Leukemia (2013) 27, 269–277 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

REVIEW Role and potential for therapeutic targeting of MYB in leukemia

DR Pattabiraman1,3 and TJ Gonda1,2

The Myb was first identified as an that causes leukemia in chickens. Since then, it has been widely associated with different types of cancers and studied in detail in myeloid leukemias. However, despite these studies, its role in the induction, pathogenesis and maintenance of AML, and other blood disorders, is still not well understood. Recent efforts to uncover its plethora of transcriptional targets have provided key insights into understanding its mechanism of action. This review evaluates our current knowledge of the role of Myb in leukemia, with a particular focus on AML, from the vast literature spanning three decades, highlighting key studies that have influenced our understanding. We discuss recent insights into its role in leukemogenesis and how these could be exploited for the therapeutic targeting of Myb, its associated co-regulators or its target , in order to improve outcomes in the treatment of a wide range of hematopoietic malignancies.

Leukemia (2013) 27, 269–277; doi:10.1038/leu.2012.225 Keywords: Myb; targeting; p300

INTRODUCTION Myb protein. The NRD is believed to act at least in part by the The Myb family recruitment of co-, such as TIF1b and Mybbp1a, that 6,17 The Myb protein is part of a family of factors first interfere with target activation. Moreover, this region is identified as the v-Myb transforming expressed by two important for several post-translational modifications of the Myb different avian leukemia viruses, the avian myeloblastosis virus protein including phosphorylation, sumoylation and ubiquitination, which have been shown to negatively regulate and the E26 virus. These viruses transform immature hemato- 18–20 poietic cells in vitro and induce acute leukemias in chickens.1,2 Myb’s activity and stability. The NRD also contains an EVES Following the discovery and molecular cloning of the v- domain, which regulates its transcriptional activity through binding of the p100 co-, or alternatively, the DBD of oncogene, it has been possible to study its normal cellular 21 counterpart, c-myb/MYB. The myb gene family consists of other Myb itself. members, mybl1 (encoding Mybl1 or A-Myb) and (encoding Mybl2 or B-Myb), which have different roles in various tissues. A Myb expression more detailed review of these other family members can be found The highest levels of c-myb mRNA expression are found 3 elsewhere. This review will focus on c-myb/MYB. predominantly in immature hematopoietic cells as demonstrated by examination of its expression among different tissues and cells 22 The Myb protein of all hematopoietic lineages. It has also been shown that levels of c-myb mRNA decrease as the cells progress towards terminal The 75 kDa c-Myb protein is a DNA-binding 22,23 that recognizes the consensus sequence PyAACG/TG.4,5 The Myb differentiation consistent with a role in cellular proliferation protein has three distinct functional domains (Figure 1), an and/or differentiation. Expression of c-myb is not solely restricted N-terminal DNA-binding domain (DBD), a centrally located to the hematopoietic lineages, as it has been detected in colonic (TAD) and a C-terminal negative regulatory epithelial cells and at high levels in malignant colonic and breast epithelia24,25 as well as in several other normal and cancer cell domain (NRD). As a transcriptional regulator, Myb interacts with 26 various co-regulators to carry out its functions. The DBD of Myb is types. known to interact with several co-repressors including N-CoR and c-Ski,6 as well as co-activators such as Mi2a and FLASH,7,8 that Myb in normal hematopoiesis have been shown to influence Myb’s transcriptional activity. The The c-myb proto-oncogene and its product are key regulators of TAD, similar to some other transactivation domains, contains hematopoietic cell proliferation and differentiation. Initial experi- clusters of acidic residues and has been shown to interact with the ments exposing normal human bone marrow mononuclear cells co-activator CBP/p300,9,10 the TIP60 co-repressor11 and Menin,12 to MYB antisense oligodeoxynucleotides showed that inhibition of all of which have an effect on its leukemogenic activity (discussed Myb expression resulted in a decrease in both colony size and further below). The negative regulatory function of the carboxy- number.27 Its importance in hematopoietic development in vivo terminal region of Myb was first demonstrated through studies, was first shown in c-myb null mice, which die by embryonic day which showed that its deletion led to increases in the DNA 15, at which time they display a failure of (definitive) fetal liver binding,13 transactivation14 and transforming abilities15,16 of the hematopoiesis resulting in severe anemia. Similarly, all other

1University of Queensland Diamantina Institute, University of Queensland, Brisbane, Queensland, Australia and 2School of Pharmacy, University of Queensland, Brisbane, Queensland, Australia. Correspondence: Professor TJ Gonda, School of Pharmacy, University of Queensland, Pharmacy Australia Centre of Excellence, 20 Cornwall Street, Woolloongabba, Brisbane, Queensland 4102, Australia. E-mail: [email protected] 3Current address: Whitehead Institute for Biomedical Research, Cambridge, MA, USA Received 10 May 2012; revised 23 July 2012; accepted 31 July 2012; accepted article preview online 9 August 2012; advance online publication, 28 August 2012 MYB in leukemia DR Pattabiraman and TJ Gonda 270 for the notion of therapeutically inhibiting Myb activity as it suggests that substantial functional inhibition could be tolerated by patients; this will be discussed further below.

