Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis

Chromosomal rearrangements at 11q23 are associated with the development of acute leukemia and lead to the discovery of lysine (K)-specific methyltransferase 2A (KMT2A also known as MLL1; refs. 1, 2). MLL translocations are found in about 10% of all patients with acute leukemia and generally associated with an unfavorable prognosis (3). MLL translocations are more frequently present in infant and pediatric patients with the highest frequency (80%) in infant acute lymphoblastic leukemia (ALL; ref. 4).

It is evident that chromatin modifications, including DNA methylation and histone modifications, enforce oncogenic gene expression programs and substantially contribute to the initiation and maintenance of leukemia cells (5, 6). Epigenomic studies utilizing in vivo and in vitro models of MLL-rearranged leukemia have revealed that direct targets of MLL fusion proteins such as HOXA cluster genes are associated with aberrantly high levels of histone 3 lysine 79 dimethylation (H3K79me2; refs. 7, 8). DOT1L was found to be the key regulator of H3K79me2, and MLL-rearranged cells were shown to be highly dependent on Dot1l for leukemia initiation and maintenance. (9–12) This discovery led to the development of small-molecule inhibitors targeting DOT1L, one of which is currently undergoing early-phase trials (13, 14). Similarly, MLL-AF9 leukemia maintenance was shown to be dependent upon expression of other chromatin regulators such as lysine (K) demethylase 1A (Kdm1a; ref. 15) and bromodomain containing 4 (Brd4; ref. 16). KDM1A is an H3K4 and H3K9 demethylase and BRD4 is a well-known member of the bromodomain family. The bromodomain family consists of epigenetic “readers,” important for recognizing posttranslational chromatin modifications and recruiting downstream effector proteins to specific loci to activate gene expression programs (17). Multiple bromodomain inhibitors are currently under investigation in early-phase clinical trials (17).

Findings such as these indicate the importance of chromatin regulation in leukemia. To identify novel druggable epigenetic targets in MLL-rearranged leukemia, we conducted a chromatin regulator–focused RNAi screen in murine MLL-AF9 leukemia cells and found hairpins targeting lysine (K) acetyltransferase 8 (Kat8, also known as Mof) and the previously identified target Brd4 (16), to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST family of lysine acetyltransferases. The MYST family is named for its founding members MOZ, YBF2, SAS2, and TIP60, proteins that all contain a MYST region with a canonical acetyl coenzyme A (CoA) binding site and C2HC-type zinc finger motif (18). MOF is one of the best-characterized MYST family proteins and was shown to be crucial for murine embryogenesis. MOF functions as a cell-type–dependent regulator of chromatin state and controls various cellular processes such as T-cell differentiation (19), DNA damage response (20–24), cell-cycle progression (20, 25), and embryonic stem cell self-renewal and pluripotency (26).

The role of MOF in tumorigenesis seems complex. Studies in breast carcinoma (27), medulloblastoma (27), and ovarian cancer (28) suggest that tumor progression is associated with downregulation of MOF and H4K16 acetylation (H4K16ac). On the other hand, studies in lung (29, 30) and oral (31) carcinoma have associated high expression of MOF with carcinogenesis and suppression of MOF with cancer cell death. This suggests that MOF may regulate tumorigenesis in a cell- and tissue-dependent manner.

While the enzymatic activity of MOF is druggable by small molecules (32) and our RNAi screen suggests a crucial role for MOF in MLL-AF9 leukemogenesis, we studied the role of Mof in detail using a conditional murine Mof knockout system (33). Our current findings indicate a strong dependency of MLL-AF9 leukemic cells on Mof. Gene expression and immunofluorescent data suggest that the importance of MOF in MLL-AF9 leukemogenesis may be through DNA damage repair. Mof knockout in MLL-AF9–transformed cells led to loss of global H4K16ac and in line with this finding, rescue experiments with histone acetyltransferase (HAT) domain–mutated MOF illustrated that the HAT activity of MOF is indispensable for MLL-AF9 leukemia maintenance. Finally, experiments with the selective MYST protein HAT inhibitor MG149 showed a strong antiproliferative effect on murine as well as human MLL-AF9 leukemia cell lines. MOF HAT activity may be a good target for novel small-molecule inhibitor development to improve the treatment of patients with MLL-rearranged leukemia.

