(2013) 27, 1793–1802 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

SPOTLIGHT REVIEW RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia

CP Koh1,6, CQ Wang1,2,6, CEL Ng1,YIto1,2, M Araki1, V Tergaonkar2, G Huang3 and M Osato1,2,4,5

A broad range of human carries RUNX1 and MLL genetic alterations. Despite such widespread involvements, the relationship between RUNX1 and MLL has never been appreciated. Recently, we showed that RUNX1 physically and functionally interacts with MLL, thereby regulating the epigenetic status of critical cis-regulatory elements for hematopoietic genes. This newly unveiled interaction between the two most prevalent leukemia genes has solved a long-standing conundrum: leukemia-associated RUNX1 N-terminal point mutants that exhibit no obvious functional abnormalities in classical assays for the assessment of transcriptional activities. These mutants turned out to be defective in MLL interaction and subsequent epigenetic modifications that can be examined by the histone-modification status of cis-regulatory elements in the target genes. RUNX1/MLL binding confirms the importance of RUNX1 function as an epigenetic regulator. Recent studies employing next-generation sequencing on human hematological malignancies identified a plethora of in epigenetic regulator genes. These new findings would enhance our understanding on the mechanistic basis for leukemia development and may provide a novel direction for therapeutic applications. This review summarizes the current knowledge about the epigenetic regulation of normal and malignant hematopoiesis by RUNX1 and MLL.

Leukemia (2013) 27, 1793–1802; doi:10.1038/leu.2013.200 Keywords: RUNX1; AML1; MLL; epigenetic regulation

INTRODUCTION Heterodimerization of RUNX1 with its partner b subunit RUNX1 and MLL are the two major genes that are frequently facilitates the DNA binding of RUNX1 in an allosteric manner altered in B33 and 19% of human acute leukemias, respectively.1–3 at the regulatory regions of target genes. In addition, b hetero- RUNX1-related leukemias account for about 32% of acute myeloid dimerization also protects RUNX1 from ubiquitin-proteasome- 16 leukemia (AML),4,5 23% of (MDS)6 and mediated degradation. DNA binding and b heterodimerization 37% of chronic myelomonocytic leukemia (CMML).7 RUNX1 of RUNX1 is mediated by an evolutionarily conserved 128 amino genetic alterations are also found in acute lymphoblastic acid (aa) Runt domain (aa 50–177) located in its N-terminal region leukemia (ALL) and chronic myeloid leukemia (CML).8,9 Hence, (Figure 1). Several other functional domains that negatively or RUNX1 abnormalities are now associated with a broad spectrum of positively modulate RUNX1 transcriptional activity are also found 17 leukemia. MLL alterations are also detected in both myeloid and in both N- and C-terminal moieties. The RUNX1 C-terminal 17,18 lymphoid leukemias, although higher incidence of MLL region contains transactivation and inhibition domains. A abnormalities has been observed in ALL patients compared with series of , such as mSin3A, p300, MOZ, YAP and TAZ, are AML patients.10 Strikingly, 80% of the infant ALL is caused by MLL reported to interact with RUNX1 (Figure 1). The interactions of rearrangement.11,12 As RUNX1 or MLL genetic alterations are those regulatory proteins facilitate post-translational modification SPOTLIGHT independently associated with distinct proportions of human and regulate lineage-specific expression in distinct cell 19 leukemia, two genes have been thought to account for different types. Phosphorylation of RUNX1 at C-terminal regions is shown subgroups of leukemia, and hence the relationship between to negatively regulate RUNX1 function in early hematopoiesis and 20 RUNX1 and MLL was not well studied. T-cell differentiation. In addition, a nuclear localization signal is found in the C-terminal border of the Runt domain.18,21 There is also a nuclear matrix-targeting signal present at the C-terminal A FACTOR RUNX1 portion of the RUNX1 to enhance nuclear matrix 22 RUNX1 belongs to a small family of Runt-related heterodimeric interaction for functional organization of the nucleus. , polyomavirus -binding protein 2 (PEBP2)/core-binding factor (CBF) that is essential for the generation and maintenance of hematopoietic stem cells.13–15 GENETIC ALTERATIONS OF RUNX1 IN HUMAN LEUKEMIA PEBP2/CBF consists of a DNA-binding a subunit, RUNX1 and The importance of RUNX1 function in hematopoiesis is highlighted a non-DNA-binding b subunit, PEBP2b/CBFb (henceforth b). by its involvement in human leukemias. Prevalent types of RUNX1

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore; 2Institute of Molecular and Cell Biology, Singapore, Singapore; 3Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 4Institute of Bioengineering and Nanotechnology, Singapore, Singapore and 5Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan. Correspondence: Assistant Professor G Huang, Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229-3039, USA or Professor M Osato, Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore 117599, Singapore. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 15 April 2013; revised 26 June 2013; accepted 26 June 2013; accepted article preview online 2 July 2013; advance online publication, 16 July 2013 RUNX1 meets MLL CP Koh et al 1794

Figure 1. Schematic diagram of RUNX1 structure. A diagram of the RUNX1 protein (AML1b/RUNX1b isoform, NP_001001890) shows functional domains, sites of phosphorylation and , and portions interacting with other indicated proteins. RD, Runt domain; AD,

SPOTLIGHT transactivation domain; ID, inhibitory domain; NRH, negative regulatory region for b heterodimerization; NRDB, negative regulatory region for DNA binding; NLS, nuclear localization signal; NMTS, nuclear matrix-targeting signal.