MYB AND LEUKEMIA MYB as a driver of leukemogenesis Studies in animal systems. As mentioned above, myb was first identified as an oncogene in retroviruses that induced leukemias. Avian myeloblastosis virus was shown to cause myeloblastic leukemia in chickens and transforms only cells of the myelomo- nocytic lineage in vitro, whereas E26 induces a mixed erythro- blastic and myeloblastic leukemia in chickens and quails and transforms immature cells of both the erythroid and myelomo- nocytic lineages in vitro.42,43 Figure 1. Schematic showing the three main functional domains of Studies in animal and cell line model systems provided further c-Myb: DBD, TAD and NRD, and the major known regulators of evidence for Myb’s leukemogenic potential. Several studies c-Myb activity and function (green: co-activators, blue: co-repres- showed that ectopic expression of Myb contributed to the sors, orange: post-translational modifications). Mybbp1a can func- transformed state by imposing a block of differentiation in tion as either a co-activator or a co-. leukemic cell lines representing several lineages.44,45 Suppression of differentiation and continued proliferation of myeloid cells by myb could also be recapitulated by ectopic expression in primary hematopoietic lineages, except the megakaryocyte lineage, were 15 significantly suppressed.28 murine cells. Moreover, the c-myb locus has been a site of recurrent retroviral insertional mutagenesis in several murine More recently, the derivation of mice bearing conditionally- 46–49 deleted (floxed) alleles of c-myb has enabled a finer analysis of its hematopoietic malignancies. Taken together, these role in hematopoietic differentiation. Such studies showed that observations underscore the potential of MYB activation to c-myb is required at different stages during thymocyte develop- contribute to leukemia in humans. ment29,30 as well as myelopoiesis,31 erythropoiesis,32 B-cell Myb’s differentiation-blocking activity was shown to be exerted development33 and maintenance of the HSC pool.34 Other by its DBD and TAD, which together were sufficient to induce a block of differentiation, with its C-terminal domain (which is studies have addressed this issue by introducing a c-myb 13,15,50,51 transgene expressed under the control of a tetracycline- truncated in v-Myb) acting as a negative regulator. Hence, regulated into a c-myb–/– embryonic stem cell line. through mechanisms including those described above, the NRD renders the c-Myb protein weaker than its retroviral and truncated This enabled controlled induction of Myb expression at various 13,15,51 stages of hematopoietic development, and showed again that counterparts in transforming hematopoietic cells, possibly appropriate levels of Myb expression are required at distinct explaining its insufficiency as a sole driver of leukemogenesis. differentiation steps of each haemopoietic cell lineage.35 Myb However, upon co-expression with other constitutive signaling molecules, it has been shown to cooperate and contribute to levels have also been manipulated in vivo through the use of a 52,53 fortuitous ‘knock-down’ allele of murine c-myb.36 These studies myeloid transformation. Indeed in the avian system at least, leukemogenesis requires activation of autocrine growth factor revealed differential effects of Myb reduction on the various 54 hematopoietic sub-lineages and importantly, showed that even production. very substantial reductions in Myb expression were compatible with adult viability (albeit at a reduced level) and thus are not Direct involvement in human leukemia. Given the compelling equivalent to total loss of c-myb. evidence for a widespread causative role in leukemias of both Other studies using the potent germline mutagen N-ethyl-N- birds and mammals, its role in human leukemogenesis has, nitrosourea (ENU) in mice have generated mice bearing point unsurprisingly, been of substantial interest. Here we will briefly mutations in c-myb. One such screen identified strains with point discuss to some of the key findings which are summarized in mutations in the DBD and NRD domains that exhibited a Table 1. myeloproliferative syndrome and supraphysiological expansion MYB mRNA is expressed at high levels in most human myeloid of megakaryocyte and platelet production in the absence of and acute lymphoid leukemias.22 Although there are earlier thrombopoietin signaling.37 Two further ENU mutagenesis studies reports of abnormalities in the MYB locus (6q22–24) in myeloid reported mouse strains harboring point mutations in the TAD of and lymphoid leukemias as well as lymphomas,55–57 such the protein.38,39 The biological consequences of these mutations alterations appeared to be rare and thus were only suggestive included thrombocytosis, megakaryocytosis, anaemia, lympho- of an important role of MYB in human leukemogenesis. This rarity penia and the absence of eosinophils, as well as a substantial may have been due, in part, to a lack of appropriately sensitive increase in the number of HSCs. Both of these Myb TAD mutants technologies to recurrently detect these or more subtle alterations were defective in their ability to interact with p300. Interestingly, (see below). The advent of more sophisticated technologies such other mouse studies describing point mutations in the KIX domain as high-resolution genome-wide array CGH enabled more detailed of p300, which directly interacts with Myb, also show and sensitive analyses of genomic alterations in human leukemias. hematopoietic abnormalities in multiple lineages,40,41 including Two groups in 2007 reported the occurrence of recurrent some that closely resemble the abnormalities seen in the c-myb chromosomal translocations and duplications in the MYB locus ENU mutants. Thus, the above-mentioned studies have generated of patients with T-cell acute lymphocytic leukemias.58,59 These hypomorphic alleles of c-myb that led to significant abnormalities translocations juxtapose the T-cell and MYB loci, defining in cells of several hematopoietic lineages. a new class of T-cell acute lymphocytic leukemias that expresses In summary, it is clear that c-myb is required for the generation higher levels of MYB, presumably due to an present in and normal function of all hematopoietic lineages. In contrast to the T-cell receptor locus. The recurrent duplications appeared to total absence, reduced Myb levels can be tolerated, as can forms be mediated by Alu elements that flank the MYB locus. These of Myb with point mutations that affect its activity. This bodes well duplications were also shown to occur at low frequency during

