Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2374 Cancer Tumor and Stem Cell Biology Research Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis Daria G. Valerio1,2, Haiming Xu1,2,3, Chun-Wei Chen1,2,3, Takayuki Hoshii1,2,3, Meghan E. Eisold1,2, Christopher Delaney1,2,3, Monica Cusan1,2, Aniruddha J. Deshpande1,4, Chun-Hao Huang2, Amaia Lujambio5, YuJun George Zheng6, Johannes Zuber7, Tej K. Pandita8, Scott W. Lowe2, and Scott A. Armstrong1,2,3 Abstract Chromatin-based mechanisms offer therapeutic targets in tumor cell genome. Rescue experiments with catalytically inac- acute myeloid leukemia (AML) that are of great current interest. tive mutants of MOF showed that its enzymatic activity was In this study, we conducted an RNAi-based screen to identify required to maintain cancer pathogenicity. In support of the druggable chromatin regulator–based targets in leukemias role of MOF in sustaining H4K16 acetylation, a small-molecule marked by oncogenic rearrangements of the MLL gene. In this inhibitor of the HAT component MYST blocked the growth of manner, we discovered the H4K16 histone acetyltransferase both murine and human MLL-AF9 leukemia cell lines. Further- (HAT) MOF to be important for leukemia cell growth. Condi- more, Mof inactivation suppressed leukemia development in tional deletion of Mof in a mouse model of MLL-AF9–driven an NUP98-HOXA9–driven AML model. Taken together, our leukemogenesis reduced tumor burden and prolonged host results establish that the HAT activity of MOF is required survival. RNA sequencing showed an expected downregulation to sustain MLL-AF9 leukemia and may be important for multiple of genes within DNA damage repair pathways that are con- AML subtypes. Blocking this activity is sufficient to stimulate DNA trolled by MOF, as correlated with a significant increase in damage, offering a rationale to pursue MOF inhibitors as a yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In targeted approach to treat MLL-rearranged leukemias. Cancer Res; parallel, Mof loss also impaired global H4K16 acetylation in the 77(7); 1–10. Ó2017 AACR. Introduction present in infant and pediatric patients with the highest frequency (80%) in infant acute lymphoblastic leukemia (ALL; ref. 4). Chromosomal rearrangements at 11q23 are associated with the It is evident that chromatin modifications, including DNA development of acute leukemia and lead to the discovery of lysine methylation and histone modifications, enforce oncogenic gene (K)-specific methyltransferase 2A (KMT2A also known as MLL1; expression programs and substantially contribute to the initiation refs. 1, 2). MLL translocations are found in about 10% of all and maintenance of leukemia cells (5, 6). Epigenomic studies patients with acute leukemia and generally associated with an utilizing in vivo and in vitro models of MLL-rearranged leukemia unfavorable prognosis (3). MLL translocations are more frequently have revealed that direct targets of MLL fusion proteins such as HOXA cluster genes are associated with aberrantly high levels of 1Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New histone 3 lysine 79 dimethylation (H3K79me2; refs. 7, 8). DOT1L York, New York. 2Cancer Biology & Genetics Program, Memorial Sloan Kettering was found to be the key regulator of H3K79me2, and MLL- 3 Cancer Center, New York, New York. Department of Pediatric Oncology, Dana- rearranged cells were shown to be highly dependent on Dot1l for Farber Cancer Institute, and Division of Hematology/Oncology, Boston Chil- leukemia initiation and maintenance. (9–12) This discovery led dren's Hospital, Harvard Medical School, Boston, Massachusetts. 4Tumor Initi- ation and Maintenance Program, Sanford Burnham Prebys Medical Discovery to the development of small-molecule inhibitors targeting Institute, La Jolla, California. 5Department of Oncological Sciences, Mount Sinai DOT1L, one of which is currently undergoing early-phase trials School of Medicine, New York, New York. 6Department of Pharmaceutical and (13, 14). Similarly, MLL-AF9 leukemia maintenance was shown Biomedical Sciences, The University of Georgia, Athens, Georgia. 