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)

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). 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,7upon 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). == Figure 1. == Alkylating Ketorolac agents induce histone H3K18 hyperacetylation through SIRT7 downregulation and HDAC3 cleavage. (a) HBL-2 cells were cultured with the indicated drugs at IC50 (doxorubicin 20 nM, dexamethasone 100 nM, 4-OHCY 1 M, chlorambucil 4 M, bendamustine 50 Mand romidepsin 2 nM) for 24 h and subjected to immunoblotting with specific antibodies against the histone H3 acetylated at lysine-18 (Cell Signaling Technology, Beverly, MA, USA), total histone H3 (Cell Signaling Technology), HDAC1 (Sigma-Aldrich, St Louis, MO, USA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). (b) HBL-2 cells were cultured with either bendamustine (provided by SymBio Pharmaceuticals, Tokyo, Japan) or 4-OHCY (provided by Shionogi, Osaka, Japan) at IC50 (50 and 1.0 M, respectively) for the indicated periods and subjected to immunoblotting with specific antibodies against the histone H3 acetylated at lysine-18 (H3K18), histone H3 and GAPDH (loading controls). (c) We cultured four MCL cell lines in the absence () or presence (+) of 50 Mbendamustine for 12 h and subjected them to immunoblotting with specific antibodies against histone H3 acetylated at lysine-18 (H3K18), histone H3 and GAPDH Ketorolac (loading controls). HBL-2 cells were treated with 2 nMromidepsin (provided by Gloucester Pharmaceuticals, Ketorolac Cambridge, MA, USA) for 12 h to serve as a positive control for H3K18 hyperacetylation.14(d) HBL-2 cells were cultured with either bendamustine or 4-OHCY at the indicated concentrations for 24 h (left panel) or at IC50 (50 and 1.0 M, respectively) for the indicated periods (right panel) and subjected to immunoblotting with specific antibodies against SIRT1, SIRT7, HDAC2, acetylated H3K18, histone H3 (Cell Signaling Technology), HDAC1, HDAC3 (Upstate Biotechnology, Lake Placid, NY, USA) and GAPDH. We also used another anti-HDAC3 antibody (BD Transduction Laboratories, San Diego, CA, USA) to detect both full-length HDAC3 (fHDAC3) and cleaved HDAC3 (asterisk).13(e) HBL-2 and SMCH-16 cells were cultured with either bendamustine or romidepsin at IC50 (50 Mand 2 nM, respectively) for 24 h and subjected to real-time quantitative RT-PCR using the TaqMan Expression Assays (Hs02621161 for NME1 and Hs00182826 for COPS2). The data were quantified with the 2Ctmethod using simultaneously amplified GAPDH (Hs01922876) as a reference. Theyaxis indicates relative gene expression with the expression levels Ketorolac of untreated control cells being set at 1.0. (f) We cultured HBL-2 cells with 50 Mbendamustine for the indicated periods and separated nuclear and cytoplasmic fractions using the Nuclear Extraction Kit (Cayman Chemical Company, Ketorolac Ann Arbor, MI, USA) according to the manufacturer’s instructions. We monitored the quality of separation using histone H1 and GAPDH as nuclear and cytoplasmic markers, respectively. The data shown are representative of multiple independent experiments. Next, we sought to clarify the mechanisms by which bendamustine and 4-OHCY specifically enhanced H3K18 acetylation in target cells. A recent study by Barberet al.3indicated 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 EPLG6 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 1dand data not shown)..