genetic alterations in human leukemia are chromosomal RUNX1 point mutations are predominantly reported in rearrangement, copy number variation and point . various myeloid malignancies.5,32–35 Several tight relationships of A large proportion of RUNX1 genetic alterations found in RUNX1 point mutations to distinct subtypes of malignancy are leukemias are attributed to chromosomal translocations.1,23 To documented: germline monoallelic RUNX1 mutations are observed date, 39 types of RUNX1 chromosomal translocations have been in FPD/AML,21,36 whereas somatic monoallelic RUNX1 mutations reported in leukemia patients (http://atlasgeneticsoncology.org). are found most frequently in CMML37 and AML with normal RUNX1 was originally cloned from the breakpoint of karyotype.4 Somatic biallelic RUNX1 point mutations are tightly 21 in t(8;21), a chromosomal translocation that accounts for associated with the AML M0 subtype.5,38 Recently, RUNX1 point B40% of patients with the AML M2 subtype.24 Subsequently, a mutations were also found in immature T-ALL, including early series of RUNX1 fusion proteins were identified: RUNX1-EVI1 T-cell precursor ALL.39–41 produced by chromosomal translocation t(3;21) was reported to A totally new mode of alteration, RUNX1 internal tandem be associated with therapy-related AML, MDS and the blast phase duplication was discovered in FPD/AML due to the technological of CML.8,25 The chromosomal translocation t(12;21) generating the advances, such as next-generation sequencing.42 TEL–RUNX1 was observed in childhood ALL.26 The RUNX1 heterodimerization partner, CBFb, is also well Notably, most RUNX1 fusion proteins retain their N-terminal and documented to be mutated in human leukemias, largely through Runt domain but lack the C-terminal regulatory region. As such, chromosome 16 inversion, inv(16) (p13q22), being observed in the fusion proteins are defective in transcriptional regulation but 12% of AML cases, particularly in almost all cases of AML-M4Eo are still capable of competing with the wild-type (WT) RUNX1 for subtype.43 This inversion generates a fusion gene, CBFB–MYH11, DNA binding. Therefore, the dominant-negative effect against encoding CBFb-smooth muscle myosin heavy chain fusion WT RUNX1 function by the RUNX1 fusion proteins is widely protein, which acts as a dominant repressor for RUNX1 function.44 believed to be the common underlying mechanism for leukemo- genesis throughout a variety of RUNX1-related chromosomal translocations. FUNCTIONAL CONSEQUENCES DUE TO RUNX1 POINT RUNX1 copy number variations are also associated with MUTATIONS malignant hematopoietic diseases. patients, Leukemia-associated RUNX1 point mutations are commonly carrying of where RUNX1 is located, are clustered within the functionally important Runt domain, often more susceptible to AML and ALL.27,28 Trisomy 21 is also detected resulting in the loss-of-RUNX1 protein function due to disruption as a sole chromosomal abnormality in sporadic cases of AML. of DNA binding and/or b heterodimerization capabilities. An Furthermore, extra copies of RUNX1 (2–8 copies) due to the electrophoretic mobility shift assay performed to examine the tandem repetition of a part of chromosome 21 including DNA binding and b heterodimerization of RUNX1 point mutants RUNX1 were also observed in ALL cells.29 Conversely, illustrated that about 50% of these RUNX1 Runt domain point deletions of 21q22.12 encompassing RUNX1 are implicated in mutants lacked DNA binding and/or b heterodimerization both hereditary and sporadic leukemias. The germline competency (Table 1, Figure 2). Mutant R174Q was totally of RUNX1 alone is associated with familial platelet disorder defective in DNA binding, whereas b heterodimerization was with predisposition to AML (FPD/AML), whereas the deletion completely lost in mutants, S67I, S67R and Q158H. Notably, these of relatively wider region encompassing RUNX1 is associated functional defects sustained by these particular point mutations with syndromic thrombocytopenia accompanied by non- are explained using the three-dimensional structure of the RUNX1 hematopoietic symptoms, such as mental retardation and heart protein, wherein aa R174 and S67/Q158 are located within the malfunction.30,31 loop or b-sheet responsible for direct interactions with DNA or

Leukemia (2013) 1793 – 1802 & 2013 Macmillan Publishers Limited RUNX1 meets MLL CP Koh et al 1795 Table 1. Functional alterations of RUNX1 mutants

RUNX1 mutants Localization Frequency (%)a DNA b-subunit Subcellular Transcriptional In vitro MLL Type of binding interaction localization activity ubiquitylation binding mutantb

WT þþ N þþþN.A. L29S N-terminal 4.5 þþ N þþÀ3 A33V N-terminal 0.5 þþ N þþÀ3 G42R N-terminal 0.9 þþ N þþÀ3 R49H N-terminal 0.5 þþ N þþÀ3 R49S N-terminal 0.5 þþ N þþÀ3 H58N Runt domain 0.9 þþ N þþÀ3 S67I Runt domain 0.5 þÀ N ± þÀ1b/3 S67R Runt domain 0 þÀ N ± þ N.A. 1b W79C Runt domain 2.7 þþ N Àþþ1 A107P Runt domain 0 ±± N À N.A. N.A. 1ab L117P Runt domain 0.5 ± þ N À ± N.A. 1a/2 T149A Runt domain 0 þþ N þþN.A. 4 I150T Runt domain 0.5 þþ N/C À ± N.A. 1c/2 P156A Runt domain 0.5 þþ N/C ÀþN.A. 1c Q158H Runt domain 0.5 þÀ N ± þ N.A. 1b I166T Runt domain 0.5 þþ N ± þ N.A. 1 R174Q Runt domain 11.3 À c þ c N/C ÀþN.A. 1ac E196G C-terminal 0.5 þþ N þþN.A. 4 R223H C-terminal 0.5 þþ N þ N.A. N.A. 4 Y260X C-terminal 0 þþ C ± þ N.A. 1c Functional consequences for the indicated mutants are shown: À , no or very weak; ±, weak; þ , normal; N, nucleus; C, cytoplasm; N.A., not available. (1) Transcription-defective type. (a) DNA-binding-defective type. (b) b-interaction-defective type. (c) Nuclear localization-defective type. (2) Ubiquitylation- defective type. (3) MLL-binding-defective type. (4) Unclassified. aMutation frequencies for particular amino acids in missense mutants of leukemia patients are obtained from http://www.sanger.ac.uk/genetics/CGP/cosmic/. bClassification of mutants. cBased on previous studies.

