Alteration of the MYB Locus in MYST3-Linked Cases

Genome proﬁling of acute myelomonocytic leukemia: alteration of the MYB locus in MYST3-linked cases

A Murati1,13, C Gervais2,13, N Carbuccia1,13, P Finetti1, N Cervera1, J Ade´laı¨de1, S Struski2, E Lippert3, F Mugneret4, I Tigaud5, D Penther6, C Bastard6, B Poppe7, F Speleman7, L Baranger8, I Luquet9, P Cornillet-Lefebvre9, N Nadal10, F Nguyen-Khac11, CPe´rot12, S Olschwang1, F Bertucci1, M Chaffanet1, M Lessard2, M-J Mozziconacci1 and D Birnbaum1 on behalf of the Groupe Francophone de Cytoge´ne´tique He´matologique (GFCH)

1De´partement d’Oncologie Molećulaire, Centre de Recherche en Cance´rologie de Marseille, Institut Paoli-Calmettes, Marseille, France; 2Laboratoire d’He´matologie, CHU de Hautepierre, Strasbourg, France; 3Laboratoire d’He´matologie, Hoˆpital Cardiologique-Haut Le´veˆque, Bordeaux, France; 4Laboratoire de Cytogeńe´tique, CHU du Bocage, Dijon, France; 5Laboratoire d’He´matologie, CHU Lyon Sud, Lyon, France; 6De´partement de Geńe´tique, Centre Henri Becquerel, Rouen, France; 7Centre de Geńe´tique Me´dicale, Ghent, Belgique; 8Laboratoire de Geńe´tique, CHU d’Angers, Angers, France; 9Service de Geńe´tique, Hoˆpital Maison Blanche, Reims, France; 10Laboratoire d’He´matologie, Hoˆpital Nord, Saint-Etienne, France; 11Laboratoire de Cytogeńe´tique He´matologique, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France and 12Laboratoire de Cytogeńe´tique Onco-He´matologique, Hoˆpital Saint-Antoine, Paris, France