A customized shRNA library (TRMPV-Neo system) focused on mouse chromatin-regulating genes, together with control shRNAs, was designed as previously described (15, 34). After sequence verification, the virus library containing 2,252 shRNAs targeting 468 genes (4 to 6 shRNAs per gene) was pooled. The virus pool was transduced into monoclonal mouse MLL-AF9 (MA9) leukemic cells (2 replicates), stably expressing rtTA3 (Tet-on system), at a viral titer that on average causes a single viral transduction per cell and at which each shRNA is represented in at least 2,000 cells. The infected cells were selected for 2 days in 1 mg/mL neomycin (G418 sulfate, Corning), and subsequently, shRNA expression was induced by adding 1 μg/mL doxycycline (Sigma-Aldrich). The shRNA-expressing cells (dsRed and Venus double positive) were sorted (T0) using a FACS ARIA (BD) and cultured for 12 days (T12; ref. 35). The integrated shRNA sequences in T0 and T12 cell samples were assessed by high-throughput sequencing (HiSeq) using the Illumina Next-Gen Sequencing HiSeq platform (Illumina), as previously described (15, 34). shRNA screen output data can be found in Supplementary Table S1.

The generation of Mof conditional knockout mouse in a C57Bl/6 background has been described (33). To generate Mx1-Cre; Mof^f/f mice, Mof^f/f mice were crossed to Mx1-Cre mice (B6.Cg-Tg(Mx1-Cre)1Cgn/J strain from Jackson Laboratory, and Cre was maintained as a heterozygous allele. Genotyping strategies were previously described (33). All transplant experiments were performed with 6- to 8-week-old female wild-type C57Bl/6 mice, purchased from Taconic. All animal experiments in this study were approved by and adhered to guidelines of the Memorial Sloan-Kettering Cancer Center Animal Care and Use Committee.

TRIzol (Invitrogen) was used to extract RNA from viable cells. RNA was QCed on the Agilent Bioanalyzer 2100 platform (Agilent) and Poly-A tail selection was performed. Sequencing (RNAseq) was done using the Illumina Next-Gen Sequencing HiSeq platform (Illumina) with 30 to 45 million 50-bp, paired-end reads.

GraphPad Prism software was used for statistical analysis. Statistical significance between 2 groups was determined by unpaired 2-tailed Student t test. The Kaplan–Meier method was applied to plot survival curves for murine leukemic transplant data and the log-rank test to determine statistical significance.

RNAseq raw reads were aligned to NCBI37/mm9 and normalized using STAR. Differential expression data were obtained using the DEseq algorithm. These analyses were done through the platform from Basepair. DEseq output tables are included in Supplementary Table S2 and raw data were deposited in GEO (GSE80671). Gene Ontology (GO) analyses were performed using the PANTHER gene analysis tool.

To identify novel druggable targets within epigenetic pathways required for MLL-AF9 (MA9) leukemia maintenance, we conducted a customized, chromatin regulator–focused RNAi screen in murine, MA9 leukemia cells (Fig. 1A). An shRNA library containing 2,252 shRNAs targeting 468 known chromatin regulators was constructed in TRMPV-Neo and transduced as one pool into Tet-on-competent, monoclonal mouse MA9 leukemic cells. After neomycin (G418) selection, shRNA expression was induced by doxycycline treatment. shRNA-expressing cells were then sorted, and changes in shRNA library representation after 12 days of culture were assessed by high-throughput sequencing of shRNA guide strands as previously described (15, 34). Using the scoring criterion of more than 256-fold depletion in each of 2 independent replicates, 20 shRNAs targeting 18 genes were strongly depleted (Fig. 1B). Only 2 of these 18 had 2 independent shRNAs that showed strong depletion, namely, Mof and the previously identified target Brd4 (Fig. 1B; Supplementary Fig. S1A and S1B; ref. 15). This RNAi screen strongly suggests that the lysine acetyltransferase MOF is crucial for MA9 leukemic cell growth.