b-subunit, respectively.45 A mutant L117P was partially defective Luciferase reporter assays performed revealed that most of the in DNA binding, as the band shifted by RUNX1 alone showed a Runt-domain point mutants studied possessed reduced transcrip- smear pattern. In addition, perturbation of both DNA binding and tional activity in comparison to the WT RUNX1 (Figure 4). b heterodimerization was observed in mutant A107P, in which Consistent with previous reports, the DNA-binding mutant band shifts by RUNX1 and RUNX1/b complex were both R174Q, which failed to localize to the nucleus faithfully, showed incomplete. a complete lack of transcriptional activity. A partially defective The nuclear localization mediated by nuclear localization signal DNA-binding mutant, L117P, also demonstrated nearly complete located around the C-terminal end of Runt domain18,21 is abrogation in transcriptional activity. Severe reduction in important for full functionality of RUNX1 as a transcription transcriptional activity was also found in mutant A107P, in which factor. To determine the nuclear localization, RUNX1 point DNA binding and b heterodimerization abilities were both partially mutants were expressed in NIH3T3 cells and their subcellular disrupted. As expected, the mutants S67I, S67R and Q158H, localization was visualized by immunofluorescent staining with all of which are completely defective in b heterodimerization, anti-RUNX1 antibody. WT RUNX1 protein was exclusively localized demonstrated reduced transcriptional activity. Consistent with a in the nucleus, and most of the RUNX1 point mutants we studied significant proportion of cells showing cytoplasmic localization of also exhibited distinct nuclear localization, with the exception of RUNX1 protein (Figure 3), a significant reduction in transcriptional three Runt domain mutants (I150T, P156A and R174Q) and one activity was observed in both I150T and P156A mutants. Decrease SPOTLIGHT C-terminal mutant (Y260X) (Figure 3). All cells expressing the in transcriptional activity was shown in Y260X, which is defective R174Q mutant exhibited RUNX1 staining in both the cytoplasm in nuclear localization. Unexpectedly, W79C and I166T mutants and the nucleus. On the other hand, we observed either distinct showed reduced transcriptional activity, although no defects were cytoplasmic or nuclear RUNX1 localization in cells expressing observed in DNA binding, b heterodimerization or nuclear I150T or P156A mutant proteins. Mutant Y260X showed pre- localization. dominant cytoplasmic localization. Hence, mutants I150T, P156A, Besides the above listed functional abnormalities, changes in R174Q and Y260X were classified as nuclear localization-defective RUNX1 protein stability might affect the RUNX1 transcriptional mutants (Table 1). Besides the above-mentioned nuclear localiza- activity. The anaphase-promoting complex (APC), a mitosis- tion signal, other multiple aa residues and domains are also regulating E3 ubiquitin ligase complex, was reported to modulate involved in the nuclear localization.18,33,46 The mutants that the protein amount of RUNX1 in cells cultured in vitro.48,49 The exhibited cytoplasmic localization are considered to contain APC-activator protein Cdh1, but not Cdc20, was found to directly defects in such additional domains. The cytoplasmic localization ubiquitylate RUNX1. To study whether RUNX1 point mutants affect patterns in I150T, P156Q and Y260X seem to be intriguing, their protein stability, in vitro ubiquitylation assay was conducted although these could be artifacts owing to the overexpression of by using APC-Cdh1 (Figure 5). Ubiquitylation rates of majority of RUNX1 mutant proteins. Recent studies showed that some the RUNX1 point mutants were comparable to that of the WT abnormally misfolded proteins are preferentially accumulated in protein. However, mutants L117P and I150T showed reduction in the endoplasmic reticulum, thereby causing endoplasmic ubiquitylation rate quantified by Typhoon Trio (Amersham reticulum-stress-associated human diseases including leukemia.47 Biosciences, Sunnyvale, CA, USA) (data not shown). Reduction of Therefore, precise investigation on the cytoplasmic localization of RUNX1 ubiquitylation rate is expected to potentially result in the RUNX1 mutant proteins might provide further insights into reduction of protein degradation rate, thereby causing accumula- pathogenic mechanisms. tion of the RUNX1 protein. Excess of RUNX1 mutant proteins may

& 2013 Macmillan Publishers Limited Leukemia (2013) 1793 – 1802 RUNX1 meets MLL CP Koh et al 1796 compete for DNA binding and b heterodimerization against the WT RUNX1 protein. For example, accumulation of mutant L117P proteins, which showed absolute impairment in transcriptional activity but still retained b heterodimerization and partial DNA binding, might compete with WT RUNX1, leading to a reduction in RUNX1-mediated transcriptional activity in the cells. Hence, protein degradation efficiency is also considered to be a key mechanism for RUNX1-mediated transcriptional activity and pathogenesis. It is of note that although majority of the mutations are defective in DNA binding, b heterodimerization, nuclear

Figure 2. DNA binding and b heterodimerization of RUNX1 mutants. RUNX1 point mutant constructs were transiently transfected into SPOTLIGHT Cos7 cells. Total cell lysates were extracted 48 h after transfection and subjected to electrophoretic mobility shift assay (EMSA) with a Figure 4. Transcriptional activity of RUNX1 mutants. Luciferase biotin-labeled probe carrying a Runx-binding site (50-ACCGCA-30). reporter assay was carried out using the pBXH2-LTR-luc reporter The ‘ þ ’ and ‘ À ’ symbols signify the presence and absence of PEBP construct.96,97 U937 cells were transfected with 0.2 mg of pBXH2-LTR- (polyomavirus enhancer-binding protein)2b, respectively. In the luc and 0.2 mg of indicated RUNX1 plasmid. Each bar indicates the lanes marked with asterisk (*), 50-fold excess of unlabeled probe was percentage of reporter activity induced by RUNX1 mutant proteins added for competition with biotin-labeled probe. b, PEBP2b; relative to that of the RUNX1 WT protein. Results shown are the WT, wild type. mean±s.d. of triplicate experiments. WT, wild type.

Figure 3. Subcellular localization of RUNX1 point mutants. Subcellular localization of RUNX1 mutants in NIH3T3 cells was examined by labeling RUNX proteins with rabbit anti-AML1 antibody (active motif), followed by Alexa Fluor 488-conjugated anti-rabbit secondary antibody (green). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Half of the total transfected cells with I150T or P156A mutant proteins showed cytoplasmic localization, whereas the remaining half of the transfected cells revealed the nuclear pattern. WT, wild type.

Leukemia (2013) 1793 – 1802 & 2013 Macmillan Publishers Limited RUNX1 meets MLL CP Koh et al 1797

