Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells

Abstract

SummaryChronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)-dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the c-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor jB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL.

Gliotoxin Targets Nuclear NOTCH2 in Human Solid Tumor Derived Cell Lines In Vitro and Inhibits Melanoma Growth in Xenograft Mouse Model

“…Moloney murine leukemia virus (M-MLV) reverse transcriptase and GoTaqPCR kits (Promega) were used for semiquantitative RT-PCR using primer sets as follows: HEY1 , forward 5′-ATACGCCTGCATTTACCAGC-3′ and reverse 5′-TCAATTGACCACTCGCACAC-3′. Primer sets for NOTCH1 , NOTCH2 , and ACTB were published elsewhere (Hubmann et al, 2013). Real-time quantitative RT-PCR (qPCR) for NOTCH2 was performed with TaqMan ® -probes (Hs01050717_m1) purchased from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, United States).…”

“…As a consequence, the nuclear NOTCH2 activity might be resistant to γ-secretase inhibitors (GSI) (Das et al, 2004), a phenomenon that we have observed in the majority of CLL cases (Hubmann et al, 2010, 2013). In light of the observation that GSI are less effective in clinical studies (Andersson and Lendahl, 2014; Lee et al, 2015), we hypothesize that GSI resistance might be a widespread characteristic of NOTCH2 associated human malignancies.…”

“…In light of the observation that GSI are less effective in clinical studies (Andersson and Lendahl, 2014; Lee et al, 2015), we hypothesize that GSI resistance might be a widespread characteristic of NOTCH2 associated human malignancies. Therefore, we tested human cell lines derived from melanoma, HCC, and pancreas-CA for their nuclear NOTCH activity by EMSA and evaluated their sensitivity to the representative GSI DAPT and to the Aspergillum derived canonical NOTCH2/CSL transactivation inhibitor gliotoxin which efficiently induced apoptosis in CLL cells (Hubmann et al, 2013). The secondary metabolite gliotoxin was identified as major virulence factor in Aspergillosis patients with immunosuppressive functions and since the discovery of its structure in 1958 (Bell et al, 1958), it became a target for extensive investigations to explore its complex mechanism of action and its multiple downstream effector molecules and for potential drug development (Gardiner et al, 2005; Dolan et al, 2015; Scharf et al, 2016).…”

The emerging roles of Notch signaling in leukemia and stem cells

“…Emerging evidence suggests that the Notch signaling network is frequently deregulated in human B-CLL with up-regulated expression of Notch1 and Notch2 as well as their ligands Jagged1 and Jagged2 [42]. Moreover, Notch signaling inhibition by the gamma-secretase inhibitors (GSIs) and the specific Notch2 down-regulation using small interfering RNA (siRNA) could promote B-CLL cell apoptosis [38,42]. It has been also reported that Notch2 is not only overexpressed in B-CLL cells but also might be related to the failure of apoptosis-oriented treatment for this disease and deregulation of Notch2 signaling is involved in the aberrant expression of CD23 in B-CLL [39-41].…”

Stromal control of chronic lymphocytic leukemia cells

“…45 γ-Secretase inhibitors I apoptotic activity was associated with a reduction in NF-κB and p53 activities. Another recent study showed that γ-secretase inhibitors I induce apoptosis through at least three mechanisms: proteasome inhibition, endoplasmic reticulum stress increase, and Notch downregulation.…”