Histone modifications have crucial roles in diverse biological processes including development, differentiation and oncogenesis. Among them, acetylation of histone H3 at the lysine-18 residue (H3K18) is particularly important, because specific deacetylation of H3K18 is indispensable for oncogenic transformation by adenovirus1 and for host responses to bacterial infection.2 Regarding the former, it has also been demonstrated that H3K18 hypoacetylation is linked to the maintenance of malignant phenotypes3 and poor prognosis4, 5 in cancer.
Given the fundamental roles of H3K18 hypoacetylation in cancer biology, we investigated the possibility of therapeutic targeting of this modification in mantle cell lymphoma (MCL), in which epigenetic alterations, including aberrant hypomethylation of the HDAC1 gene,6 are central to the disease process and in which conventional therapies are usually not effective.7 Toward this end, we first screened for the effects of various anti-cancer drugs on the levels of H3K18 acetylation in the MCL cell line HBL-2. As shown in Figure 1a, in addition to the HDAC inhibitor romidepsin, two alkylating agents, bendamustine and 4-hydroperoxy-cyclophosphamide (4-OHCY; an active metabolite of cyclophosphamide), were able to reverse H3K18 hypoacetylation, whereas doxorubicin, dexamethasone and chlorambucil failed to do so at equitoxic concentrations. The increase in the acetylation levels of H3K18 was time dependent and considerably sustained until cell death (∼72 h; Figure 1b). This observation was reproducible in other MCL cell lines, except in Granta519, which is highly resistant to chemotherapeutic agents,7 upon treatment with bendamustine (Figure 1c). Moreover, we confirmed the induction of H3K18-specific acetylation in the bendamustine-sensitive Burkitt lymphoma cell line Namalwa but not in the bendamustine-resistant myeloid leukemia cell line K-562 (Supplementary Figure 1). It is of note that neither bendamustine nor 4-OHCY alone induced hyperacetylation at other sites including H3K9, H3K27, H4K5, H4K12 and H4K16 (Supplementary Figure 1).
Next, we sought to clarify the mechanisms by which bendamustine and 4-OHCY specifically enhanced H3K18 acetylation in target cells. A recent study by Barber et al.3 indicated that SIRT7, a class III HDAC, is responsible for site-specific deacetylation at H3K18 in cancer cells. Consistent with their finding, both bendamustine and 4-OHCY were seen to reduce the expression levels of SIRT7 in a dose- and time-dependent manner in HBL-2 cells (Figure 1d). Another class III HDAC, SIRT1, was also markedly downregulated under the same condition (Figure 1d), whereas there were no changes in the expression of SIRT2 and SIRT6 (data not shown). In addition, the two drugs decreased the abundance of full-length HDAC3 (∼49 kDa) concomitantly with the appearance of the cleaved form (∼42 kDa), whereas they did not affect the expression and activities of HDAC1 and HDAC2 (Figure 1d and data not shown). It is worth noting that the abundance of cleaved HDAC3 correlated precisely with the increase in H3K18 acetylation (Figure 1d).
Bendamustine-mediated H3K18 hyperacetylation might cause upregulation of a unique subset of target genes. Indeed, Barber et al.3 reported that SIRT7-mediated hypoacetylation of H3K18 contributes to cancer development and maintenance through silencing of tumor suppressor genes such as NME1 and COPS2. As anticipated, bendamustine increased the expression of these genes stronger than did romidepsin, which could not inhibit SIRT7 activity, in HBL-2 and SMCH-16 cells in association with the induction of H3K18 hyperacetylation (Figure 1e).
It is well known that core histones are acetylated immediately after translation in the cytoplasm and deacetylation allows their nuclear import by facilitating the binding of specific transporters to positively charged N-termini.8 It is possible that, as a reciprocal process, H3K18 hyperacetylation causes cytoplasmic translocation of nuclear histone H3. Immunoblot analyses of nuclear and cytoplasmic fractions of bendamustine-treated HBL-2 cells clearly demonstrated that this was the case (Figure 1f), which may contribute to growth inhibition because a ready supply of histones is required for the assembly of newly replicated DNA to complete S-phase progression. This is fully consistent with a recent report by Qian et al.,9 in which core histones are acetylated upon DNA damage and transferred to the cytoplasm, where they are degraded by the PA200/Bim 10-containing proteasomes, to facilitate DNA repair. Taken together, these results suggest that the repression of SIRT7 and HDAC3 is mainly responsible for H3K18 hyperacetylation induced by the two alkylating agents and that downregulation of SIRT1, a nuclear and cytoplasmic deacetylase for H3K9 and H4K16 as well as multiple non-histone cytosolic proteins including p53, may be implicated in further modification of translocated histone H3 in the cytoplasm.
