Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations.

Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 & SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry.

Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p<0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p<0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p<0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p<0.01, respectively).

Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.

Wenzl:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Maurer:BMS: Consultancy, Research Funding; GenMab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Rimsza:Roche: Other: Consulting; NanoString: Other: Licensed intellectual property. Habermann:sorrento: Research Funding; Genentech: Research Funding; BMS: Research Funding. Ansell:Pfizer, Inc: Other: Contracted Research; Bristol-Myers Squibb: Other: Contracted Research; ADC Therapeutics: Other: Contracted Research; Seagen Inc: Other: Contracted Research; Takeda Pharmaceuticals USA Inc: Other: Contracted Research; Affirmed: Other: Contracted Research; Regeneron Pharmaceuticals Inc: Other: Contracted Research. Witzig:ADC: Membership on an entity's Board of Directors or advisory committees; Kura Oncology: Research Funding; Salarius Pharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding. Nowakowski:MEI Pharma: Consultancy; Incyte: Consultancy; Karyopharm Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy; Selvita Inc: Consultancy; F Hoffmann-La Roche Limited: Consultancy; TG Therapeutics: Consultancy; Debiopharm: Consultancy; Zai Lab Limited: Consultancy; Kymera Therapeutics: Consultancy; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy; Abbvie: Consultancy; Curis: Consultancy; Seagen: Consultancy; Celgene Corporation: Consultancy; Blueprint Medicines: Consultancy; Bantam Pharmaceutical LLC: Consultancy; ADC Therapeutics: Consultancy; Ryvu Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cerhan:Protagonist: Other: Safety Monitoring Committee; Genmab: Research Funding; NanoString: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding. Novak:Bristol Myers Squibb: Research Funding.

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