The t(8;16)(p11;p13) is a rare translocation involved in de novo NCOA3 at 20q12.6 MYST3-linked acute myeloid leukemias and therapy-related myelomonocytic and monocytic acute (AMLs) share specific features, such as frequent extramedullary leukemia. It fuses two genes encoding histone acetyltrans- involvement, disseminated intravascular coagulation, erythro- ferases (HATs), MYST3 located at 8p11 to CREBBP located at 7 16p13. Variant translocations involve other HAT-encoding phagocytosis and a poor prognosis. They are often secondary, 8,9 genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked therapy-related AMLs. Because of its rarity, the molecular acute myeloid leukemias (AMLs) share specific clinical and biology of the MYST3-linked AMLs remains poorly understood. biological features and a poor prognosis. Because of its rarity, A study of three cases using DNA microarrays has shown that the molecular biology of MYST3-linked AMLs remains poorly they have a distinct gene expression profile compared with other understood. We have established the genome and gene 10 expression profiles of a multicentric series of 61 M4/M5 AMLs AMLs. including 18 MYST3-linked AMLs by using array comparative Here, we have established the genome and/or gene expres- genome hybridization (aCGH) (n ¼ 52) and DNA microarrays sion profiles of a series of 61 M4/M5 AMLs including MYST3- (n ¼ 44), respectively. We show that M4/5 AMLs have a variety of linked AMLs and variants by using array comparative genome rare genomic alterations. One alteration, a gain of the MYB hybridization (aCGH) and DNA microarrays, respectively. We locus, was found recurrently and only in the MYST3-linked show that MYST3-linked AMLs have specific gene alterations AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and gene expression profiles within the M4/M5 group. and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor. Leukemia (2009) 23, 85–94; doi:10.1038/leu.2008.257; Patients and methods published online 25 September 2008 Keywords: array-CGH; acute myeloid leukemia; gene expression Patients profiling; MYB; MYST3; t(8;16) We collected a series of 53 bone marrow (BM) and eight peripheral blood samples from 61 patients at the time of diagnosis for primary or secondary AML. The characteristics of the patients and samples are summarized in Supplementary Table 1. According to FAB classification11 and World Health Introduction Organization12 criteria, the panel comprised 16 M4, 44 M5, and one case of AML with no more data available. Some cases have The t(8;16)(p11;p13) is a rare translocation involved in de novo been previously reported in detail, namely M45-1, M45-3, M45- and therapy-related myelomonocytic and monocytic acute 4, M45-5, M45-7, M45-11, M45-13, M45-16, M45-19, M45-20, leukemia (French-American-British classification (FAB) AML- M45-21, M45-23, M45-26,7 M45-052,13 M45-031,14 M45-0326 M4, M5a and M5b). It fuses two genes encoding histone M45-062 and M45-063.4 Three BM samples were used as acetyltransferases (HAT), MYST3 (also called MOZ) located at 1–3 normal controls (normal bone marrow (NBM)). They were 8p11 to CREBBP (also called CBP) located at 16p13. Variant collected from breast cancer patients without micrometastases. translocations involve other HAT-encoding genes, such as 4 5 The aCGH study was carried out on 52 of the 61 samples, EP300 at 22q13, MYST4 at 10q22, NCOA2 at 8q13 or including 18 MYST3-linked AMLs, 16 AMLs with normal karyotype, nine AMLs with 11q23 (mixed lineage leukemia Correspondence: Dr D Birnbaum, Centre de Recherche en Cance´rologie (MLL)) abnormalities, four AMLs with inv(16; CBF-b-MYH11 de Marseille, UMR891 Inserm, 27 Bd. Leı¨ Roure, 13009 Marseille, fusion), one AML with t(8;21) (RUNX1-RUNX1T1 aka AML1- France. ETO fusion) and four AMLs with trisomy 8. E-mail: [email protected] 13These authors contributed equally to this work. A total of 44 M4/M5 cases with good quality RNA were Received 6 May 2008; revised 1 August 2008; accepted 21 August selected for global gene expression profile analysis, including 2008; published online 25 September 2008 eight MYST3-linked AML and 36 other samples comprising 18 MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 86 cases with normal karyotype, six with 11q23 abnormalities, four Before hierarchical clustering, filtered data were log2 trans- with inv(16), two with trisomy 8 and six with various karyotypes. formed and submitted to the cluster program using the median- Of the 61 cases analyzed by aCGH and/or gene expression centered data on genes, Pearson correlation as similarity metric profile, mutations of nucleophosmin 1 (NPM1) were found in 11 and centroid linkage clustering. Results were displayed using cases (mostly in AMLs with normal karyotype, as expected), and TreeView program. To identify and rank genes discriminating an internal tandem duplication and mutation of the FLT3 gene in eight cases of MYST3-linked and 36 M4/M5 AMLs, a supervised four and six cases, respectively (Table 1). analysis was applied to the 26 376 genes/ESTs. A discriminating score (DS) was calculated for each gene. DS ¼ (M1ÀM2)/ (S1ÀS2) where M1 and S1, respectively, represent mean and Nucleic acids extraction s.d. of expression levels of the gene in MYST3-linked subgroup, Bone marrow aspirates or peripheral blood samples were and M2 and S2 in M4/M5 AMLs subgroup. Confidence levels collected from 61 patients at the time of diagnosis. Blasts and were estimated by 100 random permutations of samples. mononuclear cells were purified