Leukemia (2013) 269 – 277 & 2013 Macmillan Publishers Limited MYB in leukemia DR Pattabiraman and TJ Gonda 271 Table 1. Abnormalities and associations of MYB with human leukemia

Year Abnormality Disease sub-type Reference

1983 MYB overexpression Childhood myeloid leukemias 115 1984 MYB overexpression; amplification in locus ML cell lines, AML 56 1985 MYB overexpression AML and ALL 22, 116 1988 MYB overexpression T-cell leukemia cell lines—PEER and MOLT-4 117 1990 MYB overexpression T-ALL-derived cell lines 118 1990 MYB overexpression Non-Hodgkin’s lymphoma 119 1994 MYB promoter rearrangement T-ALL cell line CCRF-CEM 120 1998 MYB truncation TK-6 cell line derived from CML patient with T-cell blast crisis 57 2005 Recurrent translocation involving MYB Pediatric T-ALL 121 2007 Recurrent genomic duplication of MYB locus T-ALL 58, 59 2007 Recurrent translocation involving MYB T-ALL 58 2009 Double minute containing MYB T-cell leukemia 122 2009 Genomic gain in MYB locus AML harboring MYST3-translocations 61 2011 Recurrent translocation involving MYB Infant acute basophilic leukemia 63 2011 Recurrent translocation involving MYB AML-M5 62 Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; T-ALL, T-cell acute lymphocytic leukemias. The table lists studies that have reported abnormalities in the MYB locus across hematopoietic cell lines and primary hematological malignancies. normal thymocyte development and to be clonally selected CBFb–SMMHC and MLL-fusions—make up the majority.65 Some during the molecular pathogenesis of human T-cell acute of these translocations affect transcription factors that are critical lymphocytic leukemias.60 Subsequently, recurrent copy number for normal myeloid differentiation such as the core-binding factors alterations were also reported in the MYB locus in specific subsets (particularly Runx1/AML1 and CBFb) and RARa. On the other hand, of AML patients that harbor a chromosomal translocation MLL-fusions such as MLL–ENL and MLL–AF9 act through t(8;16)(p11;p13) involving MYST3 and CREBBP.61 More recent epigenetic mechanisms, utilizing cofactors such as the histone studies have reported the occurrence of MYB-GATA1 methyltransferase DOT1L, to induce constitutive activation of its translocations in infant acute basophilic leukemias and in one targets HOXA9 and MEIS1.67 case of acute monocytic leukemia.62,63 These genomic analyses In any case, early studies from the Gewirtz68 laboratory using reveal that recurrent abnormalities in MYB do occur in leukemia, antisense oligodeoxynucleotides showed the sensitivity of but at fairly low frequencies, particularly in myeloid leukemia. leukemia cell lines as well as primary human AML and CML While these observations and studies in animal systems clearly blasts to MYB inhibition, implying that it was necessary for show MYB’s leukemogenic potential, they leave open the question maintenance, even if it were not sufficient for induction, of of the role that MYB has in the development of majority of leukemia. Significantly, these studies also suggested that myeloid leukemias where it (apparently) remains unaltered. leukemias may be more sensitive to MYB inhibition than normal hematopoietic cells.68,69 Confirmation of these observations, and insight into the molecular mechanisms behind them, have come The emerging role of Myb as an essential cofactor in the induction only quite recently with the development of more powerful and maintenance of myeloid leukemia experimental systems and technologies. Much of what follows here will focus on myeloid leukemia, as Studies using an inducible MLL–ENL oncogene reported Myb to historically a surprisingly large proportion of studies on the role of be an essential downstream target of MLL fusion- and HoxA9/ MYB in leukemia have dealt with this subtype, despite evidence, Meis1-induced leukemic transformation,70 a finding confirmed summarized above that MYB is also causatively involved in and expanded upon by other studies.71–73 Correspondingly, lymphoid leukemia. Perhaps this is a reflection of the fact that the transformation by MLL–ENL through HoxA9/Meis1 was inhibited avian viruses carrying v-myb transform myeloid cells, by relatively modest (65%) short-hairpin RNA-mediated knock- and the discovery of frequent retroviral insertions in mouse down of c-myb. This was one of the few studies to link Myb, an myeloid leukemias. Myeloid transformation is also probably easier otherwise ‘orphaned’transcription factor, to a pathway utilized to study in vitro. during leukemic transformation. That Myb was shown to be Before discussing the ‘cofactor’ role of MYB in AML in detail, it is downstream of causative genetic events (MLL–ENL in this case) worth briefly summarizing the key features and known genetic and not the driving factor itself represented an important step in drivers of this disease. Many excellent publications including those the understanding of its role in leukemogenesis. Further studies of Dash and Gilliland64 and Kelly and Gilliland65 provide com- on MLL fusion-induced leukemogenesis showed that Myb was prehensive reviews of this disease. Leukemia is characterized by required for the maintenance of the leukemic stem cell transcrip- clonal expansion of aberrant stem/progenitor cells that show tional signature, and that Myb knock-down led to a decrease in increased proliferation, self-renewal and a block of differentiation, leukemic stem cell frequencies.72 Importantly, the ability of due to the accumulation of mutations in oncogenes and tumor MLL–ENL to induce leukemias in mice was severely impaired suppressors.64,65 The two events required for the onset of when the levels of Myb were knocked down even to half of the leukemogenesis can be broadly generalized as: firstly, acquisi- normal levels. tion of an abnormality in differentiation commitment leading to a Other recent studies on c-myb in a mouse AML model found higher number of progenitor cells, that is, a block of differentiation that Myb was downregulated upon short-hairpin RNA-mediated and secondly, acquisition of the ability to proliferate indepen- knockdown of MLL–AF9, and that the Myb promoter was occupied dently of or with reduced requirement for extrinsic growth by the fusion protein.73 Importantly, Myb was shown to be factors.66 Chromosomal translocations are the leading known required for the maintenance of MLL–AF9 leukemias, and to drivers of aberrant differentiation in the pathogenesis of AML. regulate a self-renewal program that is essential for leukemogen- Several hundred of these have been reported; however, a limited esis but more dispensable for normal hematopoiesis. Furthermore, number of recurring translocations—PML–RARa, AML1–ETO, Myb knockdown led to complete remission in the MLL–AF9 mice,

