Germinal Center-Derived Lymphomas and Plasmacytomas in Mice with Targeted Deletion of MiR-15a/16-1 in Activated B Cells
Authors: Sewastianik Tomasz , Zhao Jianjun , Jiang Meng , Wang Jianli , Pillai Vinodh , Dennis Peter S. , Lin Jianhong , Brooks Daniel , Bouxsein Mary , Pinkus Geraldine S , Jarolim Petr , Robbiani Davide , Ghobrial Irene M. , Anderson Kenneth C. , Carrasco Ruben D. 
Published: October 2019

Abstract
MicroRNA (miR)-mediated gene regulation plays critical roles in B-cell development and dysregulated expression of miRs has been implicated in the pathogenesis of various types of B-cell malignancies. Somatic deletions of chromosome 13q14, harboring miR-15a/16-1, occurs frequently in B-cell lymphomas suggesting that members of this miR family are tumor suppressors. Consistently, mice with CD19-Cre-induced deletion of miR-15a/16-1 in early B-cells and follicular B-cells develop chronic lymphocytic leukemia (CLL). Since the 13q14 deletion is observed in a broader range of B-cell malignancies, we hypothesized that the type of B-cell malignancy resulting from miR-15a/16-1 down-regulation may depend on the stage of B-cell development at which this deletion occurs. Therefore, we generated a transgenic mouse model in which conditional deletion of miR-15a/16-1 takes place at later stages of B-cell development. To delete miR-15a/16-1 in activated B-cells, miR-15a/16-1fl/fl mice were mated with AID-Cre+/+ mice to obtain AID-Cre+/-; miR-15a/16-1fl/fl compound mice that expressed Cre recombinase from the Activation-induced Cytidine Deaminase (AID promoter) gene - a gene needed for generation of somatic hypermutations in the immunoglobulin (Ig) variable region (V) genes that is highly expressed in activated B-cells and is a well-known marker for germinal center (GC) B-cells. Expression levels of both miR-15a and miR-16-1, but not miR-15b were decreased in GC B-cells of AID-Cre+/-; miR-15a/16-1fl/fl mice as compared with control AID-Cre+/- mice when evaluated by In Situ Hybridization (ISH) analysis. Given that in humans miR-15a, b and 16 are also expressed in GC B-cells, these results demonstrate the validity of this mouse model in which the biological consequences of miR-15a/16-1 deletion can be studied. Next we assessed whether miR-15a/16-1 deletion could affect proliferation and/or survival of GC B-cells. GCs in the spleens of AID-Cre+/-; miR-15a/16-1fl/fl mice at 10 weeks of age were significantly increased in both number and size, and contained a larger number of Ki-67-positive B-cells as compared with spleens of AID-Cre+/- mice. No significant differences in the number of apoptotic cells, neither in the expression of the miR-15a/16-1 putative target BCL2 were detected, indicating that miR-15a/16-1 may play important roles in the proliferation, but not survival of GC B-cells. Apart from mild splenic enlargement and increased number and size of GCs, AID-Cre+/-, miR-15a/16-1fl/fl mice where indistinguishable from AID-Cre+/- mice between 8 and 40 weeks of age as assessed by weight and posture. However, after 48 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-, miR-15a/16-1fl/fl mice but none from control cohorts (0/30) showed signs of disease. Gross pathologic examination of euthanized AID-Cre+/-; miR-15a/16-1fl/fl mice revealed enlargement of the spleen and lymph nodes. Detailed histological examination revealed in most instances an effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells, or less commonly by sheets of plasma cells in interfollicular areas. Two distinct patterns of B220+BCL6+BCL2- B-cell lymphomas were identified after detailed analysis. The most common (47%) resembled human follicular lymphoma (FL) and the next in frequency (28%) resembled human diffuse large B-cell lymphoma (DLBCL). The other group of tumors (25%) resembled human plasmacytoma (PC). All three tumor subtypes were clonal, hypermutated and associated with different degrees of preservation of the dendritic meshwork in the lymph nodes. The comparison of lymphomas arising in AID-Cre+/-; miR-15a/16-1fl/fl mice and CD19-Cre+/-; miR-15a/16-1fl/fl mice corroborated that deletion of miR-15a/16-1 at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated miR-15a/16-1 expression in human FL and PC and showed that miR-15a/16-1 abundance is significantly decreased in those malignancies when compared with nodal B-cells in reactive GCs and normal plasma cells in interfollicular areas respectively, suggesting that miR-15a/16-1 may play important roles in normal GC B-cell development as well as in the pathogenesis of FL and PC in humans.

Disclosures
Ghobrial: BMS: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.