To study the role of MOF in MA9 leukemogenesis and validate the results of our RNAi screen, we used a well-described conditional Mof knockout (KO) mouse in a C57Bl/6 background (33). For the initial in vitro experiments, Mof^f/f, Mof^f/+, and wild-type (Mof^+/+) adult mice were euthanized, and Lin⁻, SCA1⁺, cKIT⁺ cells (LSK) were purified from the bone marrow (Fig. 2A). These fresh LSKs were infected with MA9 in an MSCV-Migr1 retroviral vector containing the GFP selection marker. Following a few days of liquid culture, cells were sorted for GFP positivity. These stable MA9-transformed cells grow indefinitely and were capable of forming dense, round colonies (blast colonies, data not shown).

For in vitro excision of Mof, MA9 cells were infected with an MSCV-dTomato retrovirus containing Cre. Forty-eight hours after infection, Cre-positive Mof^f/f, Mof^f/+, and Mof^+/+ cells were sorted, counted, and immediately plated in myeloid cytokine–supplemented methylcellulose for colony-forming unit (CFU) assays. Homozygous Mof loss significantly reduced colony-forming capacity of MA9 cells (Fig. 2B and C). No difference in colony morphology was observed (Fig. 2D). Heterozygous loss of Mof (Mof^f/+) also led to a reduction of total colony number (Fig. 2C), albeit less dramatic compared with full Mof deletion, suggesting a potential gene dosage effect of Mof on the clonogenic capacity and cell growth of MA9 cells. However, Mof excision PCR data consistently illustrated that although at the time of plating, all cells in Mof^f/f and Mof^f/+ were completely excised (Fig. 2E), all Mof^f/f colonies that had formed at day 7 of the CFU assay were in fact unexcised, whereas the floxed allele in the Mof^f/+ colonies remained fully excised (Fig. 2E). These PCR data indicate strong selective pressure against homozygous, but not heterozygous, Mof loss in an MA9 leukemic setting.

To assess specificity of the observed phenotype, a Mof full-length rescue experiment was performed. Mof^f/f, Mof^f/+, and Mof^+/+ MA9 cells were transduced with either an MSCV-miCD2 retrovirus containing full-length, human influenza hemagglutinin (HA)-tagged Mof, or the empty vector (Fig. 2A). hCD2-positive cells were sorted, and subsequent Western blot analysis for the HA-tagged MOF indicated that exogenous MOF was expressed (Supplementary Fig. S2A). These cells were then transduced with Cre, and Cre-positive cells were used for CFU assays as before. Figure 2F shows full rescue of the phenotype observed in Mof^f/f, miCD2 cells by exogenous expression of full-length Mof (Mof^f/f, miCD2-Mof). Mof excision PCR analysis confirms this rescue. While Mof^f/f, miCD2 cells have fully lost excision at day 9 post-Cre infection, Mof^f/f, miCD2-Mof cells are still largely excised (Fig. 2G). Taken together, these findings demonstrate that Mof is required for the colony-forming capacity of MA9 leukemic cells.