7Research to be dependent upon expression of other chromatin regulators Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria. 8 such as lysine (K) demethylase 1A (Kdm1a; ref. 15) and bromo- Department of Radiation Oncology, Houston Methodist Research Institute, domain containing 4 (Brd4; ref. 16). KDM1A is an H3K4 and Houston, Texas. H3K9 demethylase and BRD4 is a well-known member of the Note: Supplementary data for this article are available at Cancer Research bromodomain family. The bromodomain family consists of Online (http://cancerres.aacrjournals.org/). epigenetic "readers," important for recognizing posttranslational D.G. Valerio and H. Xu contributed equally to this study. chromatin modifications and recruiting downstream effector Corresponding Author: Scott A. Armstrong, Department of Pediatric Oncology, proteins to specific loci to activate gene expression programs Dana-Farber Cancer Institute, and Division of Hematology/Oncology, Boston (17). Multiple bromodomain inhibitors are currently under Children's Hospital, Harvard Medical School, Boston, MA 02215. Phone: investigation in early-phase clinical trials (17). 617-632-3644; Fax: 617-632-4367; E-mail: [email protected] Findings such as these indicate the importance of chromatin doi: 10.1158/0008-5472.CAN-16-2374 regulation in leukemia. To identify novel druggable epigenetic Ó2017 American Association for Cancer Research. targets in MLL-rearranged leukemia, we conducted a chromatin www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2374 Valerio et al. regulator–focused RNAi screen in murine MLL-AF9 leukemia cells using the Illumina Next-Gen Sequencing HiSeq platform (Illu- and found hairpins targeting lysine (K) acetyltransferase 8 (Kat8, mina), as previously described (15, 34). shRNA screen output data also known as Mof) and the previously identified target Brd4 (16), can be found in Supplementary Table S1. to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST Mice family of lysine acetyltransferases. The MYST family is named for The generation of Mof conditional knockout mouse in a its founding members MOZ, YBF2, SAS2, and TIP60, proteins that C57Bl/6 background has been described (33). To generate all contain a MYST region with a canonical acetyl coenzyme A Mx1-Cre; Moff/f mice, Moff/f mice were crossed to Mx1-Cre mice (CoA) binding site and C2HC-type zinc finger motif (18). MOF is (B6.Cg-Tg(Mx1-Cre)1Cgn/J strain from Jackson Laboratory, and one of the best-characterized MYST family proteins and was Cre was maintained as a heterozygous allele. Genotyping strategies shown to be crucial for murine embryogenesis. MOF functions were previously described (33). All transplant experiments were as a cell-type–dependent regulator of chromatin state and con- performed with 6- to 8-week-old female wild-type C57Bl/6 mice, trols various cellular processes such as T-cell differentiation (19), purchased from Taconic. All animal experiments in this study were DNA damage response (20–24), cell-cycle progression (20, 25), approved by and adhered to guidelines of the Memorial Sloan- and embryonic stem cell self-renewal and pluripotency (26). Kettering Cancer Center Animal Care and Use Committee. The role of MOF in tumorigenesis seems complex. Studies in breast carcinoma (27), medulloblastoma (27), and ovarian can- RNA extraction and RNA sequencing cer (28) suggest that tumor progression is associated with down- TRIzol (Invitrogen) was used to extract RNA from viable regulation of MOF and H4K16 acetylation (H4K16ac). On the cells. RNA was QCed on the Agilent Bioanalyzer 2100 platform other hand, studies in lung (29, 30) and oral (31) carcinoma have (Agilent) and Poly-A tail selection was performed. Sequencing associated high expression of MOF with carcinogenesis and (RNAseq) was done using the Illumina Next-Gen Sequencing suppression of MOF with cancer cell death. This suggests that HiSeq platform (Illumina) with 30 to 45 million 50-bp, paired- MOF may regulate tumorigenesis in a cell- and tissue-dependent end reads. manner. While the enzymatic activity of MOF is druggable by small Data analysis and statistical methods molecules (32) and our RNAi screen suggests a crucial role for GraphPad Prism software was used for statistical analysis. MOF in MLL-AF9 leukemogenesis, we studied the role of Mof in Statistical significance between 2 groups was determined by detail using a conditional murine Mof knockout system (33). Our unpaired 2-tailed Student t test. The Kaplan–Meier method was fi current ndings indicate a strong dependency of MLL-AF9 leuke- applied to plot survival curves for murine leukemic transplant fl mic cells on Mof. Gene expression and immuno uorescent data data and the log-rank test to determine statistical significance. suggest that the importance of MOF in MLL-AF9 leukemogenesis RNAseq raw reads were aligned to NCBI37/mm9 and normal- – may be through DNA damage repair. Mof knockout in MLL-AF9 ized using STAR. Differential expression data were obtained using transformed cells led to loss of global H4K16ac and in line with the DEseq algorithm. These analyses were