Mouse Model of Splenic Marginal Zone Lymphoma to Decipher the Pathogenetic Implications of NOTCH2 and KLF2 Mutations

Background. Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous disease with a variable clinical course and genetic profile. Our group identified a high-risk subset of patients, termed NNK-SMZLs (58% of cases), associated with poorer survival. This genetic cluster is characterized by the co-occurrence of heterozygous mutations that activate NOTCH2 and heterozygous or homozygous genetic lesions that disrupt KLF2. To investigate the role of NOTCH2 and KLF2 genetic alterations in the development of SMZL, we created genetically engineered mouse models (GEMMs) with conditional alleles that either activated Notch2, inactivated Klf2, or combined both alterations specifically in B cells. Short-term (ST) experiments focused on monitoring changes in the B-cell compartment and understanding how Notch2 and Klf2 mutations cooperate in affecting quantity and quality of splenic B-cell subsets. Long-term (LT) experiments aimed to assess lymphoma type and penetrance.

Methods. We generated a new GEMM by crossbreeding wherein concomitant Klf2 and Notch2 mutations are under the control of Cd19Cre [Klf2^fl/+/Notch2IC^fl/+/Cd19Cre^+/- (Klf2^het/Notch2IC^het) or Klf2^fl/fl/Notch2IC^fl/+/Cd19Cre^+/- (Klf2^hom/Notch2IC^het) in the C57BL/6background]. Genotypes expressing single Notch2IC^fl/+/Cd19Cre^+/- (Notch2IC^het), Klf2^fl/+/Cd19Cre^+/- (Klf2^het), Klf2^fl/fl/Cd19Cre^+/- (Klf2^hom) or Cd19Cre^+/- (control) were also tested. In ST experiments, 3 age/sex-matched 8–12-week-old mice were immunized with NP-KLH or vehicle and sacrificed for analysis after 28 days. Spleen was analyzed for histopathologic features. Splenocytes were analyzed by high dimensional flow cytometry using the BD FACSymphony, exploiting 41 B, T and myeloid markers. Additionally, Chromium Next GEM-X Single Cell Gene Expression (scRNA-seq) on splenocyte, Visium CytAssist for FFPE Spatial Gene Expression profile across spleen compartments and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on sorted marginal zone (MZ) B cells, follicular (FO) B cells, transitional 1 B-cells and transitional 2 B-cells were performed in ST experiments, and results will be presented. In LT experiment 25 age/sex-matched 8–12-week-old mice/genotype underwent chronic immunization with sheep red blood cells and were monitored over 20 months. Spleen, liver, bone marrow and lymph node were extracted for FFPE block/single cell suspension preparation and assessed by two independent pathologists in blind. Lymphoma development was diagnosed based on morphology and phenotype following the classification of mouse lymphomas.

Results. In ST experiment Klf2^hom mice exhibited enlarged FO and MZ areas, while Notch2IC^het mice revealed reduced FO and expanded MZ with blurred marginal sinus (interphase). In turn, Klf2^hom/Notch2IC^het mice displayed features of Klf2^hom mice but also a blurred marginal sinus (interphase). FACSymphony analysis depicted increased proportion of MZ B cells at the expenses of FO B and myeloid cells in Notch2IC^het and Klf2^hom/Notch2IC^het compared to Klf2^hom and control. LT mice with either Klf2^hom/Notch2IC^het or Klf2^het/Notch2IC^het showed higher incidences of SMZL (36%, p=0.002 and 54%, p<0.0001, respectively) and DLBCL (20% and 12%, respectively) compared to single mutants (Notch2IC^het: SMZL 19%, DLBCL 14%; Klf2^hom: SMZL 10%, DLBCL 0%; Klf2^het: SMZL 13%, DLBCL 0%) or controls (SMZL: 5%; DLBCL: 3%).

Conclusions. Our data support the oncogenic roles of NOTCH2 and KLF2 in SMZL development, indicating that their co-occurrence increases lymphoma penetrance. The lymphoproliferative phenotype in double mutant mice closely models human SMZL, including aggressive progression to DLBCL.