Mof dependency in MA9 leukemia was further assessed using an in vivo secondary, murine leukemia model. Mx1-Cre; Mof^f/f and Mx1-Cre mouse bone marrow LSKs were transduced with MA9 in an MSCV-IRES-GFP retroviral vector and 48 hours after infection, GFP⁺ cells were sorted and injected into sublethally irradiated (600 cGy) C57Bl/6 mice. These mice developed acute myeloid leukemia (AML) within 4 to 8 weeks post-transplant (data not shown). Leukemic mice were sacrificed and bone marrow cells obtained. A total of 45,000 GFP⁺ primary leukemic Mx1-Cre; Mof^f/f or Mx1-Cre bone marrow cells were then injected into sublethally irradiated C57Bl/6 mice (Fig. 3A). At day 14 post-transplant, mice were bled to check engraftment (pre-pIpC, Fig. 3B). That same day, half of the mice per group (n = 10 for Mx1-Cre, n = 20 for Mx1-Cre; Mof^f/f) were injected with poly(I:C) (pIpC) to induce Cre expression. pIpC-treated mice received a total of 3 dosages, one every other day and were bled again, 7 days after the last dose. The WBC count (Fig. 3C) and GFP% in peripheral blood (Fig. 3B) illustrate the lethality of Mof loss to MA9 leukemic cells. Seven days after pIpC treatment, Mx1-Cre; Mof^f/f, pIpC+ mice had a mean WBC count of 3 K/μL versus 112 K/μL (Mx1-Cre; Mof^f/f, pIpC−) and 104 K/μL (Mx1-Cre, pIpC+) and a significantly lower GFP% of 38% versus 92% (Mx1-Cre; Mof^f/f, pIpC−) and 95% (Mx1-Cre, pIpC+). Several mice within the Mx1-Cre;Mof^f/f, pIpC+ group had an actual reduction of GFP% in the peripheral blood after receiving pIpC injections (Fig. 3D), indicating a reduction of tumor burden upon Mof loss.

All mice eventually succumbed to AML. At the time of death, mice had elevated WBC counts (data not shown) and splenomegaly (Supplementary Fig. S3A). FACS analysis showed more than 90% GFP⁺ cells in bone marrow (Supplementary Fig. S3B), spleen, and peripheral blood (data not shown). The majority of GFP⁺ cells in bone marrow, spleen, and peripheral blood expressed myeloid cell markers MAC1, GR1, and cKIT, indicating a myeloid leukemic phenotype (Supplementary Fig. S3C and S3D). Animals in all 3 control groups died with a median survival of 22 (Mx1-Cre, pIpC+) or 24 days (Mx1-Cre; Mof^f/f and Mx1-Cre, pIpC−). Mx1-Cre; Mof^f/f, pIpC+ mice lived significantly longer with a median survival of 31 days (P < 0.0001; Fig. 3E). At the time of death, GFP⁺ bone marrow cells of Mx1-Cre; Mof^f/f, pIpC+ mice showed incomplete excision (Supplementary Fig. S3E), illustrating that Mof-deficient MA9 cells have a strong disadvantage of forming leukemia in vivo. When we ended the experiment (day 60), the last living mouse in the Mx1-Cre; Mof^f/f, pIpC+ cohort did not have any GFP⁺ cells in the peripheral blood or bone marrow. Together, these in vivo data demonstrate that Mof is important for maintenance of MA9-driven AML.

Our CFU assay data show a strong dependence for Mof in MA9 cells. To study the potential underlying mechanism, we performed in vitro excision of Mof in MA9-transformed murine LSKs as described above. While the strong dependency caused a rapid loss of Mof excision (Fig. 2E and G), we first had to define the latest time point after Cre infection at which MA9 cells are still fully excised. PCR analysis indicated complete Mof excision up to 72 hours after Cre infection (Fig. 4A). We next performed RNA sequencing (RNAseq) on Mof^f/f and Mof^+/+ MA9 cells, harvested 72 hours after Cre infection (Supplementary Table S2). GO analysis comparing MA9 cells with homozygous Mof loss with the wild-type control showed a significant enrichment of cell division and DNA damage repair pathways in genes that are downregulated in MA9 Mof KO cells. To verify this finding, we stained MA9-transformed LSKs at 48 or 72 hours after Cre infection with an immunofluorescent-labeled yH2AX antibody (Supplementary Fig. S4A and S4B). Confocal microscopy revealed significantly more yH2AX foci per cell nucleus in Mof-deficient MA9 cells compared with controls (P < 0.01; Fig. 4C), indicative of more DNA damage. This increase in DNA damage was largely rescued by overexpression of full-length Mof (Fig. 4C). These experiments demonstrate that there is evidence of cell death and DNA damage in MA9 cells upon Mof loss.

