Murine modeling of cluster 1 diffuse large B-cell lymphoma reveals transformation from marginal zone lymphoma Free

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease. Our comprehensive genomic profiling of newly diagnosed DLBCLs identified 5 discrete molecular subsets (Clusters 1–5, [C1–5]). C1 DLBCLs share molecular features with transformed marginal zone lymphomas (tMZL), including NOTCH2 PEST domain hotspot mutations, which limit protein degradation, and BCL6 translocations. We developed a murine model of C1-like DLBCL to define the cell of origin and assess the role of Notch2 alterations in Bcl6-driven lymphomagenesis.

Methods: The coding sequences of Notch2 wild-type (wt) or Notch2 R2402* (mut), which encodes the most frequent PEST domain mutation, were cloned into a vector targeting the Rosa26 (R26) locus. Both Notch2 constructs included the intact extracellular ligand-binding domain to analyze Notch2 signaling in a ligand-dependent and physiologically relevant context. R26^N2wt/+ and R26^N2mut/+mice were generated via CRISPR/Cas9-mediated knock-in. B-cell specific expression of exogenous Notch2 was achieved by crossing R26^N2wt/+ and R26^N2mut/+ mice with Cd19^cre/cre animals,generating Cd19^cre/+; R26^N2wt/+ (N2wt) and Cd19^cre/+; R26^N2mut/+(N2mut) cohorts (and Cd19^cre/+[Cre] controls).

To model C1 DLBCL, the Notch2 cohorts were bred with Bcl6^tg/tg mice, generating Cd19^cre/+; R26^N2mut/+; Bcl6^tg/+(N2mutBcl6) animals and additional Cd19^cre/+; R26^N2wt/+; Bcl6^tg/+ (N2wtBcl6)and Cd19^cre/+; Bcl6^tg/+(Bcl6) cohorts. We aged 20-22 mice per genotype for 18 months (mo) to assess lymphoma development and euthanized additional asymptomatic animals at 2, 6, and 12/14 mo to evaluate aberrant B-cell evolution. Spleens from asymptomatic and tumor-bearing mice were collected for morphologic and single-cell analyses. The intact splenic microenvironment was evaluated with H&E and immunohistochemical (IHC) staining by expert pathologists. Splenocytes were also analyzed by flow cytometry (FC).

Results: Compared to Creand N2wt control mice, N2mut animals exhibited splenic MZ expansion detected by CD21 IHC. FC analysis revealed an increase in MZ B cells and a reduction in follicular B cells (p < 0.01) in N2mut splenocytes. Notch2 target genes (Hes1, Dtx1, S1pr3) were upregulated in N2mut, compared to N2wt, splenocytes (p < 0.05), confirming Notch2 pathway activation. Neither splenic enlargement nor Ig clonal rearrangements were observed in the N2wt or N2mut cohorts.

In contrast, N2wtBcl6and N2mutBcl6mice developed splenic enlargement at 6 mo, in comparison to Bcl6 mice (p = 0.03 and 0.01, respectively) and Notch2-only counterparts (N2wtand N2mut, both p < 0.01), suggesting a synergistic effect between Notch2 activation and deregulated Bcl6 expression. This phenotype persisted at 12/14 mo.

Like N2mut animals, N2mutBcl6mice showed early (2 and 6 mo) splenic MZ expansion. However, at 12/14 mo, N2mutBcl6 mice exhibited attenuated MZs, disrupted splenic follicular dendritic cell networks and compressed germinal centers, morphologically resembling human MZL. At this timepoint, all N2mutBcl6 mice, vs only 25% of the N2wtBcl6 and none of the Bcl6 animals, had clonal Ig rearrangements. Changes in N2mutBcl6 splenic architecture, including progressive attenuation of the architectural distinction between the MZ and follicle, were more pronounced at 18 mo, suggesting dynamic remodeling during disease progression. Notably, MZ B cells with elevated Ki-67+ expression were only detected inN2mutBcl6mice.

In the aging cohorts, 14/21 (67%) N2mutBcl6 mice, vs only 4/20 (20%) N2wtBcl6 and 5/22 (23%) Bcl6 animals, were euthanized for symptoms (log-rank p = 0.0001). Additionally, N2mutBcl6 mice had earlier onset disease: 5/21 (24%) N2mutBcl6 mice, vs none of N2wtBcl6 or Bcl6 mice, were symptomatic at 6 mo. Among all 20 symptomatic mice with viable intact spleens for histological analysis, 10 showed a diffuse pattern of enlarged aberrant B cells (N2mutBcl6, N2wtBcl6, Bcl6: 5, 2, 3); 6 animals exhibited white pulp/MZ expansion with small to intermediate-sized aberrant B cells (N2mutBcl6, N2wtBcl6, Bcl6:5, 1, 0); and 4 lacked morphologic evidence of lymphoma. In the 11/16 presumptive lymphoma cases evaluated to date, all had clonal Ig rearrangements.

Conclusions: We found that the Notch2 R2402* mutations synergize with Bcl6 translocationsto promote B-cell lymphomagenesis and progression from MZL to tMZL/DLBCL. This C1-like murine model provides a framework to define the pathogenesis of human MZL and C1 DLBCL.