& 2013 Macmillan Publishers Limited Leukemia (2013) 269 – 277 MYB in leukemia DR Pattabiraman and TJ Gonda 272 which was durable in most cases, in contrast to conventional identified in their study were in fact repressed by Myb. chemotherapy (Ara-C þ Doxorubicin). Importantly, several genes that have key roles in myeloid An additional role of MYB in MLL fusion-induced leukemias was differentiation such as , sfpi1 and , amongst others, revealed when the Myb protein was found to interact through its were repressed by Myb. Moreover, interaction with CBP/p300 was TAD with Menin to form a complex with MLL itself and with MLL also shown to be required for the ability of Myb to repress some fusions.12 This interaction of MLL with Myb was shown to be key targets. Given that this interaction is essential for Myb’s essential for the histone methyltransferase activity of MLL, which transforming activity,81 these findings implied a novel role in gene promotes global H3K4 methylation and is likely to be important repression (by mechanisms that are not yet clear) for this for MLL-mediated transformation. Finally, it appears from ubiquitous co-activator in leukemic transformation. structural studies with recombinant proteins that both Myb and The study of Zuber et al.73 mentioned above also examined MLL can interact simultaneously with the CBP (and genes that were differentially expressed upon Myb knockdown in presumably also p300) through the KIX domain of the latter.74–76 MLL–AF9–induced leukemic cells. These authors found that As we discuss below, p300 is an important coactivator of Myb and 440% of Myb-regulated genes that were involved in the is essential for Myb’s role in leukemic transformation. These maintenance of the leukemic program were also MLL–AF9 target findings raise the intriguing possibility that the three proteins genes, including some well-established Myb target genes such as could be involved in a complex that enables Myb target gene and bcl-2 that could contribute to leukemic transformation. regulation and MLL-mediated histone modification, which, in This is entirely consistent with the authors’ own work,80 which combination, promote leukemic transformation. Confirmation will showed an extensive overlap between Myb target genes and the await the demonstration that such a complex does in fact form signature of MLL-fusion induced leukemia-initiating cells defined in vivo with the relevant cellular proteins. Interestingly though, by Somervaille et al.72 Leu628 of the KIX domain of CBP was identified as an essential Hence, the transcriptional program controlled by Myb has an residue for interaction with the TAD of MLL using NMR important role in the initiation and maintenance of leukemias that spectroscopy.74 The nearby Leu630 residue of the p300 KIX are induced by oncogenic MLL fusion proteins. It appears that domain has been shown to be involved in interaction with Myb in other oncogenes are also able to utilize and possibly manipulate a an ENU-generated mouse pedigree, Plt6,41 consistent with the Myb-regulated transcriptional program as part of their leukemo- notion of an intimate connection in vivo. genic activities. Indeed our study on the role of Myb in AML1–ETO- Taken together, these studies on experimental MLL fusion- induced transformation and leukaemogenesis (DRP and TJG, induced AML were important in establishing the essential roles of unpublished observations) identified a set of Myb target genes MYB in transformation and the maintenance of the leukemic stem that were differentially expressed between AML1–ETO-expressing cell in this form of AML. They further implied that targeting MYB cells in the presence of WT or hypomorphic alleles of c-myb. could be efficacious in at least some forms of the disease. Moreover, some of these genes appeared to be regulated by both The requirement for MYB is not confined to MLL fusion-induced AML1–ETO and Myb. Thus even though MYB is not altered in most leukaemogenesis. Significantly, the study of Hess et al.70 also AML cases, activation and repression of key Myb target genes showed that more extensive suppression of Myb (to o10% of might be critical events required for transformation. In Figure 2, wild-type levels) prevented myeloid transformation by another we illustrate possible relationships between Myb, oncogenic oncogene, E2A–HLF. Other studies have now shown that MYB is fusion proteins and their target genes in myeloid leukemogenesis. required for BCR–ABL-induced myeloid transformation77 and In one scenario, there may be no interaction between Myb and B-cell ALL.78 Moreover, our own studies using mice carrying a the leukemia oncogene, even though genes regulated by each are hypomorphic allele of Myb (Booreana38) have shown that Myb essential for AML induction or maintenance (Figure 2a). Alter- activity is required for transformation and leukaemogenesis by natively, the leukemia oncogene might affect the activity of Myb another human myeloid oncogene, AML1-ETO (DRP and TJG, (Figure 2b); one example would be the case of MLL-fusions unpublished observations; see also below), which accounts for discussed above where these oncogenes appear to promote MYB over 10% of all cases of adult AML.79 From these studies, the expression through Meis1 and HoxA9. Finally, some target genes pioneering studies of Gewirtz and colleagues, as well as many that are critical for leukemogenesis might be co-regulated by Myb other studies with myeloid leukemia cell lines, it is becoming and the AML oncogene (Figure 2c). All of these scenarios, each of increasingly clear that MYB is essential for the initiation and/or which may be relevant for different oncogenes, can explain why maintenance of many/most myeloid leukemias. Myb appears to be indispensable for leukemogenesis as suggested by studies over 20 years ago.68 Although the entire repertoire of Myb-regulated genes that are required for this Myb target genes in leukemogenesis process has not been completely unveiled, our current knowledge To understand the role of Myb in leukemia it is essential to identify provides enough evidence to make MYB an attractive target for and characterize the genes that it regulates. A substantial number therapeutic intervention in myeloid leukemia. of Myb target genes have been identified to date,26 including genes that have diverse functions in processes such as myeloid differentiation, cellular proliferation, cell cycle, apoptosis and cell signalling. Genes that have a major role in oncogenesis such as TARGETING MYB IN HUMAN LEUKEMIAS myc and bcl-2 have been shown to be direct Myb targets.26 Suppression of Myb expression by antisense and RNA interference However, until recently, the established set of Myb-regulated The use of antisense nucleic acids against Myb was pioneered by genes could not account for its ability to impart a block of Gewirtz and colleagues who showed its efficacy in leukemia cell differentiation in hematopoietic cells and contribute to the lines and primary patient samples.68,69 They were also shown to process of leukemogenesis. A recent in-depth ChIP-Seq analysis be effective in vivo in a SCID mouse model of leukemia using the of genome-wide Myb-binding regions, in conjunction with K562 cell line,82 but only showed limited success in a pilot Phase I expression profiling, identified a large number of novel Myb clinical trial on CML patients.83 However, the advent of RNA regulated genes.80 Traditionally, Myb has been recognized as a interference made this the mode of choice for specifically transcriptional activator, however, the identification of several co- knocking-down . Suppression of Myb using repressor molecules that interact through different Myb domains short-hairpin RNA was successful in achieving a complete (see Figure 1) implied that it could act also as a repressor.6 Indeed, remission in mouse models of AML that are triggered by MLL– Zhao et al.80 found that almost half of the Myb-regulated genes AF9, with most animals remaining relapse-free.73 The shRNAs used