MOF has been identified as the major H4K16 acetyltransferase in humans, mice, and Drosophila (25, 33, 36–38). MOF contains a HAT domain with a CoA-binding site that was found to be crucial for its acetyltransferase activity (18, 36). While MOF possesses acetyltransferase activity on various histones and nucleosomes, depletion of MOF in HeLa cells was shown to lead to a dramatic decrease in H4K16ac, whereas other acetylation sites appeared to be unaffected (25).

As our data indicate a strong Mof dependence in MA9 leukemic cells, we set out to establish whether this dependence is through MOF HAT activity. When assessing changes in global H4K16ac upon Mof loss in MA9 cells, we found a significant decrease of H4K16ac (Fig. 5A), a decrease that was averted by expression of exogenous Mof (Mof^f/f, miCD2-Mof). Next, we designed 2 HA-tagged, HAT-inactivated Mof retroviral constructs (Fig. 5B) in which either the CoA-binding site was deleted (Mof-CoAdel) or a HAT-inactivating point mutation (G327E) was introduced (Mof-G327E). The HAT-inactivating point mutation in the MOF CoA-binding domain was first described in Drosophila (36) and later used in a human MOF construct where it was also found to diminish H4K16ac (39). Alignment of Drosophila, murine, and human MOF illustrates that the point mutation involves replacement of glycine by glutamic acid on position 327 (G327E) in murine as well as human MOF. The constructs were packaged in an miCD2-MSCV retroviral vector with the hCD2 selection marker (miCD2-Mof-CoAdel and miCD2-Mof-G327E).

We infected unexcised Mof^f/f and Mof^+/+ MA9 cells with miCD2-Mof, miCD2-CoAdel, miCD2-G327E, or miCD2 and sorted hCD2-positive cells. Western blot analysis for HA-tagged MOF indicated that exogenous MOF was expressed at similar levels in miCD2-Mof and miCD2-G327E, although MOF expression seemed a little lower with the miCD2-CoAdel construct (Fig. 5C). Upon in vitro Mof KO by transduction with retroviral Cre, Western blot analysis confirmed that exogenous full-length MOF was capable of restoring H4K16ac levels upon genetic Mof loss, whereas both HAT domain mutant Mof constructs were not (Fig. 5D). When using these Cre-transduced MA9 cells for a CFU assay, full-length MOF indeed rescued colony formation of Mof^f/f cells, whereas exogenous, HAT-deficient MOF could not (Fig. 5E). Mof excision PCR analysis reaffirms these findings where Mof^f/f miCD2, miCD2-CoAdel, and miCD2-G327E cells have fully lost excision at day 9 post-Cre infection, but miCD2-Mof cells are still largely excised (Fig. 5F). In summary, we found a loss of global H4K16ac upon Mof KO, and in line with this finding of H4K16ac loss, rescue experiments with HAT domain–mutated MOF illustrated that the HAT activity of MOF is indispensable for MA9 colony-forming capacity.

Our finding that MOF HAT activity is required for MA9 leukemia suggests that targeting MOF HAT activity could provide us with a new approach in treating MA9 leukemia patients. In 2011, a selective small-molecule inhibitor of MYST protein HAT activity, MG149, was designed (32). While this small molecule is not particularly potent, we decided to use this inhibitor to test its effect on cell viability in the human MA9 AML cell lines NOMO1 and MOLM13 and in murine polyclonal MA9 primary leukemia cells. A dose–response curve showed a strong antiproliferative effect on all 3 MA9 cells with an IC₅₀ of 19.6 μmol/L (murine MA9), 25.4 μmol/L (MOLM13), and 50.9 μmol/L (NOMO1). We further repeated the MG149 in vitro assay on the human non–MLL-rearranged leukemia cell lines Kasumi-1 (AML1-ETO fusion), U937 (CALM-AF10 fusion), and K562 (BCR-ABL fusion). All 3 cell lines showed an apparent sensitivity to the small-molecule MYST protein HAT inhibitor (Supplementary Fig. S5G). In addition, MG149 inhibition induced global H4K16ac loss in murine MA9 cells (Fig. 5H). Together, these results indicate that MG149 effectively targets MOF HAT activity with an antiproliferative effect on human as well as murine leukemia cells, suggesting that MOF HAT activity may be a viable target in not only MLL-AF9–driven but also various acute leukemias.

