Dual Conditional Loss of BLIMP-1 and p53 in B-Cells Drives B-Cell Lymphomagenesis

Background. p53 is a well defined tumor suppressor involved in the modulation of cell proliferation, cell cycle progression and programmed cell death. BLIMP-1 plays a crucial role in modulating B-cell differentiation towards Ig-secreting plasma cells, and it acts as a tumor suppressor, as documented in both diffuse large B-cell lymphoma and Burkitt lymphoma. Whether B-cell specific loss of both p53 and BLIMP-1 may favor a B-cell lymphoma phenotype remains unanswered. We therefore aimed to generate in vivo dual p53/BLIMP-1-floxed conditional inactivation in B-cells, and to define the functional relevance of both p53 and BLIMP-1 n B-cell lymphomagenesis in vivo

Methods.Cre recombinase under the control of CD19 promoter (C57BL/6 CD19^Cre/Cre) mice were crossed with either C57BL/6 BLIMP^flox/flox or C57BL/6 p53^flox/flox mice to achieve deletion of BLIMP or p53, respectively, in B cells. Secondly, CD19^Cre/Cre BLIMP^flox/flox mice were crossed with CD19^Cre/Cre p53^flox/flox to achieve concomitant deletion of both BLIMP and p53 in B cells (CD19^Cre/Cre BLIMP^flox/flox p53^flox/flox), referred as CD19/Bl^-/p53^- mice. Transgenic experimental mice (CD19/Bl^-/p53^-) where characterized for B cell infiltration using immunohistochemistry, flow cytometry; clonotypic immunoglobulin heavy-chain rearrangement was assessed by Southern Blotting. Whole exome sequencing was performed using DNA isolated from B220⁺ selected cells obtained from pathological lymph nodes of CD19/Bl^-/p53^- mice and from matched tail-derived tissues, used as germline (Illumina HiSeq 2500 platform; Agilent SureSelectXT). MTT assay was used to BTK-inhibitor-dependent cytotoxicity using CD19/Bl^-/p53^-derived B220 cells.

Results.We generated dual p53/BLIMP-1-floxed conditional inactivation in B-cells, using mice expressing Cre recombinase under the control of CD19 promoter. 100% of the CD19/Bl^-/p53^- mice presented with diffuse lymphadenomegalies, and splenomegaly, hepatomegaly (90.3% and 77.4%, respectively). Other clinical manifestations included presence of ascites and hind lymb paralysis (12.9% and 19.3%, respectively). The CD19/Bl^-/p53^- showed worse survival compared to Bl^-/p53^- mice non-expressing the CD19/Cre recombinase, CD19/p53^-, or CD19/Bl^- (363, 469.5, 460.5, and 770 days, respectively). H.E. staining of CD19/Bl^-/p53^--derived lymph nodes, defined a nodal architecture with a monomorphic population of large sized atypical lymphoid cells with finely clumped and dispersed chromatin, and multiple basophilic medium sized, paracentrally situated nucleoli. A "starry sky" pattern was also observed. Overall, these features are compatible with a high-grade lymphomas. IHC analysis confirmed a marked positivity for B220 staining (TdT, Bcl6, CD138 and CD4, CD8 negative). Tumors were confirmed to be B220⁺/IgM⁺, with either Igk- or Ig-lambda-restriction as demonstrated by flow cytometry; and either mono- or bi-clonal, as demonstrated by Southern blotting, thus further confirming the clonal transformation induced by dual BLIMP/p53 deletion in B cells. Whole exome sequencing was performed from B220⁺ selected cells obtained from pathological lymph nodes of CD19/Bl^-/p53^- mice and identified 143 SNVs. Among them, non-synonymous somatic mutations were mapped on genes involved in the regulation of focal adhesion, PDGF signaling, p53-downstream pathway, and lipoprotein metabolism. B220⁺ cells selected from CD19/Bl^-/p53^--derived lymph nodes were implanted subcutaneously into recipient SCID/Bg mice (n: 10), and presented with 100% engraftment, with a monomorphic lymphoid infiltration of B220⁺ and IgM⁺ cells. B220 positive cells were selected from the s.q. tumor and intravenous injected into recipient SCID/Bg (n: 10) and BL/6 mice (n: 10). Engraftment was demonstrated in all the mice, where hepatomegaly, splenomegaly and hind lymb paralysis were observed. Infiltration of B220⁺ cells was documented within bone marrow, liver and spleen. We next investigated the anti-tumor activity of BTK-inhibitor, and found that B220⁺ cells selected from lymph nodes harvested from CD19/Bl^-/p53^-mice were sensitive to ibrutinib treatment.

Conclusion. These studies demonstrate that the specific dual inactivation of p53 and BLIMP in B-cells promotes oncogenic transformation, resulting in aggressive B-cell lymphoma development.