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Histone deacetylase inhibitors are unable to synergize with ABT-737 in killing primary chronic lymphocytic leukaemia cells in vitro

Leukemia volume 26, pages 1433–1435 (2012)Cite this article

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High levels of BCL-2 universally found in chronic lymphocytic leukaemia (CLL), especially those refractory to chemotherapy, can potentially now be targeted therapeutically with ABT-737, a small molecule inhibitor of BCL-2 and BCL-XL. The structural analogue ABT-263 (Navitoclax) is under clinical development and has shown encouraging single-agent activity in vivo.1 Although single-agent efficacy has been demonstrated, responses were not complete. Thus, further therapeutic improvements remain necessary and outcomes for CLL patients may be improved by combination of ABT-737 with other agents, particularly those that activate the intrinsic apoptotic pathway. Many conventional chemotherapeutic agents that may be considered for combination with ABT-737 are genotoxic and indirectly trigger apoptosis by damaging DNA, potentially causing permanent damage that could contribute to the development of chemoresistance and/or inducing secondary malignancies. In contrast, histone deacetylase inhibitors (HDACi) effect reversible epigenetic changes and induce tumour cell apoptosis. We have found that in mouse models of B-cell lymphoma overexpressing BCL-2, HDACi sensitise these cells to sub-lethal doses of ABT-737.2, 3 These studies provided pre-clinical evidence that HDACi may be a useful agent to combine with ABT-737 and may synergise with prolonged administration of ABT-737 at doses that minimise toxicities such as thrombocytopenia. To test this hypothesis, we evaluated the sensitivity of primary CLL samples to co-incubation with increasing concentrations of ABT-737 and the HDACi, vorinostat and panobinostat.

In murine lymphoma cells, we found that the BH3-only protein BMF, a potent activator of the intrinsic apoptotic signalling pathway, was significantly upregulated within 24 h exposure to HDACi and was essential for synergy with ABT-737.3 As there was no synergistic response to ABT-737 combined with either vorinostat or panobinostat, we examined the transcriptional response of BMF to vorinostat, within the 24 h time frame. A reverse transcription–multiplex ligation-dependent probe amplification (RT–MLPA) screen of apoptotic gene expression in CLL samples versus CCRF-CEM, a human acute T-cell leukaemia line shown previously to respond to HDACi,5 was performed. This analysis indicated that, for CLL cells, 8-hr incubation in the presence of 2.5 μM vorinostat resulted in BMF mRNA levels similar to cells incubated for 8 h in the absence of vorinostat (mean fold change 1.1). In contrast, BMF expression was more than tenfold greater in vorinostat-treated CCRF–CEM cells (Figure 1f), similar to our observations in murine B lymphoma cells.3 Quantitative real-time PCR (QRT–PCR) of BMF and H1F0, another gene known to be upregulated by HDAC inhibitors,5 was performed on CLL samples and CCRF–CEM cells following incubation with 2.5 μM vorinostat for 0, 4, 8, 16 and 24 h. As in the RT–MLPA screen, a significant increase in BMF expression in response to HDACi was observed only in CCRF–CEM cells and not in any of the 12 CLL samples examined (Figure 1g). HDACi activity in CLL cells was confirmed by up-regulation of H1F0 (Figure 1g). To exclude the possibility that the lack of response to vorinostat in CLL cells was simply due to having used insufficient, but clinically achievable, doses, we performed QRT–PCR for BMF expression after exposure to 0, 2.5, 5, 10 and 25 μM vorinostat (Figure 1h). However, even when the dose of vorinostat was increased tenfold, no induction of BMF occurred in CLL cells. The induction of BMF in CCRF–CEM in response to 5, 10 and 25 μM vorinostat (fold change: 33.9±9.2, 36.8±9.1, 35.1±9.6, respectively) was not substantially increased above the level of induction observed with 2.5 μM (fold change: 28.0±5.0), indicating that pharmacologically relevant doses of vorinostat were sufficient for maximal upregulation of BMF.

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Acknowledgements

We thank members of the Dobrovic lab for useful discussions of CLL and gratefully acknowledge the National Health and Medical Research Council of Australia, Cancer Australia, the Cure Cancer Australia Foundation, and the Peter MacCallum Cancer Centre Foundation for their generous support of the work described herein. Vorinostat was kindly provided by Merck, ABT-737 was kindly provided by Abbott Laboratories. RWJ received collaborative research grants from Merck and Co. and Novartis for research involving vorinostat and panobinostat, respectively.

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  1. Cancer Therapeutics Program, Gene Regulation Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    R Ralli, K M Banks, A P Wiegmans, R W Johnstone & A E Alsop

  2. Department of Haematology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    D Carney & J F Seymour

  3. University of Melbourne, Parkville, Victoria, Australia

    J F Seymour & R W Johnstone

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  1. R Ralli
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Correspondence to R W Johnstone.

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Ralli, R., Banks, K., Wiegmans, A. et al. Histone deacetylase inhibitors are unable to synergize with ABT-737 in killing primary chronic lymphocytic leukaemia cells in vitro. Leukemia 26, 1433–1435 (2012). https://doi.org/10.1038/leu.2011.370

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