Common treatment-emergent adverse events (TEAE) were thrombocytopenia (90%), anemia (66%), and neutropenia (59%). Cytokine release syndrome occurred in 45% of patients; all were grade 1–2 and resolved after a median of 2 days. Immune effector cell-associated neurotoxicity syndrome occurred in one patient. No fatal TEAE or clinical tumor lysis syndrome were observed. Epcoritamab plus R-DHAX/C achieved deep, durable responses with manageable safety. Over half of patients proceeded to ASCT, a potentially curative treatment. These findings suggest the potential of epcoritamab combined with standard chemoimmunotherapy as an effective salvage treatment for patients with R/R DLBCL.

Abstract Resistance to Bruton tyrosine kinase inhibitors is inevitable in mantle cell lymphoma (MCL). Cyclin-dependent kinase-9 (CDK9), a key regulator of oncogenic transcription, is a promising therapeutic target. In this study, we studied a selective CDK9 inhibitor, AZD4573, in MCL. Treatment with AZD4573 thwarted growth of both parental and ibrutinib-resistant MCL cell lines and primary MCL cells and downregulated expression of MYC and MCL1. However, CDK9 inhibition enhanced basal and maximal oxygen consumption rate, as well as increased production of ATP and reactive oxygen species in ibrutinib-resistant cell lines and primary MCL cells. Whereas treatment with AZD4573 led to modest prolongation of survival in an ibrutinib-resistant MCL patient-derived xenograft mouse model, accompanied by downregulation of TNFα/NF-κB and mTORC1 signaling pathways in murine splenocytes, oxidative phosphorylation (OxPhos) was upregulated, suggesting tumor metabolic reprogramming. Single-cell RNA sequencing analysis of peripheral blood mononuclear cells (PBMC) from patients treated with AZD4573 on a clinical trial demonstrated sustained downregulation of MYC targets and OxPhos in malignant B cells from a responding patient with MCL. Conversely, two refractory patients exhibited upregulation of MYC targets and OxPhos in PBMCs. OxPhos inhibitor IACS-010759 demonstrated synergy with AZD4573 in vitro. Thus, CDK9 inhibition exhibits activity in ibrutinib-resistant MCL and can be further enhanced by cotargeting of OxPhos. Significance: Ibrutinib resistance is a challenge in MCL. Targeting CDK9 partially overcomes this resistance but results in upregulation of cell metabolism contributing to survival. Dual targeting of CDK9 and OxPhos cooperates to suppress tumor growth, providing a rationale for future exploration of combination therapies in MCL.

GSEA analysis of a patient sensitive to AZD4573 treatment. GSEA plots for hallmark MYC targets V1 and OxPhos gene sets for patient AZ01. Graphs shown are only for B_cell_1 cluster at the designated time points vs. 0 hour.

Epcoritamab, a CD3xCD20 bispecific antibody, resulted in deep, durable responses with a manageable safety profile in patients with relapsed/refractory large B-cell lymphoma (LBCL) in EPCORE® NHL-1 (NCT03625037). We report results from a 3-year follow-up. Adults with relapsed/refractory LBCL received epcoritamab until progressive disease or unacceptable toxicity. The primary endpoint was overall response rate (ORR). Median age was 64.0 years, 39