Figure 5. In vitro ubiquitylation assay of RUNX1 mutants. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and APC- activator (Cdh1) were expressed and purified from bacteria or baculovirus expression system. Anaphase-promoting complex (APC) was immunoprecipitated from Xenopus interphase extracts by anti-Cdc27, one of the APC subunits. After the APC activation by Cdh1, E1, E2 and in vitro-synthesized S35-methionine-labeled RUNX1 mutant protein were reacted in the presence of ATP. Phosphoimager was used to take images. Bands showing decreased intensities in L117P and I150T are highlighted by vertical lines. localization or stability of protein level, there still remain many genetic alterations arise from chromosomal translocation. The uncharacterized mutants in which none of the experiments causative factors of MLL chromosomal translocation are either conducted have successfully identified any defects. Most spontaneous or therapy-related. Cancer patients treated with importantly, N-terminal point mutants, including a relatively topoisomerase II inhibitor, such as etoposide, show high common mutant L29S, are all classified as such uncharacterized occurrence of MLL chromosomal translocation.60,61 More than 70 mutants. Hence, the involvement of these RUNX1 N-terminal types of partner genes have been found to generate fusion point mutations in leukemogenesis has been a long-standing proteins by chromosomal translocations with MLL.59 The common conundrum in the field. MLL fusion partners are AF4, ENL, AF9, AF6, ELL and AF10.62 The most prevalent MLL-AF4 is associated with infant pro-B ALL,63,64 whereas the MLL–ENL fusion protein is observed in both AML and AN EPIGENETIC REGULATOR MLL ALL patients. Chromosomal translocations t(9;11) and t(6;11), MLL is a histone methyltransferase that belongs to the Trithorax- generating MLL-AF9 and MLL-AF6, respectively, are mainly found group protein family,50 and functions as a transcriptional activator, in AML patients. MLL fusion proteins share some common regulating during early development and features: most of them retain MLL N-terminal region and lack hematopoiesis. MLL is also known as MLL1 and there are four C-terminal moiety containing the SET domain, which was reported to be responsible for H3K4 methylation in the regions of other distinct MLL family genes, MLL2–MLL5. MLL works as a 58 protein complex to modulate chromatin modification via HOX genes (Figure 6). HOX genes have a key role in the regulation of hematopoietic development and aberrant HOX methylation, acetylation and nucleosome remodeling. MLL is 65–67 cleaved into N-terminal (MLLN) and C-terminal (MLLC) fragments expression is known to be associated with leukemogenesis. 51 N Inappropriate HOX gene expression is frequently correlated with by aspartic protease, namely taspase (Figure 6). MLL has a role 68,69 in DNA binding and recognition. Menin is recruited by MLLN to MLL fusion protein in leukemia. In fact, upregulation of HOXA5, HOXA7 and HOXA9 were observed in MLL-related human form an interaction domain for LEDGF and contacts chromatin via 70,71 a PWWP domain.52 MLLN also includes an AT-hooks domain, which leukemias. As such, deregulation of HOX genes due to MLL preferentially recognizes distorted DNA. Another functional fusion protein appears to be one of the underlying mechanisms domain in the N-terminal is a CxxC domain that discriminates for MLL-related leukemogenesis. SPOTLIGHT the methylation status of DNA.53 Four plant homeodomains Besides higher occurrence of the MLL fusion protein in human (PHD) and one Bromo domain, responsible for protein–protein leukemia, MLL–PTD, which generates an in-frame repeat within the MLL N-terminal region (Figure 6), was also observed in 2.7% of interaction and recognition of modified histone marks, are located 3,72–76 N C myelodysplastic syndrome and 6.4% of AML patients, around the center of MLL . MLL , on the other hand, has a role in 3,76,77 chromatin remodeling for efficient transcription by modulating particularly in adult cytogenetically normal AML patients. the efficiency of histone methylation and acetylation, through a AML characterized by MLL–PTD is likely molecularly different from leukemias carrying MLL-translocation mutants, as deregulation of SET domain, which holds histone H3K4 methyltransferase activity, 69 and is responsible for interacting with at least four important HOX genes were not observed. Interestingly, RUNX1 point proteins, MOF, WDR5, ASH2L and RBBP5.54,55 Transcription factors, mutations and MLL–PTD were simultaneously found in the same AML patients, constituting a distinct clinical entity with unfavor- such as and b-catenin, are shown to recruit MLL complex to 2,78 initiate transcription,54,56 and specific genes, such as the HOX able prognosis. We recently reported that the mice carrying genes, are highly dependent on the MLL complex for chromatin MLL–PTD exhibit abnormal hematopoiesis with enhanced 57,58 self-renewal, lineage bias and blockages modification during transcription. Polymerase-associated 79 factor complex (PAFc), HCF1/2, CYP33, CBP/p300, INI1 and in myeloid differentiation when stressed. DPY30 are also shown to interact with MLL.59 All such interacting molecules are summarized in Figure 6. RUNX1 MEETS MLL Besides its crucial role as a transcription factor, RUNX1 is also GENETIC ALTERATIONS OF MLL IN HUMAN LEUKEMIA documented to mediate epigenetic regulation in order to facilitate The deregulation of MLL by chromosomal translocation is or inhibit multiple regulatory regions in the target genes.80–83 As frequently observed in both AML and ALL. Around 70% of MLL RUNX1 itself does not possess any -related enzymatic

& 2013 Macmillan Publishers Limited Leukemia (2013) 1793 – 1802 RUNX1 meets MLL CP Koh et al 1798

Figure 6. Schematic diagram of MLL structure. A diagram of the MLL1 protein (NP_005924.2) shows functional domains, sites of phosphorylation and acetylation, and portions interacting with other indicated proteins. Two solid arrowheads, a dotted and a solid line on MLL represent taspase cleavage sites, a breakpoint cluster region (BCR), a partial tandem duplication (PTD) portion, respectively. AT, AT-hooks domain; CXXC, CXXC-type zinc-finger domain; PHD, plant homeodomain; BD, Bromo domain; TAD, transactivation domain; SET, SET domain.

SPOTLIGHT activities, the interaction of RUNX1 with other epigenetic regulators is expected to underlie RUNX1-associated epigenetic regulation. Indeed, RUNX1 has been shown to interact with histone acetyl transferases (HAT), such as coactivators CBP, p300 and MOZ,80,81 and components of the (HDAC) complex, such as Groucho/TLE and mSin3A.84,85 The interaction of RUNX1 with HAT acetylates chromatin-associated histones, which change the chromatin conformation and activate transcriptional activities. In contrast, HDACs act as repressors for gene expression by deacetylating nucleosomal core histone tails, which lead to tight chromatin conformation. RUNX1 also interact with distinct types of histone methyltransferases, such as SUV39H186 and Figure 7. Physical interaction between MLL and RUNX1. The PRMT6.83 The binding of SUV39H1 followed by RUNX1 MLL C-terminal region (aa 2720–3969) interacts with RUNX1 methylation in the N-terminal-negative regulatory region N-terminal region (aa 25–106). Solid lines represent essential revokes RUNX1 function.86 RUNX1 and PRMT6 interaction was domains for the interaction between RUNX1 and MLL, whereas a dotted line indicates a weak interacting portion. AT, AT-hooks reported to enhance H3R2 dimethylation, which inhibits 71 domain; CXXC, CXXC-type zinc-finger domain; PHD, plant homeo- transcriptional activity of RUNX1. The dissociation of RUNX1 domain; BD, Bromo domain; TAD, transactivation domain; SET, SET and PRMT6 interaction conversely promotes the interaction domain; RD, Runt domain; AD, transactivation domain; ID, between RUNX1 and WDR5, which is a key component of the inhibitory domain. MLL complex, thereby initiating the expression of a subset of genes to induce differentiation of hematopoietic cells.83 RUNX1 and MLL proteins are both transcriptional regulators aa 33 onwards in RUNX1c. Therefore, MLL-binding affinities with involved in epigenetic changes to govern proper hematopoiesis. the two RUNX1 N-terminal variants are likely to be comparable, MLL–PTD and RUNX1 mutations were observed simultaneously in as the differences are located outside the MLL-binding domain a subtype of AML patients.78 Therefore, it is anticipated that (aa 25–106). interaction between RUNX1 and MLL may exist in the regulation Notably, the interaction between MLL and RUNX1 was found of multiple hematopoietic genes.87–90 Indeed, we have recently to be responsible for H3K4 tri-methylation in the upstream demonstrated that RUNX1 physically and functionally interacts regulatory element (URE) and promoter regions of the PU.1 with MLL.91 (SPI-1) gene, which is an ETS family member essential for the The RUNX1 N-terminal region was found to directly bind to the hematopoietic stem cell maintenance and development of MLL C-terminal region (Figure 7). Immunoprecipitation assay macrophages and B cells.91 Pu.1 is reported to be a major showed that the elimination of RUNX1 aa 1–106 completely downstream target of Runx1 in adult mouse hematopoiesis.92 abolished MLL interaction, while this interaction gradually Three and two RUNX-binding sites were identified at the PU.1 URE diminished after the elimination of RUNX1 aa 25 onwards. and promoter regions, respectively. The binding of RUNX1 at PU.1 Therefore, RUNX1 aa 25–106 is considered to constitute a domain URE positively and negatively regulates PU.1 expression in a cell for MLL interaction, although aa 25–50 contributed to the context-dependent manner. Knockdown and rescue assays of MLL interaction to a lesser extent (dotted line in Figure 7). On the or RUNX1 were performed independently by using the early other hand, MLL C-terminal region aa 2720–3969 was sufficient for myeloid murine progenitor cell line 416B. Knockdown of MLL or RUNX1 binding, indicating that MLL aa 2720–3969 region appears RUNX1 showed less H3K4 tri-methylation, whereas reintroduction to be the RUNX1 interaction domain.91 It is known that two of MLL or RUNX1 restored H3K4 tri-methylation to normal levels, distinct RUNX1 promoters generate two different N-terminal ends indicating that both genes are important to mediate H3K4 of RUNX1 proteins starting with MAS or MRIP, full length of which tri-methylation in the PU.1 URE and promoter regions.91 are RUNX1c or RUNX1b, respectively. These two isoforms share the RUNX1 interaction with MLL is also shown to stabilize the common aa sequences from position 6 onwards in RUNX1b and RUNX1 protein in 293T cells.91 MLL binding protected RUNX1