Given that human leukemia cell lines with various translocations are sensitive to MOF HAT inhibitor MG149, we then tested whether MOF is required for non–MLL-rearranged leukemogenesis. To assess this question, we performed Mof knockout experiments in a NUP98-HOXA9 leukemia model. Similar to what we found for MA9 cells, loss of Mof in NUP98-HOXA9–transformed cells led to impaired colony formation (Supplementary Fig. S5A–S5E). An in vivo secondary NUP98-HOXA9/Meis1a leukemia experiment, in set-up similar to the MA9 in vivo experiment (Fig. 3), illustrated that significantly prolonged survival in the Mof-deficient group (Mx1-Cre; Mof^f/f, pIpC+, median survival, 117 days; P < 0.001) compared with all 5 control groups that died with a median survival of 28 to 39 days (Supplementary Fig. S5F). These results indicate that Mof is required for maintenance of NUP98-HOXA9–transformed cells in vitro and NUP98-HOXA9 driven AML in vivo. The indispensable role of Mof in NUP98-HOXA9–driven AML demonstrated here is in line with our recent findings that the NUP98 fusions physically interact with MLL1 and the nonspecific lethal (NSL) histone-modifying complexes (40). It will be interesting to further examine whether and how MOF affects the oncogenic program in NUP98 fusion–driven leukemia.

To identify novel druggable targets in MLL-rearranged leukemia, we performed an RNAi screen focused on chromatin regulators. This resulted in the identification of Mof as a critical gene for cell growth in a murine MA9 leukemia model. We found that homozygous Mof loss led to a significant decrease in colony-forming ability, reduced tumor burden, and prolonged survival in mice. RNAseq of Mof-deficient MA9 cells showed significant downregulation of genes associated with DNA damage repair pathways, and upon validation by immunofluorescence, we indeed found a significant increase of yH2AX foci in Mof-deficient MA9 cells. In addition, we uncovered a loss of global H4K16ac upon Mof KO. Rescue experiments with HAT domain–mutated MOF illustrated that MOF HAT activity is indispensable for MA9 leukemia maintenance. This new insight could be utilized in the development of a small-molecule inhibitor for the treatment of patients with leukemia carrying an MLL translocation.

Gene expression analysis in Mof-deficient MA9 cells indicated that MOF loss does not lead to clear downregulation of MA9 target genes such as Meis1 and Hoxa cluster genes. Our RNAseq data suggested that Mof loss leads to impairment of more general biologic processes required for cellular integrity implying that Mof may be required not only for MA9 leukemogenesis but also for a broad range of acute leukemias, This is further supported by the facts that Mof is required for NUP98-HOXA9–driven leukemia and that non–MLL-rearranged human leukemia cells are also sensitive to HAT inhibitor MG149. We identified DNA damage repair as a possible mechanism of cell death in Mof-deficient leukemia cells (Fig. 4C), although our findings are not conclusive with regard to the mechanism of Mof dependence. Mof-null murine embryonic fibroblasts (MEF) were previously shown to be deficient in DNA damage repair after ionizing radiation (22). In wild-type MEFs, radiation-induced DNA damage led to an increase of global H4K16ac (22), suggesting that a MOF-mediated increase of H4K16ac may be essential for an appropriate DNA damage response. Unacetylated H4K16 is required to achieve the maximum tendency of in vitro nucleosome arrays to fold into secondary or tertiary chromatin structures (41). In contrast, 30% H4K16 acetylation alleviates compaction of the chromatin fiber (42). Therefore, it may be that the MOF loss–induced global H4K16ac depletion influences the DNA damage response and/or chromatin integrity by increasing chromatin compaction.

