Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.