after density gradient centrifu- Functional processes and pathways were identified by using gation of BM aspiration or whole blood (cases M45-3, M45-4, Ingenuity software (Ingenuity Systems, Redwood City, CA, USA). M45-11, M45-16, M45-010, M45-035, M45-036 and M45- 037), and processed immediately or cryoconserved at À80 1C. High-quality total RNA and DNA was extracted by the Allprep Results DNA/RNA isolation kit (Qiagen, Germany) from blood or BM blast cells. RNA quality and purity were assessed with Agilent aCGH profiling Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Using genome-wide, high-density arrays, we established the NBM samples were processed similarly. aCGH profiles of 52 samples. Examples of profiles are shown in Figure 1 and results are summarized in Table 1. Three main types of profiles were observed. In 60% of the cases (33/52), the Array comparative genomic hybridization profile was said ‘normal-like’ because no alteration was Genomic imbalances were analyzed by aCGH using 244K CGH detected. A second type of profiles showed gains or losses Microarrays (Hu-244A, Agilent Technologies, Massy, France) visible on the karyotype and affecting large regions of the following a previously described protocol.15 Scanning was genome, such as trisomy 8, gains of 11q23-qter (Figure 1a), performed with Agilent Autofocus Dynamic Scanner (G2565BA, deletions of 12p (including the CDKN1B locus; Figure 1b), Agilent Technologies). Data analysis was performed as des- rearrangement of chromosome 13 (Figure 1c), gain of 3q26-qter cribed previously15 and visualized with CGH analytics 3.4 (Figure 1d) or 6q22–25 (case 4). Finally, few profiles showed software (Agilent Technologies). Extraction data (log2 ratio) was rare and limited gains or losses that affected few or single genes, performed from CGH analytics, whereas normalized and filtered such as deletions encompassing CTNNA1 and CXXC5 at 5q31 log2 ratio were obtained from ‘feature extraction’ software (case 011, normal karyotype, Figure 1e), reminiscent of a (Agilent Technologies). Data generated by probes mapped to 5q-syndrome,18 CXXC4 at 4q24 (case 23, MYST3, Figure 1f), X and Y chromosomes were eliminated. The final data set IKZF1/Ikaros at 7p12 (case 055) or a cluster of chemokines at contained 225 388 unique probes covering 22 509 genes and 17q12 (case 010). Among these alterations, a gain of MYB at intergenic part following the hg17 human genome mapping. 6q23 was detected in seven MYST3-linked AML samples Copy number changes were characterized as reported pre- (Figure 2). In six cases, only MYB and its neighbor gene AHI1 viously.15 Results were displayed using TreeView program. were gained, whereas in the seventh case (4) the MYB gain was Identification of copy-number variations was based on a included in a larger gained region also detected by karyotype. previous study.15 The frequency of alterations was computed for each probe locus as the proportion of samples showing an aberration therein. Alteration frequencies were evaluated by Expression profiling Fisher’s exact test and false discovery rate was applied to correct To determine how the presence or absence of genome the hypothesis of multiple testing. alterations influenced the phenotype of AMLs, we profiled 44 M4/M5 samples by using DNA microarrays. An unsupervised analysis was performed on 44 AML and Gene expression profiling three NBM samples. After filtering, 15 618 genes/ESTs were We used Affymetrix U133 Plus 2.0 human oligonucleotide retained. Before hierarchical clustering, data were log2 microarrays. Preparation of cRNA, hybridizations, washes and transformed and submitted to the cluster program using the detection were performed as recommended by the supplier and median-centered data on genes, Pearson correlation as similar- as described previously.16 Data were analyzed by the Robust ity metric and centroid linkage clustering. Results are displayed multichip average method in R using the Bioconductor (http:// in Figure 3 using the TreeView program. Two main clusters of www.bioconductor.org/) and associated package17 as described samples (I and II) were distinguished in the dendrogram. Two previously.16 Before analysis, a first filtering process removed subclusters (a and b) were recognized in branch II. Subcluster IIa the genes, from the data set, with low and poorly measured contained six MYST3-linked AMLs, suggesting that this type of expression as defined by an expression value inferior to 100 U in AML is rather homogeneous. The other cases clustering with all 44 samples retaining 26 376 genes/expressed sequence tags MYST3-linked AMLs in IIa were AMLs with normal karyotype. (ESTs). A second filter, based on the intensity of s.d., was applied MLL-linked AMLs were distributed within different subclusters. to exclude genes showing low expression variation across the Three CBF AMLs were grouped together in cluster I. The three analyses. Standard deviation was calculated on log2 transformed NBM samples clustered together in cluster IIb. data, in which the lowest values were first floored to a minimal On the basis of QT clustering method and visual inspection, value of 100 U, that is, the background intensity, retaining seven clusters of genes (k1–k7) were identified (Figure 3a). Gene 15 618 genes/ESTs with s.d. superior to 0.4. An unsupervised cluster k1, overexpressed in the NBM samples (Figure 3b) and analysis was performed on 44 samples on 15 618 probe sets. in a subcluster of cluster I, contained genes associated with