Leukemia (2013) 269 – 277 & 2013 Macmillan Publishers Limited MYB in leukemia DR Pattabiraman and TJ Gonda 273

Gene 1 Leukemogenic Leukemogenic Gene 1 Fusion Protein Fusion Protein

Gene 2 Gene 2

Gene 3 Gene 3

AML AML

Gene 4 Gene 4 p300 p300 Gene 5 Gene 5 Myb Myb Gene 6 Gene 6

Gene 1 Leukemogenic Fusion Protein

Gene 2

Gene 3

Gene 4 AML

Gene 5 p300 Gene 6 Myb Gene 7 Figure 2. Models illustrating three possible relationships between, and coordinate regulation of transcriptional programs required for myeloid leukemogenesis by, Myb and leukemia-inducing oncogenic fusion proteins. (a) Myb and the fusion oncoprotein regulate separate sets of genes. (b) Myb expression is regulated by the fusion oncoprotein (red arrow) while in (c), genes required for AML are co-regulated (red arrows) by the two transcription factors. See text for explanation and discussion. enabled knockdown of Myb to almost 10% of the endogenous cells; these have reported involvement of complexes of proteins levels, mimicking a hypomorphic phenotype. Surprisingly, this such as NF-kB (RelB) and c-Jun.85,89 Targeting interactions level of endogenous Myb was shown to be sufficient for between complexes, which act to relieve the elongation block, reconstitution of normal hematopoiesis in transplanted animals; and the intronic region of c-myb, could be a strategy that might however, it was clearly not enough to support leukemic prove efficacious in leukemia as well as other types of cancer. transformation by MLL–AF9. This indicates that there may be a Similarly, targeting those protein complexes themselves may be window that could be exploited therapeutically, whereby suppres- able to suppress MYB expression. Transcription factor–DNA sion of Myb would preferentially target the leukemic cells, as these complexes have been targeted previously by the use of small appear to be more dependent on high MYB expression levels. molecules inhibitors in the case of Hif-1,90 while a modified However, while the use of RNA interference in clinical therapy is peptide has been used to suppress protein–protein interactions in still under intense study and development, it has been hindered the case of Notch.91 to date by problems with delivery and toxicity.84 Another attractive strategy would be to use small molecules to inhibit the activity of components of the transcriptional elongation machinery. In particular, Flavopiridol inhibits the Targeting transcriptional elongation regulation of Myb activity of the Cdk9 subunit of P-TEFb (positive transcription Productive transcription of the MYB gene is predominantly elongation factor b), which we have recently shown to be critical regulated by elongation control.85 The first intron of MYB for -mediated promotion of MYB transcriptional contains two adjacent motifs that appear to be responsible for a elongation,92 and may be similarly required for MYB expression in blockade of transcription through this region. The first of these is a leukemia. Alternatively, it is interesting to speculate that the poly (dT) tract of 19–23 residues that is preceded by a second protein Brd4, which is known to recruit P-TEFb,93 sequence that potentially encodes a transcript capable of forming may also be involved in controlling MYB transcriptional an RNA stem loop sequence.86,87 It has been proposed that this elongation. Zuber et al.73 have reported an extensive overlap leads to RNA polymerase II stalling and the generation of an RNA between genes regulated by Myb and those affected by treatment stem loop that can serve as a docking site for proteins involved in with the small molecule bromodomain inhibitor JQ1,94 suggesting elongation control. In colon cancer, the poly (dT) tract is shortened that the latter may also be effective in suppressing MYB and/or the sequence that encodes the stem loop is mutated, transcription. allowing an increase in transcriptional read-through.86 In breast cancer cells, the estrogen receptor, when bound to estradiol, interacts with the gene to relieve the elongation arrest.88 Several Targeting Myb’s interaction with p300 and other partner proteins studies have looked into the mechanism of the elongation block CBP/p300 was the first co-activator identified to interact with Myb, in hematopoietic cells and how it is overcome in MYB-expressing and influence its transcriptional activity.9,10 Initial studies showed

& 2013 Macmillan Publishers Limited Leukemia (2013) 269 – 277 MYB in leukemia DR Pattabiraman and TJ Gonda 274 that mice harboring ‘knocked-in’ mutations in the KIX domain of Targeting downstream effectors of MYB p300, but not that of CBP, exhibited several hematopoietic Another approach for exploiting the dependency of leukemia on 40 abnormalities. These studies implicated the Myb-p300 MYB would be to identify and target essential downstream interaction as being critical for hematopoietic development. effectors of Myb, which may include some of those discussed Further studies using ENU mutagenesis have supported this above. However the main advantage of such an approach would 39,41 observation, with the latter showing that interaction of p300 is lie in the identification of critical Myb targets for which inhibitors required for the hematopoietic stem cell maintenance function of already exist or which are more readily ‘druggable’ than Myb itself. Myb. We have also shown that this interaction is essential for the In fact our studies and those of others have already made progress ability of Myb to impose a block of differentiation in primary in this direction.73,80 For example, as mentioned above, there are 81 hematopoietic cells. A more recent study isolated another ENU already small molecule inhibitors of the well-documented Myb pedigree, Booreana, which also harbors a point mutation in Myb target, Bcl-2. Several studies have reported Myc to be a target of 38 (E308G) that disrupts its interaction with p300, and which Myb in myeloid cell lines, the activity of the former being essential exhibited similar hematopoietic abnormalities to those reported for that of the latter.106,107 Targeting Myc—another transcription 39 previously. However, despite no detectable interaction between factor—could pose similar or greater challenges than targeting Myb and KIX, such mice are able to survive for up to one year, Myb; however, recent studies have shown promise in targeting indicating that hematopoietic development could occur despite the regulator Brd4, which is required to sustain the major disruption to the interaction between these two proteins. ability of Myc to impose aberrant self-renewal.108 As more Myb Hematopoietic cells from these mice are unable to be transformed targets that are essential for its role in leukemogenesis are in vitro by MLL-fusions or AML1–ETO (DRP and TJG, unpublished). identified, additional avenues for its therapeutic targeting in We have also observed that these mice, unlike wild-type mice, are specific sub-types of AML will emerge. resistant to developing AML when transplanted with hemato- poietic stem/progenitor cells expressing AML1–ETO. These obser- vations imply that critical components of the Myb-dependent Other approaches to targeting MYB transcriptional program that are essential for leukemic transforma- High-throughput screens using libraries of small-molecules have tion (as discussed above) specifically require the interaction of routinely been used for the discovery of novel inhibitors, Myb with p300. Hence, targeting this specific protein–protein especially those targeting enzymes such as kinases. Such screens interaction could have therapeutic benefit in multiple subtypes could be undertaken to screen for small molecules that could of AML. inhibit the sequence-specific binding of Myb to the promoters of The interacting region between the Myb TAD and the KIX target genes. A similar approach has previously been successful domain of CBP/p300 has been studied in depth by NMR, providing for the identification of Echinomycin, an inhibitor of HIF-1 DNA- 90 detailed information about essential residues and binding binding activity. As mentioned above, the increased sensitivity surfaces.95 Based on this, the development of small molecule of leukemic cells to reduced levels of Myb activity could be inhibitors or peptides could be designed, first for proof-of- exploited to define a targetable therapeutic window. The success principle studies, and eventually for development for use in the of such screening strategies could greatly depend upon the assays clinic. However, protein–protein interactions have generally been used as a readout. A recent study generated a fluorescence-based regarded as not constituting desirable drug targets, unlike screening system using a cell line containing a reporter with the tyrosine kinases whose enzymatic activity is readily amenable to coding region of eGFP placed downstream of the Myb-responsive 109 inhibition by small molecules. Nevertheless, overcoming a mim-1 promoter. Using this strategy, the authors were able to complex disease like cancer by relying on targeting a single class identify mexicanin-I, a sesquiterpene lactone, as an effective of molecules is unlikely to be successful in many cases. For suppressor of Myb target genes, which consequently resulted in example, inhibitors of FLT3, which is activated in B25% of all inhibition of proliferation of leukemic cells. AMLs, have met with limited clinical success as single agents.96 Other strategies could include inhibition of upstream regulators Moreover, the notion of targeting protein–protein interactions is of Myb, even though relatively few of these have been now being considered seriously based on recent technical conclusively identified to date. For example, previous work has advances and discoveries.97 Several inhibitors of protein–protein shown that Myb activity can be enhanced by Pim-1 mediated 110 interactions like ABT-263, a small molecule that mimics interaction phosphorylation. Although more follow-up studies are required between the pro-survival proteins Bcl-2/Bcl-XL and BH3-only to understand the role of Pim-1 in Myb’s transforming ability, family of proteins98,99 and Nutlin family of small molecule inhibition of the former’s kinase activity might present an effective inhibitors of the -MDM2 interaction,100 are currently in way to combat the Myb-induced transcriptional program that has clinical trials for various cancers.101,102 Other strategies for an essential role in leukemogenesis. Moreover, Pim-1 kinase targeting protein–protein interactions are also being developed; inhibitors have recently been shown to be effective in various cell 111 for example, ‘stapled’ peptides have been successfully used for lines and primary leukemias. In similar vein, strategies that blocking the interactions between Bcl-2 family members103 and target regulators of Myb protein stability could be developed. Like for components of the Notch transcription factor complex91 in many proteins Myb is degraded by the ubiquitin/ leukemia cells. These examples show that our view of what can be system; one of the proteins that target Myb for ubiquitylation is 112 therapeutically targeted is changing and that targeting of the Fbw7, the binding of which appears to be regulated by Myb interaction of Myb with p300 may well be feasible. It should also phosphorylation. This in turn raises the prospect of modulating be noted that Myb interacts with many other proteins6–8,17,104 and the kinase pathways that regulate Myb phosphorylation as a that targeting these interactions may also be therapeutically therapeutic strategy. Indeed, it has been reported that, somewhat useful. For example Myb is known to interact with Menin, which in counter-intuitively, small-molecule inhibition of GSK3b promotes 113 turn recruits MLL.12 This interaction appears to be important for Myb degradation, although some apparently contradictory 114 leukemic transformation by MLL–ENL,12 and thus it is worth findings have also been reported. noting that recently small molecules that block the Menin–MLL interaction have been reported.105 If development of molecules that disrupt interactions between Myb and its partner proteins CONCLUSIONS proves successful, such molecules should be tested both singly While the important roles of MYB in hematopoiesis and its and in combination with conventional chemotherapeutics or multiple links to leukaemogenesis have been known for some molecularly targeted drugs. time, it has only recently started to become clear that many

