The splicing inhibitor Spliceostatin A downmodulates Mcl-1 expression and induces a potent apoptotic response in Chronic Lymphocytic Leukaemia cells

Marta Larrayoz1,Stuart J Blakemore1,Rachel Dobson1,Matthew JJ Rose-Zerilli1,Renata Walewska2,Matthew D Blunt1,Minoru Yoshida3,David Oscier2,Mark S Cragg1,Andrew J Steele1,Jonathan C Strefford1

1Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK,2Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK,3Cehmical Genomics Group, RIKEN Center for Sustainable Resource Science, Wako, Japan

Presenting date: Wednesday 4 November
Presenting time: 10.30-10.45

Background

The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukaemia (CLL), with high protein expression correlated with progressive disease. Nonetheless, Mcl-1 antagonists have shown limited effects. The SF3B1 inhibitor spliceostatin A (SSA), is known to regulate Mcl-1, so we assessed the ability of SSA to elicit apoptosis in CLL.

Method

Primary CLL cells, normal lymphocytes and Ramos cells were exposed to increasing concentrations of SSA and apoptosis and downstream signalling evaluated by flow cytometry and immunoblotting, respectively. Next, we explored whether Bcl-xL expression was a resistance mechanism for SSA by inducing the protein with IL-4/CD40L and whether the combination of SSA with the Bcl-2/Bcl-xL inhibitor ABT-263 presented any additive/synergistic effects within the CLL cells.

Results

SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose-, time- and caspase-dependent manner, but independently of SF3B1 mutational status and progressive disease markers. However, normal B and T cells were significantly more resistant to SSA treatment than CLL samples (P=0.002). SSA-induced apoptosis was proceeded via an increase in MCL-1 gene splicing from the MCL-1L isoform into the MCL-1S isoform which resulted in a subsequent decrease in Mcl-1L protein expression (P?0.001). The role of Mcl-1 in regulating SSA-induced apoptosis was also observed in Ramos cells where over-expression of non-spliceable Mcl-1L, significantly protected them from SSA-induced apoptosis. SSA-induced apoptosis of CLL cells treated with CD40L and IL-4, factors known to induce Mcl-1 and prosurvival Bcl-xL, was significantly reduced. Therefore, to explore possible strategies for overcoming this resistance, we investigated combining SSA with ABT-263 and showed that dual exposure to both inhibitors significantly increased apoptosis, overcoming the protective effects of IL-4/CD40L.

Conclusion

SSA has a potent apoptotic effect on CLL cells through Mcl-1 inhibition. SSA alone or in combination with Bcl-2/Bcl-xL antagonists may have therapeutic utility for this incurable leukaemia.