In vivo data and validation of MV4-11_LUC cell models

Knock-In mutations to specific N-linked glycosylation sites results in higher CD79B but not immunoglobulin MFI by flow cytometry

7063 Background: Genetic subtypes of diffuse large B-cell lymphoma (DLBCL) capture biological differences between tumors that influence the response to immunochemotherapy (Schmitz et al., NEJM 2018). However, nearly 40% of DLBCL cases remain unclassified. The addition of gene expression signatures can accelerate classification and inform therapeutic intervention. Methods: We generated single-cell RNA sequencing of 103 DLBCL patient biopsies from Weill Cornell Medicine, New York Presbyterian Hospital, and the National Institutes of Health. We also utilized bulk genomic data from a discovery cohort (n=311, Ennishi et al., J Clin Oncol 2019) and a validation cohort (n=574, Schmitz et al., NEJM 2018). Results: Single cell sequencing allowed us to isolate the malignant B cells and develop genetic subtype signatures. The MCD signature was associated with poor overall survival (p<10 -6 ), as was the BN2 signature within ABC tumors (p<10 -3 ). When applied to unclassified tumors, the subtype signatures identified characteristic genetic alterations including SLC1A5 mutations in MCD (p<10 -5 ), UBE2A mutations in BN2 (p<10 -13 ), C10orf12 truncations and copy number loss in EZB (p<10 -6 ), and SGK1 mutations in ST2 (p<10 -7 ). Moreover, we discovered that most DLBCL tumors (80%) contained two or more genetic subclones (median 2, range 1-5) based on DNA copy number differences. The genetic subclones had distinct phenotypes based on expression of six recurrent gene expression meta-signatures, herein termed themes. The germinal center (GC) B cell, memory B cell, plasma cell, and pan-B cell themes reflect B cell differentiation whereas two other themes – cell cycle and cell growth – reflect proliferative and metabolic states that are independent of the differentiation states. Surprisingly, 23% of DLBCL (24/103) harbored genetic subclones expressing B cell differentiation themes that distinguished them from other malignant cells in the same tumor. The GC B cell theme was associated with a favorable response to R-CHOP chemotherapy (p<0.02), as expected, while the cell growth theme (but not the cell cycle theme) was associated with adverse survival (p<0.02). Conclusions: Our study revealed that genetic subtypes have distinct gene expression signatures. We further demonstrated a role for tumor subclones in generating intra-tumoral biological diversity. We developed signatures of inter and intra-tumoral heterogeneity that are associated with overall survival.

Supplementary Table S7, Table showing source data and statistics for non-high-throughput experiments such as flow cytometry, quantitative PCR, protein experiments and other molecular and cellular assays.

Mutation of CD79A and CD79B glycosylated residues does not alter sensitivity to MMAE

Diffuse large B-cell lymphoma (DLBCL) is clinically and genetically heterogeneous. The existence of genetically defined subtypes with therapeutic implications is clear. Additional considerations that are yet to be fully resolved include defining boundaries between discrete classes, relevant molecular data types, and handling of unclassifiable/composite cases. In this review, we juxtapose points of view on the topic from experts in the field, outline practical workflow considerations, and distill lessons from clinical trials. These synthesize experimental design and analytic considerations that inform the development and evaluation of clinically relevant DLBCL subtyping approaches. SIGNIFICANCE:The most meaningful way to stratify DLBCL remains unresolved, and this represents a significant barrier to adopting a precision medicine paradigm.

CD79B Segregation Score and Pola-V CSS for Golgi glycosylation genes shows specific sugar regulators of surface CD79B and Pola-V sensitization

Supplementary Table S3, Quantitative whole proteome analysis of pooled sorted GCB-cells from Fbxo45 homozygous knockout mice compared with identically isolated cells from wild-type littermates.

Small-molecule sialyltransferase inhibition synergizes with Pola-V in ABC and GCB DLBCL

Disease score with corresponding features for the top disease per gene

MV4-11 and NB4 BioID_ATP1B1 and BioID_ATP1B3 results

Candidate gene selection based on the introduced features

DLBCL mutations targeting the CD79B intracellular domain and their effect on surface BCR levels

Knockout of SLC35A1, SLC35A2, and ST6GAL1 decreases surface a2,6-linked sialic acid.