Leukemia (2013) 269 – 277 & 2013 Macmillan Publishers Limited MYB in leukemia DR Pattabiraman and TJ Gonda 275 leukemias seem to be particularly dependent on this gene. This 17 Tavner FJ, Simpson R, Tashiro S, Favier D, Jenkins NA, Gilbert DJ et al. Molecular immediately highlights its value as a potential target, and several cloning reveals that the p160 Myb-binding protein is a novel, predominantly studies now suggest there may be a significant therapeutic nucleolar protein which may play a role in transactivation by Myb. Mol Cell Biol window for agents that block MYB and/or its leukemia-promoting 1998; 18: 989–1002. functions. Moreover, while transcription factors have traditionally 18 Aziz N, Miglarese MR, Hendrickson RC, Shabanowitz J, Sturgill TW, Hunt DF et al. been considered difficult or impossible to target in a therapeutic Modulation of c-Myb-induced transcription activation by a phosphorylation site near the negative regulatory domain. Proc Natl Acad Sci USA 1995; 92: 6429–6433. context, this conceptual barrier is gradually being eroded. New 19 Bies J, Wolff L. Oncogenic activation of c-Myb by carboxyl-terminal truncation insights into MYB itself, as well as advances in technology and leads to decreased proteolysis by the ubiquitin-26S proteasome pathway. emerging examples of successful targeting of transcription factors Oncogene 1997; 14: 203–212. with peptides or small molecules, all augur well for the 20 Saether T, Pattabiraman DR, Alm-Kristiansen AH, Vogt-Kielland LT, Gonda TJ, development of therapeutics that suppress MYB activity. It is to Gabrielsen OS. A functional SUMO-interacting motif in the transactivation be hoped that one or more of these approaches bears fruit and domain of c-Myb regulates its myeloid transforming ability. Oncogene 2011; 30: eventually helps to reduce the morbidity and mortality due to 212–222. leukemia. 21 Dash AB, Orrico FC, Ness SA. The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb. Genes Dev 1996; 10: 1858–1869. 22 Westin EH, Gallo RC, Arya SK, Eva A, Souza LM, Baluda MA et al. Differential expression of the amv gene in human hematopoietic cells. Proc Natl Acad Sci CONFLICT OF INTEREST USA 1982; 79: 2194–2198. The authors declare no conflict of interest. 23 Gonda TJ, Metcalf D. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia. Nature 1984; 310: 249–251. 24 Guerin M, Sheng ZM, Andrieu N, Riou G. Strong association between c-myb ACKNOWLEDGEMENTS and oestrogen-receptor expression in human breast cancer. Oncogene 1990; 5: 131–135. DRP was supported by a PhD Scholarship from the Leukaemia Foundation (Australia). 25 Ramsay RG, Thompson MA, Hayman JA, Reid G, Gonda TJ, Whitehead RH. Work in the Gonda laboratory is and/or has been supported by funding from the Myb expression is higher in malignant human colonic carcinoma and pre- National Health and Medical Research Council (Australia), the Australian Research malignant adenomatous polyps than in normal mucosa. Cell Growth Differ 1992; Council and the Leukaemia Foundation (Australia). 3: 723–730. 26 Ramsay RG, Gonda TJ. MYB function in normal and cancer cells. Nat Rev Cancer 2008; 8: 523–534. REFERENCES 27 Gewirtz AM, Calabretta BA. c-myb antisense oligodeoxynucleotide inhibits 1 Beug H, von Kirchbach A, Doderlein G, Conscience JF, Graf T. Chicken hemato- normal human hematopoiesis in vitro. Science 1988; 242: 1303–1306. poietic cells transformed by seven strains of defective avian leukemia viruses 28 Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA et al. A display three distinct phenotypes of differentiation. Cell 1979; 18: 375–390. functional c-myb gene is required for normal murine fetal hepatic hematopoi- 2 Roussel M, Saule S, Lagrou C, Rommens C, Beug H, Graf T et al. Three new types esis. Cell 1991; 65: 677–689. of viral oncogene of cellular origin specific for haematopoietic cell transforma- 29 Allen 3rd RD , Bender TP, Siu G. c-Myb is essential for early T cell development. tion. Nature 1979; 281: 452–455. Genes Dev 1999; 13: 1073–1078. 3 Oh IH, Reddy EP. The myb gene family in cell growth, differentiation and 30 Pearson R, Weston K. c-Myb regulates the proliferation of immature thymocytes apoptosis. Oncogene 1999; 18: 3017–3033. following beta-selection. Embo J 2000; 19: 6112–6120. 4 Biedenkapp H, Borgmeyer U, Sippel AE, Klempnauer KH. Viral myb oncogene 31 Sumner R, Crawford A, Mucenski M, Frampton J. Initiation of adult myelopoiesis encodes a sequence-specific DNA-binding activity. Nature 1988; 335: 835–837. can occur in the absence of c-Myb whereas subsequent development is strictly 5 Boyle WJ, Lipsick JS, Reddy EP, Baluda MA. Identification of the leukemogenic dependent on the transcription factor. Oncogene 2000; 19: 3335–3342. protein of avian myeloblastosis virus and of its normal cellular homologue. Proc 32 Vegiopoulos A, Garcia P, Emambokus N, Frampton J. Coordination of Natl Acad Sci USA 1983; 80: 2834–2838. erythropoiesis by the transcription factor c-Myb. Blood 2006; 107: 4703–4710. 6 Nomura T, Tanikawa J, Akimaru H, Kanei-Ishii C, Ichikawa-Iwata E, Khan MM et al. 33 Thomas MD, Kremer CS, Ravichandran KS, Rajewsky K, Bender TP. c-Myb is Oncogenic activation of c-Myb correlates with a loss of negative regulation by critical for B cell development and maintenance of follicular B cells. Immunity TIF1beta and Ski. J Biol Chem 2004; 279: 16715–16726. 2005; 23: 275–286. 7 Alm-Kristiansen AH, Saether T, Matre V, Gilfillan S, Dahle O, Gabrielsen OS. FLASH 34 Lieu YK, Reddy EP. Conditional c-myb knockout in adult hematopoietic stem acts as a co-activator of the transcription factor c-Myb and localizes to active cells leads to loss of self-renewal due to impaired proliferation and accelerated RNA polymerase II foci. Oncogene 2008; 27: 4644–4656. differentiation. Proc Natl Acad Sci USA 2009; 106: 21689–21694. 8 Saether T, Berge T, Ledsaak M, Matre V, Alm-Kristiansen AH, Dahle O et al. The 35 Sakamoto H, Dai G, Tsujino K, Hashimoto K, Huang X, Fujimoto T et al. Proper chromatin remodeling factor Mi-2alpha acts as a novel co-activator for human levels of c-Myb are discretely defined at distinct steps of hematopoietic cell c-Myb. J Biol Chem 2007; 282: 13994–14005. development. Blood 2006; 108: 896–903. 9 Dai P, Akimaru H, Tanaka Y, Hou DX, Yasukawa T, Kanei-Ishii C et al. CBP as a 36 Emambokus N, Vegiopoulos A, Harman B, Jenkinson E, Anderson G, Frampton J. transcriptional coactivator of c-Myb. Genes Dev 1996; 10: 528–540. Progression through key stages of haemopoiesis is dependent on distinct 10 Oelgeschlager M, Janknecht R, Krieg J, Schreek S, Luscher B. Interaction of the threshold levels of c-Myb. Embo J 2003; 22: 4478–4488. co-activator CBP with Myb proteins: effects on Myb-specific transactivation and 37 Carpinelli MR, Hilton DJ, Metcalf D, Antonchuk JL, Hyland CD, Mifsud SL et al. on the cooperativity with NF-M. EMBO J 1996; 15: 2771–2780. Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological 11 Zhao H, Jin S, Gewirtz AM. The histone acetyltransferase TIP60 interacts with production of platelets in the absence of thrombopoietin signaling. Proc Natl c-Myb and inactivates its transcriptional activity in human leukemia. J Biol Chem Acad Sci USA 2004; 101: 6553–6558. 2012; 287: 925–934. 38 Papathanasiou P, Tunningley R, Pattabiraman DR, Ye P, Gonda TJ, Whittle B et al. 12 Jin S, Zhao H, Yi Y, Nakata Y, Kalota A, Gewirtz AM. c-Myb binds MLL through A recessive screen for genes regulating hematopoietic stem cells. Blood 2010; menin in human leukemia cells and is an important driver of MLL-associated 116: 5849–5858. leukemogenesis. J Clin Invest 2010; 120: 593–606. 39 Sandberg ML, Sutton SE, Pletcher MT, Wiltshire T, Tarantino LM, Hogenesch JB 13 Hu YL, Ramsay RG, Kanei-Ishii C, Ishii S, Gonda TJ. Transformation by carboxyl- et al. c-Myb and p300 regulate hematopoietic stem cell proliferation and dif- deleted Myb reflects increased transactivating capacity and disruption of a ferentiation. Dev Cell 2005; 8: 153–166. negative regulatory domain. Oncogene 1991; 6: 1549–1553. 40 Kasper LH, Boussouar F, Ney PA, Jackson CW, Rehg J, van Deursen JM et al. A 14 Sakura H, Kanei-Ishii C, Nagase T, Nakagoshi H, Gonda TJ, Ishii S. Delineation of transcription-factor-binding surface of coactivator p300 is required for haema- three functional domains of the transcriptional activator encoded by the c-myb topoiesis. Nature 2002; 419: 738–743. protooncogene. Proc Natl Acad Sci USA 1989; 86: 5758–5762. 41 Kauppi M, Murphy JM, de Graaf CA, Hyland CD, Greig KT, Metcalf D et al. 15 Gonda TJ, Buckmaster C, Ramsay RG. Activation of c-myb by carboxy-terminal Point mutation in the gene encoding p300 suppresses thrombocytopenia in truncation: relationship to transformation of murine haemopoietic cells in vitro. Mpl-/- mice. Blood 2008; 112: 3148–3153. Embo J 1989; 8: 1777–1783. 42 Moscovici C, Samarut J, Gazzolo L, Moscovici MG. Myeloid and erythroid neo- 16 Grasser FA, Graf T, Lipsick JS. Protein truncation is required for the activation of plastic responses to avian defective leukemia viruses in chickens and in quail. the c-myb proto-oncogene. Mol Cell Biol 1991; 11: 3987–3996. Virology 1981; 113: 765–768.