Leukemia (2013) 1793 – 1802 & 2013 Macmillan Publishers Limited RUNX1 meets MLL CP Koh et al 1799 from proteasome-mediated protein degradation by masking transcription of the PU.1 gene. On the other hand, RUNX1 RUNX1–proteasome interaction domain and also possibly by N-terminal point mutants, which are defective in binding to MLL, methylating residues. fail to recruit the MLL complex to the targeted chromatin region, hence resulting in the continuation of silent mode for transcription. N-TERMINAL RUNX1 POINT MUTANTS ARE DEFECTIVE IN MLL MLL fusion proteins, lacking RUNX1-interacting C-terminal BINDING moiety, are considered to be defective in RUNX1 binding, leading Interestingly, we found in the study that most of the to subsequent epigenetic deregulation. It is therefore plausible uncharacterized RUNX1 N-terminal point mutants were defective that the defective interaction between RUNX1 and MLL in MLL binding and incapable of H3K4 tri-methylation in the PU.1 may be one of the underlying mechanisms for the prevalent URE and promoter regions. To examine whether RUNX1 MLL fusion-associated leukemias as well. N-terminal point mutants were involved in the deregulation of H3K4 tri-methylation mediated by MLL, two RUNX1 N-terminal point mutants, L29S and H58N, were selected for performing chromatin immunoprecipitation assay in 416B cells. Mutants FUTURE PERSPECTIVES L29S and H58N were expressed in 416B cells, followed by Given that RUNX1 is shown to be involved in epigenetic immunoprecipitation using antibodies against MLL and regulation, classical transcriptional assays are apparently insuffi- H3K4me3. Quantitative PCR was performed to quantify the PU.1 cient to examine a molecular basis for leukemogenesis caused by URE DNA-binding fragments pulled down by the antibodies. In the RUNX1 malfunction. Many RUNX1 N-terminal mutants discovered presence of mutant L29S or H58N, PU.1 URE DNA-fragment in patient samples have never been characterized due to the lack amplification was nearly abolished,91 suggesting that defective of methods to assess the epigenetic regulation by RUNX1. Our epigenetic regulation occurred probably due to the failure of MLL recent study clearly proves that cis-regulatory elements validated recruitment to the chromatin by RUNX1 N-terminal point mutants. in vivo, such as PU.1 URE, serve as an experimental platform to This epigenetic deregulation is considered to be a basis for examine epigenetic controls. A newly identified RUNX1 intronic leukemogenesis in RUNX1 N-terminal mutants. We classify those enhancer, which is specifically active in long-term hematopoietic 93 RUNX1 N-terminal point mutants, which carry a defect in MLL stem cells, may also serve as another useful platform to assess binding, as ‘MLL-binding-defective type’ (Table 1). epigenetic functions. Our current proposed model for RUNX1–MLL-mediated RUNX1 Runt domain and C-terminal region are shown to epigenetic regulation at the regulatory regions of target genes, interact with many epigenetic modifiers, such as Bmi1, p300 such as PU.1, is depicted in Figure 8. Under normal conditions, and MOZ (Figure 1). Recent studies reported that epigenetic RUNX1 first binds to the PU.1 URE region and recruits the MLL regulators, such as TET2, IDH1/2, EZH2, ASXL1 and DNMT3A, 94 complex to open up part of the compact chromatin structure. are frequently mutated in myeloid malignancies. Currently Subsequently, RUNX1 binds to the partially relaxed PU.1 promoter unclassified RUNX1 mutants (class 4 mutants in Table 1) may be region and recruits the MLL complex to further distort compact defective in interacting with these epigenetic factors, although it DNA structure. The relaxed form of PU.1 promoter region allows remains to be proven whether these leukemia-associated for the assembly of other transcriptional cofactors and initiates epigenetic regulators interact with RUNX1. SPOTLIGHT

Figure 8. A proposed model for leukemogenesis of MLL-binding defective RUNX1 N-terminal point mutants. (a) RUNX1 wild-type protein first binds to the PU.1 URE region and recruits the MLL complex to open up part of the compact chromatin structure. The partially relaxed chromatin allows the binding of another RUNX1 at the PU.1 promoter region to further distort compact DNA structure. The relaxed form of chromatin facilitates the accumulation of transcription factors and cofactors to initiate transcriptional activity. (b) MLL-binding-defective RUNX1 N-terminal point mutants are incapable of relaxing compact chromatin structure and subsequent transcription. Such deregulation may lead to leukemogenesis.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1793 – 1802 RUNX1 meets MLL CP Koh et al 1800 RUNX1 mutations were recently observed in various solid 17 Ito Y. Molecular basis of tissue-specific gene expression mediated by the runt tumors, such as esophagus and breast cancers besides hemato- domain transcription factor PEBP2/CBF. Genes Cells 1999; 4: 685–696. 95 logical malignancies. Alterations in MLL family genes were also 18 Kanno T, Kanno Y, Chen LF, Ogawa E, Kim WY, Ito Y. Intrinsic transcriptional reported in solid tumors. Hence, the usage of RUNX1–MLL activation-inhibition domains of the polyomavirus enhancer binding protein epigenetics-related axis could be more widespread than pre- 2/ alpha subunit revealed in the presence of the beta subunit. viously thought. These new findings greatly enhance our under- Mol Cell Biol 1998; 18: 2444–2454. standing of the molecular mechanisms involved in cancer 19 Ito Y. Oncogenic potential of the RUNX gene family: ’overview’. Oncogene 2004; development and may provide a novel direction for future 23: 4198–4208. 20 Yoshimi M, Goyama S, Kawazu M, Nakagawa M, Ichikawa M, Imai Y et al. Multiple therapeutic applications. phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation. Eur J Immunol 2012; 42: 1044–1050. 21 Michaud J, Wu F, Osato M, Cottles GM, Yanagida M, Asou N et al. In vitro CONFLICT OF INTEREST analyses of known and novel RUNX1/AML1 mutations in dominant familial The authors declare no conflict of interest. platelet disorder with predisposition to acute myelogenous leukemia: implications for mechanisms of pathogenesis. Blood 2002; 99: 1364–1372. 22 Zeng C, van Wijnen AJ, Stein JL, Meyers S, Sun W, Shopland L et al. Identification of a nuclear matrix targeting signal in the leukemia and ACKNOWLEDGEMENTS bone-related AML/CBF-alpha transcription factors. Proc Natl Acad Sci USA 1997; We thank Stephen D Nimer and Osato laboratory members for their helpful 94: 6746–6751. discussion. We apologize to colleagues whose work could not be cited due to 23 Speck NA, Gilliland DG. Core-binding factors in and leukaemia. space limitations. This work was supported by the Singapore National Research Nat Rev Cancer 2002; 2: 502–513. Foundation and the Ministry of Education under the Research Center of Excellence 24 Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki M. t(8;21) breakpoints on Programme, BMRC (Biomedical Research Council) and NMRC (National Medical chromosome 21 in are clustered within a limited region Research Council). of a single gene, AML1. Proc Natl Acad Sci USA 1991; 88: 10431–10434. 25 Nucifora G, Begy CR, Erickson P, Drabkin HA, Rowley JD. The 3;21 translocation in myelodysplasia results in a fusion transcript between the AML1 gene and the gene for EAP, a highly conserved protein associated with the Epstein-Barr virus

