Conditional Knockout of Sfpi1 in Post GC B and Plasma Cells Induces B Cell Lymphoma and Plasma Cell Neoplasm

PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream enhancer region (URE) located in 14 kb 5’ of the murine PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Therefore, down-regulation of PU.1 in myeloid and B cell lineages results in hematological malignancies.

We previously reported that PU.1 is down-regulated in 5 out of 7 myeloma cell lines as well as primary myeloma cells from a subset of myeloma patients; that the promoter and the URE located in 17 kb 5’ of the human PU.1 gene that is homologous to that in 14 kb 5’ of murine PU.1 gene are highly methylated in these cell lines; and that conditionally expressed PU.1 with tet-off system induces cell growth arrest and apoptosis in myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth.

Here, to evaluate tumor suppressor activity of PU.1 in mature B and plasma cells in vivo, we generated Cγ1-Cre PU.1 knockout mice by crossing Cγ1-Cre and PU.1-loxP mice. We confirmed that PU.1 alleles were both conditionally deleted in the maturation stages of B cells from post germinal center B to plasma cells. By 18-24 months of age, about 77.7% (10 of 13) of the knockout mice had developed serum M proteins. To induce B cell differentiation to plasma cells, those mice were immunized with NP-CGG and 76.9% (20 of 26) of the mice developed serum M protein. ELISA of sera from those mice revealed that IgG was not elevated compared to those from the PU.1-loxP mice, which was thought because Cγ1-Cre locus fails to produce IgG1. Instead, a small number (5 of 20) of the mice showed relatively large amounts of IgM and/or IgA. When 11 such mice were sacrificed, 7 had developed splenomegaly and/or intestinal B cell lymphoma. Immunostaining revealed that B220⁺ cells had infiltrated into the tumors and various organs including the spleen, liver, and bone marrow. Those cells were monoclonal for κ chain and partly CD138 positive. When we transplanted those tumor cells into Rag2^-/- Jak3^-/- immunedeficient mice, all the mice died within 3 weeks. Thus, PU.1 apparently functions as a tumor suppressor in mature B cells and its deletion in late B cell maturation stages produces B cell lymphoma with M proteinemia. The remaining 4 mice developed high titer IgM and/or IgA levels and flow cytometry of bone marrow cells and splenocytes revealed that those cells were monoclonal for κ chain and positive for B220 and IgM and/or IgA, suggesting that those mice suffered from multiple myeloma or monoclonal gammopathy with undetermined sighnificance (MGUS). These data strongly suggest that conditional knockout of PU.1 in post germinal center B and plasma cells results in B cell lymphoma and plasma cell neoplasms related to multiple myeloma.