& 2013 Macmillan Publishers Limited Leukemia (2013) 269 – 277 MYB in leukemia DR Pattabiraman and TJ Gonda 276 43 Radke K, Beug H, Kornfeld S, Graf T. Transformation of both erythroid and 69 Calabretta B, Sims RB, Valtieri M, Caracciolo D, Szczylik C, Venturelli D et al. myeloid cells by E26, an avian leukemia virus that contains the myb gene. Cell Normal and leukemic hematopoietic cells manifest differential sensitivity to 1982; 31(Pt 2): 643–653. inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study 44 Clarke MF, Kukowska-Latallo JF, Westin E, Smith M, Prochownik EV. Constitutive relevant to bone marrow purging. Proc Natl Acad Sci USA 1991; 88: 2351–2355. expression of a c-myb cDNA blocks Friend murine erythroleukemia cell 70 Hess JL, Bittner CB, Zeisig DT, Bach C, Fuchs U, Borkhardt A et al. c-Myb is an differentiation. Mol Cell Biol 1988; 8: 884–892. essential downstream target for -mediated transformation of hema- 45 Selvakumaran M, Liebermann DA, Hoffman-Liebermann B. Deregulated c-myb topoietic cells. Blood 2006; 108: 297–304. disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid 71 Huang Y, Sitwala K, Bronstein J, Sanders D, Dandekar M, Collins C et al. Identi- differentiation prior to c-myc: role in leukemogenesis. Mol Cell Biol 1992; 12: fication and characterization of Hoxa9 binding sites in hematopoietic cells. Blood 2493–2500. 2012; 119: 388–398. 46 Mushinski JF, Potter M, Bauer SR, Reddy EP. DNA rearrangement and altered RNA 72 Somervaille TC, Matheny CJ, Spencer GJ, Iwasaki M, Rinn JL, Witten DM et al. expression of the c-myb oncogene in mouse plasmacytoid lymphosarcomas. Hierarchical maintenance of MLL myeloid leukemia stem cells employs a tran- Science 1983; 220: 795–798. scriptional program shared with embryonic rather than adult stem cells. Cell 47 Shen-Ong GL, Morse 3rd HC , Potter M, Mushinski JF. Two modes of c-myb Stem Cell 2009; 4: 129–140. activation in virus-induced mouse myeloid tumors. Mol Cell Biol 1986; 6: 380–392. 73 Zuber J, Rappaport AR, Luo W, Wang E, Chen C, Vaseva AV et al. An integrated 48 Belli B, Wolff L, Nazarov V, Fan H. Proviral activation of the c-myb proto-onco- approach to dissecting oncogene addiction implicates a Myb-coordinated self- gene is detectable in preleukemic mice infected neonatally with Moloney renewal program as essential for leukemia maintenance. Genes Dev 2011; 25: murine leukemia virus but not in resulting end stage T lymphomas. J Virol 1995; 1628–1640. 69: 5138–5141. 74 Arai M, Dyson HJ, Wright PE. Leu628 of the KIX domain of CBP is a key residue for 49 Li J, Shen H, Himmel KL, Dupuy AJ, Largaespada DA, Nakamura T et al. Leu- the interaction with the MLL transactivation domain. FEBS Lett 2010; 584: 4500–4504. kaemia disease genes: large-scale cloning and pathway predictions. Nat Genet 75 De Guzman RN, Goto NK, Dyson HJ, Wright PE. Structural basis for cooperative 1999; 23: 348–353. transcription factor binding to the CBP coactivator. J Mol Biol 2006; 355: 50 Kanei-Ishii C, MacMillan EM, Nomura T, Sarai A, Ramsay RG, Aimoto S et al. 1005–1013. Transactivation and transformation by Myb are negatively regulated by a 76 Goto NK, Zor T, Martinez-Yamout M, Dyson HJ, Wright PE. Cooperativity in leucine-zipper structure. Proc Natl Acad Sci USA 1992; 89: 3088–3092. transcription factor binding to the coactivator CREB-binding protein (CBP). The 51 Ibanez CE, Lipsick JS. Structural and functional domains of the myb oncogene: mixed lineage leukemia protein (MLL) activation domain binds to an allosteric requirements for nuclear transport, myeloid transformation, and colony site on the KIX domain. J Biol Chem 2002; 277: 43168–43174. formation. J Virol 1988; 62: 1981–1988. 77 Lidonnici MR, Corradini F, Waldron T, Bender TP, Calabretta B. Requirement of 52 Ferrao P, Gonda TJ, Ashman LK. Expression of constitutively activated human c-Myb for p210(BCR/ABL)-dependent transformation of hematopoietic pro- c-Kit in Myb transformed early myeloid cells leads to factor independence, genitors and leukemogenesis. Blood 2008; 111: 4771–4779. histiocytic differentiation, and tumorigenicity. Blood 1997; 90: 4539–4552. 78 Waldron T, De Dominici M, Soliera AR, Audia A, Iacobucci I, Lonetti A et al. c-Myb 53 Gonda TJ, Ramsay RG, Johnson GR. Murine myeloid cell lines derived by in vitro and its target Bmi1 are required for p190BCR/ABL leukemogenesis in mouse and infection with recombinant c-myb retroviruses express myb from rearranged human cells. Leukemia 2011; Sep:30. vector proviruses. Embo J 1989; 8: 1767–1775. 79 Nucifora G, Rowley JD. AML1 and the 8;21 and 3;21 translocations in acute and 54 Metz T, Graf T, Leutz A. Activation of cMGF expression is a critical step in avian chronic myeloid leukemia. Blood 1995; 86: 1–14. myeloid leukemogenesis. Embo J 1991; 10: 837–844. 80 Zhao L, Glazov EA, Pattabiraman DR, Al-Owaidi F, Zhang P, Brown MA et al. 55 Barletta C, Pelicci PG, Kenyon LC, Smith SD, Dalla-Favera R. Relationship between Integrated genome-wide chromatin occupancy and expression analyses identify the c-myb locus and the 6q-chromosomal aberration in leukemias and lym- key myeloid pro-differentiation transcription factors repressed by Myb. Nucleic phomas. Science 1987; 235: 1064–1067. Acids Res 2011; 39: 4664–4679. 56 Pelicci PG, Lanfrancone L, Brathwaite MD, Wolman SR, Dalla-Favera R. Amplifi- 81 Pattabiraman DR, Sun J, Dowhan DH, Ishii S, Gonda TJ. Mutations in multiple cation of the c-myb oncogene in a case of human acute myelogenous leukemia. domains of c-Myb disrupt interaction with CBP/p300 and abrogate myeloid Science 1984; 224: 1117–1121. transforming ability. Mol Cancer Res 2009; 7: 1477–1486. 57 Tomita A, Watanabe T, Kosugi H, Ohashi H, Uchida T, Kinoshita T et al. Truncated 82 Ratajczak MZ, Kant JA, Luger SM, Hijiya N, Zhang J, Zon G et al. In vivo treatment c-Myb expression in the human leukemia cell line TK-6. Leukemia 1998; 12: of human leukemia in a scid mouse model with c-myb antisense oligodeox- 1422–1429. ynucleotides. Proc Natl Acad Sci USA 1992; 89: 11823–11827. 58 Clappier E, Cuccuini W, Kalota A, Crinquette A, Cayuela JM, Dik WA et al. The 83 Gewirtz AM, Sokol DL, Ratajczak MZ. Nucleic acid therapeutics: state of the art C-MYB locus is involved in chromosomal translocation and genomic duplications and future prospects. Blood 1998; 92: 712–736. in human T-cell acute leukemia (T-ALL), the translocation defining a new T-ALL 84 Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv subtype in very young children. Blood 2007; 110: 1251–1261. Drug Deliv Rev 2007; 59: 75–86. 59 Lahortiga I, De Keersmaecker K, Van Vlierberghe P, Graux C, Cauwelier B, 85 Bender TP, Thompson CB, Kuehl WM. Differential expression of c-myb mRNA in Lambert F et al. Duplication of the MYB oncogene in T cell acute lymphoblastic murine B lymphomas by a block to transcription elongation. Science 1987; 237: leukemia. Nat Genet 2007; 39: 593–595. 1473–1476. 60 O’Neil J, Tchinda J, Gutierrez A, Moreau L, Maser RS, Wong KK et al. Alu elements 86 Hugo H, Cures A, Suraweera N, Drabsch Y, Purcell D, Mantamadiotis T et al. mediate MYB gene tandem duplication in human T-ALL. J Exp Med 2007; 204: Mutations in the MYB intron I regulatory sequence increase transcription in 3059–3066. colon cancers. Genes Cancer 2006; 45: 1143–1154. 61 Murati A, Gervais C, Carbuccia N, Finetti P, Cervera N, Adelaide J et al. Genome 87 Thompson MA, Flegg R, Westin EH, Ramsay RG. Microsatellite deletions in the profiling of acute myelomonocytic leukemia: alteration of the MYB locus in c-myb transcriptional attenuator region associated with over-expression in colon MYST3-linked cases. Leukemia 2009; 23: 85–94. tumour cell lines. Oncogene 1997; 14: 1715–1723. 62 Belloni E, Shing D, Tapinassi C, Viale A, Mancuso P, Malazzi O et al. In vivo 88 Drabsch Y, Hugo H, Zhang R, Dowhan DH, Miao YR, Gewirtz AM et al. Mechanism expression of an aberrant MYB-GATA1 fusion induces leukemia in the presence of and requirement for estrogen-regulated MYB expression in estrogen-recep- of GATA1 reduced levels. Leukemia 2011; 25: 733–736. tor-positive breast cancer cells. Proc Natl Acad Sci USA 2007; 104: 13762–13767. 63 Quelen C, Lippert E, Struski S, Demur C, Soler G, Prade N et al. Identification of a 89 Suhasini M, Pilz RB. Transcriptional elongation of c-myb is regulated by transforming MYB-GATA1 fusion gene in acute basophilic leukemia: a new entity NF-kappaB (p50/RelB). Oncogene 1999; 18: 7360–7369. in male infants. Blood 2011; 117: 5719–5722. 90 Kong D, Park EJ, Stephen AG, Calvani M, Cardellina JH, Monks A et al. Echino- 64 Dash A, Gilliland DG. Molecular genetics of acute myeloid leukaemia. Best Pract mycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding Res Clin Haematol 2001; 14: 49–64. activity. Cancer Res 2005; 65: 9047–9055. 65 Kelly LM, Gilliland DG. Genetics of myeloid leukemias. Annu Rev Genomics Hum 91 Moellering RE, Cornejo M, Davis TN, Del Bianco C, Aster JC, Blacklow SC et al. Genet 2002; 3: 179–198. Direct inhibition of the NOTCH transcription factor complex. Nature 2009; 462: 66 Gilliland DG. Molecular genetics of human leukemias: new insights into therapy. 182–188. Semin Hematol 2002; 39(Suppl 3): 6–11. 92 Mitra P, Pereira LA, Drabsch Y, Ramsay RG, Gonda TJ. Estrogen receptor-alpha 67 Slany RK. The molecular biology of mixed lineage leukemia. Haematologica 2009; recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene. 94: 984–993. Nucleic Acids Res 2012; 40: 5988–6000. 68 Anfossi G, Gewirtz AM, Calabretta B. An oligomer complementary to c-myb- 93 Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K. The bromodomain encoded mRNA inhibits proliferation of human myeloid leukemia cell lines. Proc protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA Natl Acad Sci USA 1989; 86: 3379–3383. polymerase II-dependent transcription. Mol Cell 2005; 19: 523–534.

