FcmR Shapes BCR Signaling in IgM-Positive Leukemia
Authors: da Palma Guerreiro Alexandra , Dorweiler Cornelia , Halmer Ismini , Merkel Olaf , Hartmann Elena Maria , Berg Valeska , Reinart Nina , Fingerle-Rowson Günter , Knittel Gero , Pallasch Christian , Rosenwald Andreas , Reinhardt Christian , Wendtner Clemens-Martin , Hallek Michael , Frenzel Lukas P. 
Published: October 2019

Abstract
Background: The Fc receptor for IgM (FcmR/ TOSO) is significantly overexpressed on chronic lymphocytic leukemia (CLL) cells from peripheral blood, but becomes down-regulated in the tumor microenvironment by e.g. CD40:CD40L interaction. Since the functional role of FcmR on lymphomagenesis is still not understood, we developed a conditional knockout mouse with B cell-specific FcmR-depletion. These mice were crossbred with the Eµ-TCL1 murine model, which develops a CLL-like phenotype.
Results: The depletion of FcmR/TOSO in TCL1 mice (Eµ-Tcl1tg/wt FcmRfl/fl CD19cre/wt; further on called TCT) revealed a significantly shorter overall survival (296 days; n=40) compared to the TOSO expressing control mice (Eµ-Tcl1tg/wt FcmRwt/wt CD19cre/wt; TC; 344 days; n=106; Log-rank p<0.0001). In addition, these mice show a significantly higher blood leukocyte count and lower platelet and erythrocyte count. Leukocytes could be identified as CLL-characteristic leukemic CD19+/CD5+ B cells. Altogether TCT exhibited a faster progress of disease. Spleen immunohistochemistry revealed the transformation of most TCT (14/17 transformed) into an even more aggressive phenotype with increased splenomegaly and change in tissue and cell morphology compared to TC (9/9 not transformed). While characterizing these cells by flow cytometry, we identified a significantly higher expression of IgM on malignant B cells from TCT in comparison to TC mice. This finding indicates that the BCR itself might have a different contribution to lymphomagenesis in FcmR knock-out settings.
Therefore, to validate the functional role of FcmR in the process of lymphomagenesis, we performed transcriptome profiling by RNA-Seq using splenic leukemic cells (CD19+ CD5+) from 36-week old TC (n=4) and TCT (n=4) mice. 2089 genes were found to be significantly modulated in the malignant cells of TCT mice, from which 1221 were downregulated and 868 showed an upregulation (significant change in mean expression; p<0.05). To investigate the role of IgM on TCT mice, purified malignant B cells were incubated for two hours with F(ab')2 goat anti-mouse IgM. Strikingly, TCT mice showed 3941 genes (2054 downregulated, 1887 upregulated) with significant difference in expression compared to TC (p<0.05). The gene expression profiles of the anti-IgM treated mice revealed a stronger regulation of BCR signalling in TCT mice, suggesting that FcmR represents an important factor in these processes. We examined the gene expression profiles, using Ingenuity Pathway Analysis Software. Analysis revealed that the most deregulated functions include interferon-signalling, recruitment of leukocytes, infection of cells and cellular movement.
Conclusion: Here we present functional evidence that loss of FcmR results in increased IgM/BCR on the surface of non-switched leukemia. Moreover, malignant cells with loss of FcmR are more susceptible to BCR stimulation and show a signature of signalling pathways, which contribute to inflammation in B cell malignancies.

Disclosures
Fingerle-Rowson: MorphoSys: Employment. Pallasch:Gilead: Research Funding. Wendtner:Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding.