PRMT5 Transgenic Mice Develop Aggressive Lymphoblastic Lymphomas

Introduction: Emerging data collected from whole genome and epigenomic studies in solid and blood cancers has pointed toward dysregulation of chromatin remodelers as a unique class of cancer drivers. Next generation sequencing of lymphoma has identified several mutations affecting enzymes that regulate epigenetic control of gene expression. The epigenetic modifier protein arginine methyltransferase 5 (PRMT5) that has been shown to be essential for Epstein-Barr virus-driven B-cell transformation, is overexpressed in several histologic subtypes of B-cell non-Hodgkin's lymphomas (NHL) and is required for the driver activity of oncogenes such as MYC and NOTCH. While these findings suggest that PRMT5 may act as a driver of lymphomagenesis, definitive experiments to address its driver activity have yet to be performed. To address this question, we developed a transgenic mouse model by immunoglobulin m heavy chain enhancer/promoter (Em)-driven PRMT5 over expression in the lymphoid compartment of FVB/N mice.

Methods: Eµ-hPRMT5 transgenic mice were created by injecting a vector containing floxed human PRMT5 under the control of the Eµ enhancer/promoter into FVB/N pronuclei that were implanted into pseudo-pregnant FVB/N mice. We obtained 5 founder lines demonstrating the presence of transgene construct by genotype PCR analysis of tail snip DNA. Founder mice were crossed with wild type FVB/N mice to obtain a F1 generation. Mice were followed clinically in standard pathogen-free housing until exhibiting phenotypic features at which time necropsy was performed. Immunophenotypic analysis was performed by flow cytometry, clonality by T cell receptor (TCR) Vb PCR, and pathology by hematoxylin-eosin staining and tissue micro-arrays developed for immunohistochemical staining (IHCS). Statistical significance was determined using a two-tail t-test and survival analysis conducted using Kaplan Meier curves.

Results: F1 generation Eµ-hPRMT5 mice significantly overexpressed PRMT5 mRNA in unpurified splenocytes or bone marrow relative to non-transgenic mice (p-value < 0.001). Sorting B (CD19), NK (NK1.1) and T-cell (CD3) mononuclear subsets from splenocytes collected from Eµ-hPRMT5 mice (n=3/group) revealed PRMT5 mRNA to be overexpressed 37-fold (p-value <0.01), 7-fold (p-value <0.01) and 6-fold (p-value <0.05), respectively compared to WT FVB/N mice. All 5 founder lines were found to develop aggressive lymphomas at a statistically significant higher incidence compared to wild type (WT) FVB/N mice (range 10.7-34.6% lymphomagenesis). Gross anatomical characterization of Lymphoma bearing mice demonstrated focal lymphoid tumors, lymphadenopathy, organomegaly (liver, spleen, kidney), and malignant atypical lymphocytosis. Flow cytometric and IHCS studies showed features consistent with immature pre B and T lymphoblastic lymphomas (LL). Pre B LLs were characterized by high surface IgM, TdT and CD19 expression as analyzed by flow cytometry. Pre T LL demonstrated cytoplasmic CD3, TdT, and CD43 expression. We successfully developed a T LL cell line (Tg813) from a pre T-LL tumor isolated from a thymic tumor. Tg813 was clonal (Vb-17), demonstrated complex cytogenetic features, and over-expressed PRMT5, CYCLIN D1, CYCLIN D3, C-MYC transcript and protein, and the PRMT5 histone mark, symmetric (Me2)-H4R3. Inhibition of PRMT5 with a small molecule inhibitor, shRNA or genetic deletion using CRISPR/CAS9 PRMT5-specific gRNA (targeting exon 2) led to reduced proliferation, apoptosis and loss of CYCLIN D1 and C-MYC expression in Tg813. Engraftment of the Tg813 LL into both SCID and immunocompetent FVB/N mice led to disseminated lymphomas 21 days post-engraftment. In vivo induced expression of PRMT5 gRNA in CAS9+ Tg813 tumors is currently underway.

Conclusions:The spontaneous lymphomagenesis observed in the Eµ-hPRMT5 transgenic mouse model supports the hypothesis that PRMT5 over-expression can provide sufficient driver activity for this disease. We describe a novel in vivo and in vitro model of PRMT5-driven LL that provides a useful platform for studying the biologic role of this epigenetic modifier in cancer and for development of PRMT5 targeted therapeutic approaches for lymphoma.