GO analysis on our RNAseq data suggested that loss of Mof in MA9 leukemic cells may lead not only to DNA damage but also to general chromosomal instability. This could contribute to the rapid cell death we observed in Mof-deficient MA9 cells. Genetic and biochemical data underscore the importance of unacetylated acidic histone tails in gene silencing (43). Interestingly, the yeast ortholog of MOF, SAS2, was previously shown to lead to telomeric silencing (44), and it is well established that telomeric dysfunction can lead to chromosomal instability (45). Future experiments will be required to assess how Mof loss–induced H4K16ac depletion may contribute to chromosomal instability in Mof-deficient MA9 cells and whether telomeric silencing is involved.

MOF was shown to functionally and physically interact with the histone methyltransferase MLL1. The interaction between MOF and MLL1 is important for the chromatin regulatory function of both enzymes (46). In normal hematopoiesis, Mll1 is essential for development and maintenance of both embryonic and adult progenitors and hematopoietic stem cells (47). In addition, it has been suggested that wild-type MLL1 may play a role in MLL-rearranged leukemogenesis (48). However, in both normal and malignant hematopoiesis, the H3K4 methyltransferase activity of MLL1 is dispensable (49). Given the identified MA9 leukemia dependence on the enzymatic activity of MOF, it may be that, in the setting of MLL-rearranged leukemia, it is in fact the HAT activity of MOF that is required for leukemogenesis, and MLL1 merely functions as a scaffolding protein, recruiting MOF to the targets of the oncogenic fusion.

Over the last decade, many advances have been made in the field of cancer epigenomics. Our vastly expanding knowledge on the role of chromatin regulation in cancer has led to the development of various drug compounds that target the cancer epigenome, several of which are currently in clinical trials (5, 50). Here, we have established that MOF HAT activity is required for MA9 leukemia maintenance and that loss of MOF HAT activity leads to elevated DNA damage. In addition, we have successfully inhibited cell growth of not only human and murine MA9 leukemia cells but also other fusion-driven leukemias by using a first-generation small-molecule MYST protein HAT inhibitor. On the basis of our findings, we believe that inhibiting MOF HAT activity by small molecules may prove to be an effective, novel approach for the treatment of patients with MLL-rearranged and perhaps other leukemias.

A.J. Deshpande is a consultant/advisory board member for Salgomed Inc. and A2A Pharma. S.A. Armstrong is a consultant for Epizyme Inc. and Imago Biosciences. No potential conflicts of interest were disclosed by the other authors.

Conception and design: D.G. Valerio, H. Xu, C.-W. Chen, S.A. Armstrong

Development of methodology: D.G. Valerio, H. Xu, C.-W. Chen, M. Cusan, A. Lujambio, J. Zuber, T.K. Pandita, S.W. Lowe

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.G. Valerio, H. Xu, C.-W. Chen, T. Hoshii, C. Delaney, A.J. Deshpande, S.W. Lowe

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.G. Valerio, H. Xu, C.-W. Chen, A. Lujambio, S.A. Armstrong

Writing, review, and/or revision of the manuscript: D.G. Valerio, H. Xu, A. Lujambio, S.A. Armstrong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.G. Valerio, M.E. Eisold, C.-H. Huang, Y.G. Zheng, J. Zuber, T.K. Pandita, S.A. Armstrong

Study supervision: D.G. Valerio, S.A. Armstrong

We would like to thank C.M. Woolthuis for critically reading this article and Z. Feng for administrative assistance.

This work was supported by a CURE Childhood Cancer Research Grant (D.G. Valerio); by a DoD Bone Marrow Failure Postdoctoral Fellowship Award (W81XWH-12-1-0568 to H. Xu); NIH grants PO1 CA66996 and R01 CA140575, the Leukemia and Lymphoma Society, and Gabrielle's Angel Research Foundation (S.A. Armstrong); NIH RO1 CA129537 and RO1 GM109768 (T.K. Pandita); and an NIH Memorial Sloan Kettering Cancer Center Support Grant (P30 CA008748).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.