As bendamustine and 4-OHCY appear to suppress the expression of SIRT1, SIRT7 and HDAC3, the combination with authentic HDAC inhibitors, which primarily target the catalytic activity of class I HDACs especially HDAC1 and HDAC2,10 may produce synergistic effects in terms of histone acetylation and overall cytotoxicity. To substantiate this hypothesis, we first checked the acetylation status of histone tails in MCL cell lines treated with romidepsin and bendamustine simultaneously. As anticipated, romidepsin enhanced bendamustine-induced H3K18 acetylation in HBL-2 and SMCH-16 but not in Granta519 cells (Figure 2a). In addition, the two drugs synergistically induced hyperacetylation of other sites such as histones H3K9, H4K5, H4K12 and H4K16 (Figure 2a and data not shown). Drug combination analysis with isobolograms revealed that bendamustine and 4-OHCY were additive to synergistic cytotoxicity in combination with romidepsin against HBL-2 cells, whereas the combination with vincristine was rather antagonistic in vitro (Figure 2b). Next, we confirmed the synergistic effects of alkylating agents and HDAC inhibitors in vivo using a mouse xenograft model of MCL. As shown in Figure 2c, the combination of cyclophosphamide and romidepsin significantly retarded the growth of HBL-2 cells inoculated subcutaneously into immunodeficient mice at concentrations that did not affect tumor growth as single agents. Because romidepsin almost equally inhibits the enzymatic activity of HDAC1, 2 and 3,10 we attempted to clarify the direct target(s) responsible for the synergism with alkylating agents using knockdown approaches. shRNA against HDAC3 but not HDAC1 and 2 showed favorable effects in combination with bendamustine and 4-OHCY in HBL-2 and SMCH-16 cells (Figure 2d and data not shown), consistent with our finding that alkylating agents downregulated HDAC3 without affecting the expression and activities of HDAC1 and 2.
Among class I HDACs, HDAC3 has distinct properties despite evolutionarily conserved structures with other members.10 For instance, it has been reported that HDAC3 has critical roles in tumor cell viability, chromosomal stability, S-phase progression and the DNA damage response.11 Another unique feature of HDAC3 is conditional cleavage by extrinsic stimuli. Xia et al.12 demonstrated that HDAC3 cleaved by osmotic stress is more active than full-length HDAC3 and decreases histone acetylation at promoter regions of the c-Jun gene. On the other hand, cleaved HDAC3 is exported to the cytoplasm during the apoptotic process because of the loss of nuclear localization signals, resulting in increased histone acetylation at promoter regions of apoptotic genes such as Fas and Bax, whereas global histone acetylation decreases probably because of concurrent inactivation of histone acetyltransferases.13 In the present study, alkylator-cleaved HDAC3 remained in the nucleus (data not shown) but could not retain the ability of H3K18 deacetylation, implying that HDAC3 is inactivated by alkylating agents upon cleavage. These data suggest that alkylating agents cleave HDAC3 via distinct mechanisms from osmotic stress12 and apoptotic insults such as Fas ligand and ultraviolet exposure.13 The underlying mechanisms are currently under investigation in our laboratory.
In summary, this study uncovered a novel unexpected function of alkylating agents targeting cancer-specific histone modification (H3K18 hypoacetylation), which provides a rationale for the combination with HDAC inhibitors to potentiate each anti-tumor activity against intractable malignancies by epigenetic means, as exemplified here in MCL.
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We thank Professor Martin JS Dyer (MRC Toxicology Unit, Leicester University, Leicester, UK) for providing NCEB-1 and Granta519 cell lines. This work was supported in part by the High-Tech Research Center Project for Private Universities: Matching Fund Subsidy from MEXT, a grant-in-aid for Scientific Research from JSPS (to YF and JK), and research grants from The Naito Foundation, The Yasuda Medical Foundation, The Uehara Memorial Foundation (to YF), Japan Leukemia Research Fund and Takeda Science Foundation (to JK). NH is a winner of the Young Scientist Award of Jichi Medical University.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on Blood Cancer Journal website
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Hiraoka, N., Kikuchi, J., Koyama, D. et al. Alkylating agents induce histone H3K18 hyperacetylation and potentiate HDAC inhibitor-mediated global histone acetylation and cytotoxicity in mantle cell lymphoma. Blood Cancer Journal 3, e169 (2013) doi:10.1038/bcj.2013.66
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