Leukemia (2013) 269 – 277 & 2013 Macmillan Publishers Limited MYB in leukemia DR Pattabiraman and TJ Gonda 277 94 Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O et al. Selective 110 Leverson JD, Koskinen PJ, Orrico FC, Rainio EM, Jalkanen KJ, Dash AB et al. inhibition of BET . Nature 2010; 468: 1067–1073. Pim-1 kinase and p100 cooperate to enhance c-Myb activity. Mol Cell 1998; 2: 95 Zor T, De Guzman RN, Dyson HJ, Wright PE. Solution structure of the KIX domain of 417–425. CBP bound to the transactivation domain of c-Myb. JMolBiol2004; 337: 521–534. 111 Chen LS, Redkar S, Taverna P, Cortes JE, Gandhi V. Mechanisms of cytotoxicity 96 Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML: still to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia. Blood 2011; 118: challenging after all these years. Blood 2010; 116: 5089–5102. 693–702. 97 Mullard A. Protein-protein interaction inhibitors get into the groove. Nat Rev 112 Kitagawa K, Hiramatsu Y, Uchida C, Isobe T, Hattori T, Oda T et al. Fbw7 promotes Drug Discov 2012; 11: 173–175. ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated 98 Chipuk JE, Fisher JC, Dillon CP, Kriwacki RW, Kuwana T, Green DR. Mechanism of phosphorylation of Thr-572 in mouse c-Myb. Oncogene 2009; 28: 2393–2405. apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins. Proc Natl 113 Zhou F, Zhang L, van Laar T, van Dam H, Ten Dijke P. GSK3beta inactivation Acad Sci USA 2008; 105: 20327–20332. induces apoptosis of leukemia cells by repressing the function of c-Myb. Mol Biol 99 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. Cell 2011; 22: 3533–3540. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 114 Kitagawa K, Kotake Y, Hiramatsu Y, Liu N, Suzuki S, Nakamura S et al. GSK3 2005; 435: 677–681. regulates the expressions of human and mouse c-Myb via different mechanisms. 100 Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z et al. In vivo Cell Div 2010; 5:27. activation of the p53 pathway by small-molecule antagonists of MDM2. Science 115 Rosson D, Tereba A. Transcription of hematopoietic-associated oncogenes in 2004; 303: 844–848. childhood leukemia. Cancer Res 1983; 43: 3912–3918. 101 Hoffman-LaRoche. A Study of RO5045337 [RG7112] in Patients With Hematologic 116 Ferrari S, Torelli U, Selleri L, Donelli A, Venturelli D, Narni F et al. Study Neoplasms Bethesda, MD: ClinicalTrials.gov, 2011. of the levels of expression of two oncogenes, c-myc and c-myb, in acute 102 Hoffman-LaRoche. A Study of RO5045337 [RG7112] in Patients With Advanced and chronic leukemias of both lymphoid and myeloid lineage. Leuk Res 1985; 9: Solid Tumors Bethesda, MD: ClinicalTrials.gov, 2012. 833–842. 103 Walensky LD, Kung AL, Escher I, Malia TJ, Barbuto S, Wright RD et al. Activation 117 Ohyashiki K, Ohyashiki JH, Kinniburgh AJ, Toyama K, Ito H, Minowada J et al. myb of apoptosis in vivo by a hydrocarbon-stapled BH3 helix. Science 2004; 305: oncogene in human hematopoietic neoplasia with 6q- anomaly. Cancer Genet 1466–1470. Cytogenet 1988; 33: 83–92. 104 Ness SA. Myb binding proteins: regulators and cohorts in transformation. 118 Siegert W, Beutler C, Langmach K, Keitel C, Schmidt CA. Differential expression of Oncogene 1999; 18: 3039–3046. the oncoproteins c-myc and c-myb in human lymphoproliferative disorders. Eur 105 Grembecka J, He S, Shi A, Purohit T, Muntean AG, Sorenson RJ et al. Menin-MLL J Cancer 1990; 26: 733–737. inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia. Nat 119 Okada M, Tada M, Kanda N, Masuda M, Mizoguchi H, Kazuma M et al. c-myb Chem Biol 2012; 8: 277–284. gene analysis in T-cell malignancies with del(6q). Cancer Genet Cytogenet 1990; 106 Kumar A, Lee CM, Reddy EP. c-Myc is essential but not sufficient for c-Myb- 48: 229–236. mediated block of granulocytic differentiation. J Biol Chem 2003; 278: 120 Jacobs SM, Gorse KM, Kennedy SJ, Westin EH. Characterization of a rearrange- 11480–11488. ment in the c-MYB promoter in the acute lymphoblastic leukemia cell line 107 Schmidt M, Nazarov V, Stevens L, Watson R, Wolff L. Regulation of the resident CCRF-CEM. Cancer Genet Cytogenet 1994; 75: 31–39. chromosomal copy of c-myc by c-Myb is involved in myeloid leukemogenesis. 121 Sinclair P, Harrison CJ, Jarosova M, Foroni L. Analysis of balanced rearrangements Mol Cell Biol 2000; 20: 1970–1981. of in acute leukemia: clustered breakpoints in q22-q23 and 108 Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA et al. RNAi screen possible involvement of c-MYB in a new recurrent translocation, t(6;7)(q23;q32 identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 2011; through 36). Haematologica 2005; 90: 602–611. 478: 524–528. 122 Kawamata N, Zhang L, Ogawa S, Nannya Y, Dashti A, Lu D et al. Double minute 109 Bujnicki T, Wilczek C, Schomburg C, Feldmann F, Schlenke P, Muller-Tidow C chromosomes containing MYB gene and NUP214-ABL1 fusion gene in T-cell et al. Inhibition of Myb-dependent gene expression by the sesquiterpene leukemia detected by single nucleotide polymorphism DNA microarray and lactone mexicanin-I. Leukemia 2011; 26: 615–702. fluorescence in situ hybridization. Leuk Res 2009; 33: 569–571.

& 2013 Macmillan Publishers Limited Leukemia (2013) 269 – 277