ATP1B1 expression in CCLE highlighting Ewing sarcoma cell lines

Abstract Introduction: Oral targeted agents are active in MCL; however, most fail to induce deep or durable remissions as monotherapy. We developed the multi-agent ViPOR regimen to target B-cell receptor (BCR) signaling, BCL2, and CD20, and showed it to be safe and able to induce durable remissions in R/R DLBCL (Melani et al. NEJM. 2024). We hypothesized that fixed-duration ViPOR would be able to induce durable remissions in pts with MCL due to its dependence on BCR signaling and BCL2. Methods: TN and R/R MCL pts with adequate organ function were eligible. Venetoclax 400mg was given PO D2-14 on C2-6 with an initial 12d ramp-up on C2, with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide 15mg PO D1-14 on C1-6. ViPOR q21d x 6C was given without maintenance. TLS prophylaxis was given to all pts and R/R pts received G-CSF. Baseline CT, PET, BM, and tumor biopsy were performed with CT after C1, 2, 4, and 6 and PET after C6. CT was then performed q3m x 1y, q4m x 1y, q6m x 1y, and q12m x 2y. MRD was assessed in ctDNA from paired plasma and PBMC samples using clonoSEQ at baseline, during treatment, and in f/u. Results: 41 pts (21 R/R & 20 TN) were enrolled. Median (range) age was 67y (41-82) with 71% male. Blastoid morphology, Ki-67 >30%, and TP53 aberration was noted in 27%, 37%, and 38%, respectively. High-risk MIPI and MCL35 proliferation score was seen in 32% and 20%, respectively. Median (range) prior therapy in R/R was 3 (1-7), with prior BTKi in 43% and 52% of pts refractory to last treatment. Among all pts, heme AEs included G3-4 (% cycles) thrombocytopenia (14%), neutropenia (13%), and anemia (10%), with no febrile neutropenia observed across 216 cycles. G3 non-heme AEs in >10% pts included hypokalemia (28%) and rash (13%). Common any grade non-heme AEs (% pts) included hypokalemia (92%), diarrhea (65%), and rash (54%). G3 A.fib and G3 VTE occurred in 2 pts each, with no major bleeding noted. No TLS or treatment-related mortality occurred. Dose reductions occurred in 27%, and 90% of pts completed all 6C. ORR and CR rate were 100% (21/21) and 95% (20/21), respectively, in R/R and both 100% (20/20) in TN MCL. CRs occurred across all MIPI and MCL35 risk groups and high-risk subsets. With a median f/u of 43m, 80% of CRs are ongoing, with a 3y TTP and PFS of 70% and 53%, respectively, in R/R, and both 95% in TN MCL. Among all pts, 3y TTP was 60% in pts with elevated Ki-67 >30%, 70% in blastoid, and 71% in pts with TP53 aberration. MRD was evaluable in 98% (40/41) of pts, with undetectable MRD (uMRD) achieved in 18%, 79%, and 95% of pts after C1, C2, and at EOT, respectively. Median (range) duration of uMRD was 24m (0-53), and all 5 pts with relapse after CR experienced molecular relapse prior to imaging, with a median (range) lead-time of 4m (0-6). Conclusions: Fixed-duration ViPOR x 6C induces a high rate of uMRD CRs in R/R and TN MCL, including high-risk subsets. ViPOR with a 12d venetoclax ramp-up on C2 is safe in MCL pts of all ages without significant TLS or febrile neutropenia. Citation Format: Christopher Melani, Rahul Lakhotia, Stefania Pittaluga, James D. Phelan, Jagan Muppidi, Max Gordon, Yandan Yang, Weihong Xu, Theresa Davies-Hill, Da Wei Huang, Craig J. Thomas, Michele Ceribelli, Frances A. Tosto, Anna M. Juanitez, Amynah Pradhan, Atekelt Tadese, Colleen A. Ramsower, Lisa M. Rimsza, Allison Jacob, Elaine S. Jaffe, Mark Roschewski, Louis M. Staudt, Wyndham H. Wilson. Combination targeted therapy with ViPOR in relapsed/refractory (R/R) and treatment-naïve (TN) mantle cell lymphoma (MCL): Analysis of safety, efficacy, and minimal residual disease (MRD) [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A027.

Supplementary Table S2, Mass spectrometric (MS) identification of GEF-H1-derived peptides in FBXO45 immunoprecipitation. FLAG-tagged FBXO45 expressing B-cell lymphoma cell lines were subjected to immunoprecipitation (IP) with anti-FLAG resin. MS analysis of co-purified endogenous proteins revealed the presence of numerous GEF-H1-derived peptides.

The clinical and molecular heterogeneity of diffuse large B cell lymphoma (DLBCL) is incompletely understood. By integrating proteomic, transcriptomic, and genomic data from 478 DLBCL tumors, we identify seven DLBCL proteogenotypes (PGs) reflecting specific pathophysiological features that span known molecular subtypes. PG4 is associated with poor outcome independent of established risk factors such as cell-of-origin, international prognostic index, or genetic features. PG4 contains activated B cell-like and germinal center B cell-like tumors and genetically unclassified cases. It shares a dark-zone-related B cell phenotype and shows enrichment for BTG1 mutations that can activate MYC. Single-cell sequencing and spatial transcriptomics reveal enhanced MYC and TCF3/4 transcriptional activity irrespective of MYC translocations. The PG4 tumor microenvironment is characterized by exhausted CD8+ T cells. Our study identifies common oncogenic themes underlying high-risk DLBCL tumors and provides a proteogenomic framework for future diagnostic and therapeutic approaches.