Page 132 - Haematologica May 2022

S. Napoli et al.
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genes belonged to the Wnt signaling pathway, cell growth and differentiation (Figure 5B). RNA-Seq data after GECPAR knockdown showed modulation of three pathways associated with development, differentiation and proliferation and known to cross-talk with the Wnt pathway, such as TGF β, NF-kB and MAPK (Figure 5C).45- 47 Negative regulators of TGF-β pathways including SMAD7, SMURF1 and SMURF2 (Online Supplementary Figure S6A) and negative regulators of MAPK signaling, DUSP1, DUSP8 and DUSP10 (Online Supplementary Figure S6B), were downregulated, after GECPAR silencing. Some of the downregulated genes belonging to the afore- mentioned pathways are also negatively regulated by NF- kB (Online Supplementary Figure S6C). Notably, WNT and MAPK pathways were also affected in SUDHL2 cells overexpressing GECPAR (Figure 5D).
Intersection of CHARTseq and RNA-Seq data for U2932 cells with GECPAR knockdown identified MYC and PRDM1 among seven genes negatively regulated by GEC- PAR, indicating that the eRNA influenced both the prolif- erative capability, reducing MYC, and the terminal differ-
Figure 6. Wnt inhibitor sensitivity in activated B cell-like diffuse large B- cell lymphoma cell lines in depend- ence of GECPAR expression. (A) Anticorrelation between AZ6102 log half-maximal inhibitory concentration (IC50) and GECPAR expression, left, or tankirase protein level, right, in acti- vated B cell-like diffuse large B-cell lymphoma cell lines. (B) Cell cycle analysis in two different GECPAR over- expressing SUDHL2 clones and rela- tive controls, exposed to dimethyl sul- foxide (DMSO) or 5 μM of AZ6102 for 48 hours.
entiation to plasma cells, reducing PRDM1, the genes cod- ing for BLIMP1. Interestingly, 21 direct GECPAR upregulat- ed targets were positively correlated with GECPAR expres- sion also in GCB-DLBCL specimens (Figure 5E). Among them there were KLF6, NOTCH2, components of BMP, cAMP and TNF-a pathways. Strikingly, we also identified TLE4 (Groucho), which forms a corepressor complex with TCF/LEF1 and recruits HDAC to inhibit transactivation of TCF/LEF1 target genes.48
Our identification of GECPAR involvement in Wnt sig- naling prompted us to evaluate the activity of the tankyrase 1/2 (TNKS1/2) inhibitor, AZ6102, that pre- vents nuclear translocation of β-catenin.49 For the four ABC-DLBCL cell lines we tested, GECPAR expression and sensitivity to AZ6102 were significantly anticorrelat- ed (Figure 6A), suggesting that expression of GECPAR sensitized cells to Wnt pathway inhibition. All seven GCB-DLBCL cell lines tested where equally sensitive to Wnt pathway inhibition (Online Supplementary Figure S7). The differential sensitivity to AZ6102 in ABC-DLBCL was not related to tankyrase expression, since protein
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