Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

J-Y Yoon,W Xiao,S B Gibson,D Szwajcer,G Ishdorj,P Benjaminson,R Kumar,J B Johnston

doi:10.1038/bcj.2013.50

Abstract

Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins.

Highlights

Fludarabine-containing regimens are typically used in the frontline treatment of chronic lymphocytic leukaemia (CLL), but relapse is common and response rate with re-treatment is generally inferior to response as first-line treatment.[1,2,3,4] There is a need to develop new and non-cross-resistant drug combinations for relapsing patients
We describe the role of cathepsin B in mediating valproic acid (VPA)- and fludarabineinduced apoptosis in primary CLL cells
Using sublethal dose of VPA (1 mM), the addition of VPA increased fludarabine cytotoxicity in all three cell lines (Figure 1a). This increased apoptosis was not observed between fludarabine and valpromide, the amide analogue of VPA that lacks the histone deacetylases (HDACs) inhibition activity,[36] suggesting that HDAC inhibition is important for synergy (Figure 1a)

Summary

Introduction

Fludarabine-containing regimens are typically used in the frontline treatment of chronic lymphocytic leukaemia (CLL), but relapse is common and response rate with re-treatment is generally inferior to response as first-line treatment.[1,2,3,4] There is a need to develop new and non-cross-resistant drug combinations for relapsing patients. In vitro results with HDAC inhibitors (HDIs) using depsipeptide, LBH589 and MS-275 were promising, implicating a number of different mechanisms associated with the inhibition of HDACs in CLL cells.[5,6,7,8] in the clinic, HDI monotherapy trials in CLL have been disappointing. Both depsipeptide and MGCD0103 were tolerable, haematological grade 4 toxicity was observed with both drugs.[9,10]

Methods

Results

Conclusion