SFRP4-Mediated Inhibition of Canonical Wnt-Signaling in DLBCL

Introduction: Wnt-signaling enhances proliferation in a variety of malignancies including diffuse large B-cell lymphoma (DLBCL) and exosomes, small secreted microvesicles, are known to carry active wnt-molecules to mediate wnt-signaling. We found consistent suppression of SFRP4, an inhibitor of canonical wnt-signaling in DLBCL, elucidated it's mechanism of action and addressed it´s impact on regulating proliferation of diffuse large B-cell lymphomas. We conducted a screen of chemical wnt-inhibitors and assessed wnt-addiction in DLBCL cell lines.

Material & Methods: The Cancer Cell Line Encyclopedia (CCLE) was used to assess the transcript abundace of SFRP4 in 18 DLBCL cell lines. SFRP4-expression was confirmed using western blot and immunofluorescence in representative DLBCL cell lines. Purified exosomes of DLBCL cell lines, recombinant human SFRP4 and SFRP4-shRNA were used to address the biological mechanism of SFRP4 action. 5-Azacytidine was used to restore SFRP4-expression. Furthermore shRNA mediated knockdown of beta-catenin and pharmacological inhibition of canonical wnt-signaling using XAV-939, IWP-2, C59 and UM206 were used to address wnt-addiction in DLBCL cell lines.

Results: We found SFRP4 to be significantly suppressed in a variety of malignancies, including DLBCL, with promotor methylation as a known mechanism of suppression. SFRP4 was able to suppress exosomal mediated canonical wnt-signaling. SFRP4 inhibited proliferation in DLBCL cell lines and enhanced cytotoxicity of doxorubicin. Treatment with 5-Azacytidine restored expression of SFRP4, induced decline of beta-catenin and suppressed cell proliferation. Knock down of beta-catenin and pharmacological perturbation of canonical wnt-signaling identified wnt-addiction in beta-catenin expressing cell lines in the absence of a MYC translocation or amplification.

Conclusions: SFRP4 suppresses proliferation of DLBCL cell lines by antagonizing exosomal mediated canonical wnt-signaling. Pharmacological inhibition of canonical wnt-signaling significantly decreased proliferation of DLBCL cell lines in the absence of genomic alterations involving MYC.