Abstract PR003: BTG1 mutation induces an age-associated B cell precursor population with extranodal and brain-infiltrating potential in diffuse large B cell lymphoma via interleukin-21 hypersensitivity

Abstract

The MCD/C5 subtype of diffuse large B cell lymphoma (DLBCL) features frequent extranodal dissemination to immune-privileged sites such as the central nervous system (CNS), poor clinical outcomes, and recurrent BTG1 genetic alterations in up to 70% of cases. However, the cellular origin and mechanisms underlying this dissemination remain unknown. Genomic analysis revealed that BTG1 is the third most enriched mutated gene in CNS versus nodal DLBCL (27% vs 9%, p<0.001). Furthermore, BTG1-mutated MCD-DLBCL patients exhibit significantly worse overall survival (p<0.001). Building on previous finding that the most frequent Btg1 Q36H mutation facilitates MYC protein synthesis, we generated a lymphoma mouse model combining Btg1 Q36H and Myc conditional knock-in with Cgamma1-Cre, restricting expression mostly to germinal center B cells (GCBCs). Myc+Btg1 Q36H expression led to spontaneous large mesenteric lymph node tumors, which were not observed in Myc-only mice. After two serial transplantations into Rag1 knockout mice, Myc+Btg1 Q36H cells rapidly disseminated to extranodal tissues (kidney, liver, lung) and the CNS (5-week latency). Myc+Btg1 Q36H tumor cell suspensions were able to grow ex vivo as a cell line with a 50-h doubling time. Using Xenium spatial RNA sequencing, we found that over 40% of tumor cells in the spleen, liver and lung, and 20% in the brain, phenotypically resembled age/autoimmune-associated B cells (AiBCs), suggesting AiBCs as the cell with extranodal and CNS infiltration potential. Combining Btg1 Q36H with the MCD-DLBCL hallmark Myd88 L265P mutation, we measured higher proportions of AiBCs (CD23-CD21-CD11c+) in the spleen of aged non-immunized Myd88 L265P+Btg1 Q36H mice vs Myd88 L265P mice. Aged non-immunized Btg1 Q36H mice also showed a higher proportion of spleen-resident and circulating AiBCs vs wild-type controls. Ex vivo stimulation of naïve B cells revealed that Btg1 Q36H cells differentiate more readily into AiBCs, a process reliant on T follicular helper (Tfh) cell signals such as IL-21. Mechanistically, Btg1Q36H GCBCs displayed a Tfh cell-dependent competitive advantage and hypersensitivity to limiting IL-21 levels, evidenced by enhanced STAT3 phosphorylation at low IL-21 concentrations. Btg1 Q36H Myc+ GCBCs exhibited elevated IL-21 receptor (IL-21R) expression. Specific knockdown of IL-21R in Btg1 Q36H cells abrogated their competitive advantage. Finally, in human DLBCL bulk RNAseq datasets (n=347), BTG1 mutant cases were enriched for upregulated IL-21-induced transcriptional signatures (GSEA NES=1.87, FDR<0.001). The GSVA score for IL-21 activated signature in DLBCL cases highly correlated with a MYC-driven malignant AiBC signature (R=0.887, p<0.001), and patients enriched for both exhibited significantly higher tumor content compared to other patients (n=99 vs 248, p<0.0003). Collectively, our data point to a role for BTG1 mutations in driving extranodal dissemination and CNS infiltration in MCD DLBCLs, by promoting an AiBC-like cellular state fueled by hypersensitivity to IL-21.