SPOTLIGHT REFERENCES small RNA EBER 1. Proc Natl Acad Sci USA 1993; 90: 7784–7788. 1 Look AT. Oncogenic transcription factors in the human acute leukemias. 26 Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P et al. Science 1997; 278: 1059–1064. Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lym- 2 Grossmann V, Schnittger S, Kohlmann A, Eder C, Roller A, Dicker F et al. A novel phoblastic leukemia. Proc Natl Acad Sci USA 1995; 92: 4917–4921. hierarchical prognostic model of AML solely based on molecular mutations. Blood 27 Osato M, Ito Y. Increased dosage of the RUNX1/AML1 gene: a third mode of RUNX 2012; 120: 2963–2972. leukemia? Crit Rev Eukar Gene Expr 2005; 15: 217–228. 3 Steudel C, Wermke M, Schaich M, Schakel U, Illmer T, Ehninger G et al. 28 Watson MS, Carroll AJ, Shuster JJ, Steuber CP, Borowitz MJ, Behm FG et al. Trisomy Comparative analysis of MLL partial tandem duplication and FLT3 internal tan- 21 in childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study dem duplication mutations in 956 adult patients with acute myeloid leukemia. (8602). Blood 1993; 82: 3098–3102. Genes Cancer 2003; 37: 237–251. 29 Ferro MT, Hernaez R, Sordo MT, Garcia-Sagredo JM, Garcia-Miguel P, Fernandez 4 Schnittger S, Dicker F, Kern W, Wendland N, Sundermann J, Alpermann T et al. Guijarro M et al. Chromosome 21 tandem repetition and AML1 (RUNX1) gene RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and amplification. Cancer Genet Cytogenet 2004; 149: 11–16. confer an unfavorable prognosis. Blood 2011; 117: 2348–2357. 30 Shinawi M, Erez A, Shardy DL, Lee B, Naeem R, Weissenberger G et al. 5 Osato M. Point mutations in the RUNX1/AML1 gene: another actor in RUNX Syndromic thrombocytopenia and predisposition to acute myelogenous leukemia leukemia. Oncogene 2004; 23: 4284–4296. caused by constitutional microdeletions on chromosome 21q. Blood 2008; 112: 6 Harada H, Harada Y, Niimi H, Kyo T, Kimura A, Inaba T. High incidence of 1042–1047. somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and 31 Lindstrand A, Malmgren H, Sahlen S, Schoumans J, Nordgren A, Ergander U et al. low blast percentage myeloid leukemia with myelodysplasia. Blood 2004; 103: Detailed molecular and clinical characterization of three patients with 21q 2316–2324. deletions. Clin Genet 2010; 77: 145–154. 7 Kuo MC, Liang DC, Huang CF, Shih YS, Wu JH, Lin TL et al. RUNX1 mutations are 32 Osato M, Asou N, Abdalla E, Hoshino K, Yamasaki H, Okubo T et al. frequent in chronic myelomonocytic leukemia and mutations at the C-terminal Biallelic and heterozygous point mutations in the runt domain of the region might predict acute myeloid leukemia transformation. Leukemia 2009; 23: AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood 1999; 93: 1426–1431. 1817–1824. 8 Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M, Mano H et al. Generation of 33 Osato M, Yanagida M, Shigesada K, Ito Y. Point mutations of the the AML1-EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic RUNx1/AML1 gene in sporadic and familial myeloid leukemias. Int J Hematol 2001; myelocytic leukemia. EMBO J 1994; 13: 504–510. 74: 245–251. 9 Romana SP, Mauchauffe M, Le Coniat M, Chumakov I, Le Paslier D, Berger R et al. 34 Niimi H, Harada H, Harada Y, Ding Y, Imagawa J, Inaba T et al. Hyperactivation of The t(12;21) of acute lymphoblastic leukemia results in a tel-AML1 gene fusion. the RAS signaling pathway in myelodysplastic syndrome with AML1/RUNX1 point Blood 1995; 85: 3662–3670. mutations. Leukemia 2006; 20: 635–644. 10 Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J et al. New 35 Taketani T, Taki T, Takita J, Ono R, Horikoshi Y, Kaneko Y et al. Mutation of the insights to the MLL recombinome of acute leukemias. Leukemia 2009; 23: AML1/RUNX1 gene in a transient myeloproliferative disorder patient with Down 1490–1499. syndrome. Leukemia 2002; 16: 1866–1867. 11 Biondi A, Cimino G, Pieters R, Pui CH. Biological and therapeutic aspects of infant 36 Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D et al. leukemia. Blood 2000; 96: 24–33. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to 12 Taki T, Ida K, Bessho F, Hanada R, Kikuchi A, Yamamoto K et al. Frequency and develop acute myelogenous leukaemia. Nat Genet 1999; 23: 166–175. clinical significance of the MLL gene rearrangements in infant acute leukemia. 37 Gelsi-Boyer V, Trouplin V, Adelaide J, Aceto N, Remy V, Pinson S et al. Genome Leukemia 1996; 10: 1303–1307. profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and 13 Motoda L, Osato M, Yamashita N, Jacob B, Chen LQ, Yanagida M et al. Runx1 RUNX1 genes. BMC Cancer 2008; 8: 299. protects hematopoietic stem/progenitor cells from oncogenic insult. Stem Cells 38 Roumier C, Fenaux P, Lafage M, Imbert M, Eclache V, Preudhomme C. 2007; 25: 2976–2986. New mechanisms of AML1 gene alteration in hematological malignancies. 14 Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of Leukemia 2003; 17: 9–16. multiple chromosomal translocations in human leukemia, is essential for normal 39 Della Gatta G, Palomero T, Perez-Garcia A, Ambesi-Impiombato A, Bansal M, fetal liver hematopoiesis. Cell 1996; 84: 321–330. Carpenter ZW et al. Reverse engineering of TLX oncogenic transcriptional 15 Jacob B, Osato M. Stem cell exhaustion and leukemogenesis. J Cell Biochem 2009; networks identifies RUNX1 as tumor suppressor in T-ALL. Nat Med 2012; 18: 107: 393–399. 436–440. 16 Huang G, Shigesada K, Ito K, Wee HJ, Yokomizo T, Ito Y. Dimerization with 40 Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D et al. The genetic PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012; 481: degradation. EMBO J 2001; 20: 723–733. 157–163.

