Inhibition of NOTCH Activity by GLIOTOXIN Induces Apoptosis in CLL Cells and Overcomes the Supportive Effect of Primary Bone Marrow Stromal Cells

Abstract 4609

Active NOTCH signaling is observed in an increasing number of human neoplasias including chronic lymphocytic leukemia (CLL) and represents a potential therapeutic target. We have recently reported that the γ-secretase inhibitor DAPT may not be effective in all cases of CLL (Hubmann et al; BJH 2010). The aim of this study was to evaluate the therapeutic value of a newly identified NOTCH transactivation inhibitor (gliotoxin) and to compare its efficiency with the highly selective γ-secretase inhibitor (GSI) DAPT in CLL cells.

Electrophoretic mobility shift assays (EMSA) showed that gliotoxin completely blocked the formation of DNA-bound NOTCH2 in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signaling by gliotoxin was associated with down regulation of its target gene CD23 and induction of apoptosis. The IC₅₀ of Gliotoxin was variable between patients (n=20) and ranged between 50–100 nM irrespective to their sensitivity to GSI. Short term (4 hours) exposure of CLL cells to gliotoxin revealed that gliotoxin modulates the mRNA expression of several NOTCH-related genes including JAG1, PRKCD (PKC-δ), and NR4A1. We (Shehata et al; BLOOD 2010) and others have reported on the supportive effect of primary bone marrow stromal cells (BMSC) for CLL cells. Therefore, we tested whether gliotoxin may overcome this supportive effect. FACS analysis and MTT assays showed that gliotoxin abolished the supportive effect of BMSC under co-culture conditions. The effect of gliotoxin was dose dependent and selectively induced apoptosis in CLL cells accompanied by down regulation of NOTCH2 and CD23 transcription. Western blotting analysis demonstrated that gliotoxin also decreased the phosphorylation of Akt-pSer473 suggesting a link to PI3-K signaling. NOTCH1 has been recently shown to be affected by “gain of function” mutations in a subset of CLL patients. We observed that co-culture of CLL cells with BMSC was associated with increased NOTCH1 mRNA expression which could be decreased upon exposure to gliotoxin. In addition, gliotoxin inhibited DNA-bound NOTCH1 complexes in the T-ALL cell line SupT1 which is known to express high NOTCH1 activity. This indicates that gliotoxin may target both NOTCH1 and NOTCH2 isoforms.

In conclusion, the data show that gliotoxin effectively targets NOTCH activity in CLL cells in a mechanism which is independent of γ-secretase. Thus, gliotoxin might have a beneficial effect in a wider range of CLL patients and warrants further evaluation.