Leukemia (2013) 1793 – 1802 & 2013 Macmillan Publishers Limited RUNX1 meets MLL CP Koh et al 1801 41 Grossmann V, Kern W, Harbich S, Alpermann T, Jeromin S, Schnittger S et al. 65 Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Hoxa9 Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia. transforms primary bone marrow cells through specific collaboration with Meis1a Haematologica 2011; 96: 1874–1877. but not Pbx1b. EMBO J 1998; 17: 3714–3725. 42 Jongmans MC, Kuiper RP, Carmichael CL, Wilkins EJ, Dors N, Carmagnac A et al. 66 Magli MC, Largman C, Lawrence HJ. Effects of HOX genes in blood cell Novel RUNX1 mutations in familial platelet disorder with enhanced risk for acute differentiation. J Cell Physiol 1997; 173: 168–177. myeloid leukemia: clues for improved identification of the FPD/AML syndrome. 67 Drabkin HA, Parsy C, Ferguson K, Guilhot F, Lacotte L, Roy L et al. Quantitative Leukemia 2010; 24: 242–246. HOX expression in chromosomally defined subsets of acute myelogenous 43 Liu P, Tarle SA, Hajra A, Claxton DF, Marlton P, Freedman M et al. Fusion between leukemia. Leukemia 2002; 16: 186–195. transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute 68 Schraets D, Lehmann T, Dingermann T, Marschalek R. MLL-mediated transcrip- myeloid leukemia. Science 1993; 261: 1041–1044. tional gene regulation investigated by gene expression profiling. Oncogene 2003; 44 Castilla LH, Wijmenga C, Wang Q, Stacy T, Speck NA, Eckhaus M et al. 22: 3655–3668. Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos 69 Ross ME, Mahfouz R, Onciu M, Liu HC, Zhou X, Song G et al. Gene heterozygous for a knocked-in leukemia gene CBFB-MYH11. Cell 1996; 87: expression profiling of pediatric acute myelogenous leukemia. Blood 2004; 104: 687–696. 3679–3687. 45 Tahirov TH, Inoue-Bungo T, Morii H, Fujikawa A, Sasaki M, Kimura K et al. 70 Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML, Minden MD et al. Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its MLL translocations specify a distinct gene expression profile that distinguishes a allosteric control by CBFbeta. Cell 2001; 104: 755–767. unique leukemia. Nat Genet 2002; 30: 41–47. 46 Yoshida T, Kanegane H, Osato M, Yanagida M, Miyawaki T, Ito Y et al. 71 Rozovskaia T, Feinstein E, Mor O, Foa R, Blechman J, Nakamura T et al. Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4: dysplasia demonstrates novel genotype-phenotype correlations. Am J Hum Genet 11) abnormality. Oncogene 2001; 20: 874–878. 2002; 71: 724–738. 72 Caligiuri MA, Strout MP, Schichman SA, Mrozek K, Arthur DC, Herzig GP et al. 47 Khan M. Interplay of protein misfolding pathway and unfolded-protein response Partial tandem duplication of ALL1 as a recurrent molecular defect in acute in acute promyelocytic leukemia. Expert Rev Proteomics 2010; 7: 591–600. myeloid leukemia with trisomy 11. Cancer Res 1996; 56: 1418–1425. 48 Biggs JR, Peterson LF, Zhang Y, Kraft AS, Zhang DE. AML1/RUNX1 73 Schichman SA, Caligiuri MA, Gu Y, Strout MP, Canaani E, Bloomfield CD et al. phosphorylation by cyclin-dependent kinases regulates the degradation of ALL-1 partial duplication in acute leukemia. Proc Natl Acad Sci USA 1994; 91: AML1/RUNX1 by the anaphase-promoting complex. Mol Cell Biol 2006; 26: 6236–6239. 7420–7429. 74 Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T et al. 49 Wang S, Zhang Y, Soosairajah J, Kraft AS. Regulation of RUNX1/AML1 during the Screening for MLL tandem duplication in 387 unselected patients with AML G2/M transition. Leuk Res 2007; 31: 839–851. identify a prognostically unfavorable subset of AML. Leukemia 2000; 14: 796–804. 50 Francis NJ, Kingston RE. Mechanisms of transcriptional memory. Nat Rev Mol Cell 75 Bacher U, Haferlach T, Kern W, Haferlach C, Schnittger S. A comparative study of Biol 2001; 2: 409–421. molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 51 Takeda S, Chen DY, Westergard TD, Fisher JK, Rubens JA, Sasagawa S et al. patients with acute myeloid leukemia. Haematologica 2007; 92: 744–752. of MLL family proteins is essential for taspase1-orchestrated cell cycle 76 Shih LY, Liang DC, Fu JF, Wu JH, Wang PN, Lin TL et al. Characterization of fusion progression. Genes Dev 2006; 20: 2397–2409. partner genes in 114 patients with de novo acute myeloid leukemia and MLL 52 Yokoyama A, Cleary ML. Menin critically links MLL proteins with LEDGF on cancer- rearrangement. Leukemia 2006; 20: 218–223. associated target genes. Cancer Cell 2008; 14: 36–46. 77 Basecke J, Whelan JT, Griesinger F, Bertrand FE. The MLL partial tandem 53 Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K, Titgemeyer F, Slany RK. The MT duplication in acute myeloid leukaemia. Brit J Haematol 2006; 135: 438–449. domain of the proto-oncoprotein MLL binds to CpG-containing DNA and dis- 78 Tang JL, Hou HA, Chen CY, Liu CY, Chou WC, Tseng MH et al. AML1/RUNX1 criminates against methylation. Nucleic Acids Res 2002; 30: 958–965. mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic 54 Dou Y, Milne TA, Tackett AJ, Smith ER, Fukuda A, Wysocka J et al. Physical implication and interaction with other gene alterations. Blood 2009; 114: association and coordinate function of the H3 K4 methyltransferase MLL1 and the 5352–5361. H4 K16 acetyltransferase MOF. Cell 2005; 121: 873–885. 79 Zhang Y, Yan X, Sashida G, Zhao X, Rao Y, Goyama S et al. Stress hematopoiesis 55 Southall SM, Wong PS, Odho Z, Roe SM, Wilson JR. Structural basis for the reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation requirement of additional factors for MLL1 SET domain activity and recognition of in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells. epigenetic marks. Mol Cell 2009; 33: 181–191. Blood 2012; 120: 1118–1129. 56 Sierra J, Yoshida T, Joazeiro CA, Jones KA. The APC tumor suppressor counteracts 80 Kitabayashi I, Aikawa Y, Nguyen LA, Yokoyama A, Ohki M. Activation of beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev AML1-mediated transcription by MOZ and inhibition by the MOZ-CBP fusion 2006; 20: 586–600. protein. EMBO J 2001; 20: 7184–7196. 57 Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I et al. 81 Kitabayashi I, Yokoyama A, Shimizu K, Ohki M. Interaction and functional Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell complex with menin to regulate Hox gene expression. Mol Cell Biol 2004; 24: differentiation. EMBO J 1998; 17: 2994–3004. 5639–5649. 82 Taniuchi I, Littman DR. Epigenetic gene silencing by Runx proteins. Oncogene SPOTLIGHT 58 Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD et al. MLL targets SET 2004; 23: 4341–4345. domain methyltransferase activity to Hox gene promoters. Mol Cell 2002; 10: 83 Herglotz J, Kuvardina ON, Kolodziej S, Kumar A, Hussong H, Grez M et al. Histone 1107–1117. arginine methylation keeps RUNX1 target genes in an intermediate state. Onco- 59 Zhang Y, Chen A, Yan XM, Huang G. Disordered epigenetic regulation in MLL- gene 2012; 32:1–11. related leukemia. Int J Hematol 2012; 96: 428–437. 84 Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S et al. 60 Super HJ, McCabe NR, Thirman MJ, Larson RA, Le Beau MM, Pedersen-Bjergaard J Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho et al. Rearrangements of the MLL gene in therapy-related acute myeloid leukemia . Proc Natl Acad Sci USA 1998; 95: 11590–11595. in patients previously treated with agents targeting DNA-topoisomerase II. Blood 85 Lutterbach B, Westendorf JJ, Linggi B, Isaac S, Seto E, Hiebert SW. A mechanism of 1993; 82: 3705–3711. repression by acute myeloid leukemia-1, the target of multiple chromosomal 61 Domer PH, Head DR, Renganathan N, Raimondi SC, Yang E, Atlas M. Molecular translocations in acute leukemia. J Biol Chem 2000; 275: 651–656. analysis of 13 cases of MLL/11q23 secondary acute leukemia and identification of 86 Chakraborty S, Sinha KK, Senyuk V, Nucifora G. SUV39H1 interacts with AML1 and topoisomerase II consensus-binding sequences near the chromosomal break- abrogates AML1 transactivity. AML1 is methylated in vivo. Oncogene 2003; 22: point of a secondary leukemia with the t(4;11). Leukemia 1995; 9: 1305–1312. 5229–5237. 62 Meyer C, Schneider B, Jakob S, Strehl S, Attarbaschi A, Schnittger S et al. The MLL 87 Gutierrez MI, Siraj AK, Bhargava M, Ozbek U, Banavali S, Chaudhary MA et al. recombinome of acute leukemias. Leukemia 2006; 20: 777–784. Concurrent methylation of multiple genes in childhood ALL: Correlation with 63 Johansson B, Moorman AV, Haas OA, Watmore AE, Cheung KL, Swanton S et al. phenotype and molecular subgroup. Leukemia 2003; 17: 1845–1850. Hematologic malignancies with t(4;11)(q21;q23)—a cytogenetic, morphologic, 88 Maiques-Diaz A, Chou FS, Wunderlich M, Gomez-Lopez G, Jacinto FV, immunophenotypic and clinical study of 183 cases. European 11q23 Workshop Rodriguez-Perales S et al. Chromatin modifications induced by the AML1-ETO participants. Leukemia 1998; 12: 779–787. fusion protein reversibly silence its genomic targets through AML1 and Sp1 64 Nakamura T, Alder H, Gu Y, Prasad R, Canaani O, Kamada N et al. Genes on binding motifs. Leukemia 2012; 26: 1329–1337. chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia 89 Stumpel DJ, Schneider P, Seslija L, Osaki H, Williams O, Pieters R et al. Connectivity share and/or common motifs. Proc Natl Acad Sci USA 1993; mapping identifies HDAC inhibitors for the treatment of t(4;11)-positive infant 90: 4631–4635. acute lymphoblastic leukemia. Leukemia 2012; 26: 682–692.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1793 – 1802 RUNX1 meets MLL CP Koh et al 1802 90 Lasa A, Carnicer MJ, Aventin A, Estivill C, Brunet S, Sierra J et al. MEIS 1 expression 94 Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic is downregulated through promoter hypermethylation in AML1-ETO acute regulators in myeloid malignancies. Nat Rev Cancer 2012; 12: 599–612. myeloid leukemias. Leukemia 2004; 18: 1231–1237. 95 Taniuchi I, Osato M, Ito Y. Runx1: no longer just for leukemia. EMBO J 2012; 31: 91 Huang G, Zhao X, Wang L, Elf S, Xu H, Sashida G et al. The ability of MLL to 4098–4099. bind RUNX1 and methylate H3K4 at PU.1 regulatory regions is impaired by 96 Asou N, Yanagida M, Huang L, Yamamoto M, Shigesada K, Mitsuya H et al. MDS/AML-associated RUNX1/AML1 mutations. Blood 2011; 118: 6544–6552. Concurrent transcriptional deregulation of AML1/RUNX1 and GATA factors by the 92 Huang G, Zhang P, Hirai H, Elf S, Yan X, Chen Z et al. PU.1 is a major downstream AML1-TRPS1 chimeric gene in t(8;21)(q24;q22) acute myeloid leukemia. Blood target of AML1 (RUNX1) in adult mouse hematopoiesis. Nat Genet 2008; 40:51–60. 2007; 109: 4023–4027. 93 Ng CE, Yokomizo T, Yamashita N, Cirovic B, Jin H, Wen Z et al. A Runx1 intronic 97 Huang L, Osato M, Yanagida M, Yamashita N, Ito Y. The myeloid features of BXH2 enhancer marks hemogenic endothelial cells and hematopoietic stem cells. Stem leukemias may result from the lack of one copy of the repetitive sequence in the Cells 2010; 28: 1869–1881. long terminal repeat viral enhancer. Int J Hematol 2007; 85: 170–172. SPOTLIGHT

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