Leukemia Table 1 Cytogenetical and molecular features of the 61 AML cases

No. Karyotype Fusion transcripts aCGH Mutation

FLT3-ITD FLT3 NPM1

M45-1 46–47,XY,t(7;18)(?;?),+8,t(8;16)(p11;p13),der(9)t(8;9)(?;p2?), MYST3-CREBBP type IV/V 12p12–13 loss, 17p12-ter loss No No No add(11) (p1?4),del(12)(p1?2),der(17)t(7;17)(?;p1?1)[cp29]/46,XY[9] M45-3 46,XX,t(8;16)(p11;p13)[22] MYST3-CREBBP type I 6q23 gain (MYB) No No No M45-4 46,XY,dup(6)(q2?4),t(8;16)(p11;p13)[2]/46,sl, MYST3-CREBBP type I 6q22–25 gain (MYB), 7q11–32 loss, À10, No No No add(21)(p1?)[4]/45,sl,del(9)(q13q31),À18[3]/46,XY[14] M45-5 46,XX,t(8;16)(p11;p13)[4]/47,idem,+8[5]/46,XX[1] MYST3-CREBBP type I 1q32-ter gain, 6q23 gain (MYB), 7q31-ter loss, No No No trisomy 8 M45-7 46,XX,t(8;16)(p11;p13),t(11;19)(p12–13;p13 or MYST3-CREBBP type IV 2p23–25 loss, 3q13–24 loss, 3q26-ter gain, No No No q13)[5]/46,sl,del(3)(q21)[11]/47,sdl1,+i(8)(q10)[2]/46,XX[2] 6q23 gain (MYB), +8q, 10p12-ter loss M45-11 46,XX,t(8;16)(p11;p13)[10] MYST3-CREBBP type I Normal-like No No No M45-13 46,XX,t(8;16)(p11;p13)[19]/46,XX[1] MYST3-CREBBP type I Normal-like No No No M45-16 46,XY,t(8;16)(p11;p13)[25] MYST3-CREBBP type I 6q23 gain (MYB) No No No M45-19 46,XX,t(8;16)(p11;p13)[26] MYST3-CREBBP type I 6q23 gain (MYB) No D835V No M45-20 46,XX,t(8;16)(p11;p13)[10]/46,XX[4] MYST3-CREBBP type I 6q23 gain (MYB) No No No M45-21 46,XY,t(8;16)(p11;p13)[19] MYST3-CREBBP type I Normal-like No No No M45-23 46,XX,t(8;16)(p11;p13)[17]/46,XX[10] MYST3-CREBBP type I Normal-like No No No M45-26 46,XX,t(8;16)(p11;p13),ider(13)(q10)del(13)(q13q22)[8]/46,XX[44] MYST3-CREBBP type IV 4q24 loss (CXXC4), 13q losses No No No M45-031 46,XX,inv(8)(p11q13) [7]/46,XX [16] MYST3-NCOA2 Normal-like ND ND ND M45-052 46,XX,inv(8)(p11q24) [20] MYST3-CREBBP cryptic Normal-like No D835Y No translocation M45-062 ND MYST3-CREBBP Trisomy 8, trisomy 13 M45-032 46,XX,t(8;20)(p11;q13)[18] MYST3-NCOA3 Normal-like Yes No No M45-063 46,XY,t(8;22)(p11;q13)[8]/47,XY,t(8;22)(p11;q13),+der(8)t(8;22) MYST3-EP300 Normal-like Murati A MYB

(p11;q13),del(17)(p11)[3] leukemias myeloid acute MYST3-linked in gain

M45-010 46, XY,del(9)(q22q34),add(11)(q23),del(12)(p11p13) [20] 9q21–31 loss, 11q23-ter gain (MLL), No No No al et 12p11–13 loss, 17q12 gain (CCL1, ACCN1) M45-020 46, X, t(X;10;11)(q13;p12;q23),t(1;5)(p36;q35)[20] Normal-like No No No M45-021 46,XY,t(6;11)(q27;q23)[20] Normal-like No No No M45-022 46,XY,t(9;11)(p22;q23),inv(9)(p12q12)?c [20] Normal-like No No No M45-023 46, XX,t(9;11)(p21-22;q23) [6] Normal-like No No No M45-040 46,XY,t(1;11)(q21;q23)[20] Normal-like No No No M45-047 46,XX,t(9;11)(p22;q23)[20] ND 1q43 gain (EFCAB2) ND ND ND M45-059 50,XX,+8,+8,+14,+19[20] Trisomy 8, trisomy 14, trisomy19 No D835H No M45-060 46, XX, t(9;11)(p22;q23)[14]/46,idem,i(21)(q10)[6] ND Normal-like No No No M45-034 46, XY,inv(16)(p13q22)[20] CBFB-MYH11 Normal-like No No No M45-035 46, XY,inv(16)(p13q22)[20] CBFB-MYH11 Normal-like No No No M45-036 46, XX,inv(16)(p13q22)[17] CBFB-MYH11 Normal-like No No No M45-037 46, XY,inv(16)(p13q22)[20] CBFB-MYH11 Normal-like No No No M45-055 46,XY,t(8;21)(q22;q22)[19] AML1-ETO 7p12 loss (Ikaros) M45-02 46,XX[20] ND ND ND ND M45-03 46,XX[20] Normal-like No No Yes M45-05 46,XX[20] Normal-like Yes No ND M45-06 46,XX[20] ND ND ND ND M45-07 46,XX[20] Normal-like No No Yes M45-08 46,XX[20] Normal-like No No Yes M45-09 46,XX[20] Normal-like No No Yes M45-011 46,XX[20] 5q31 loss (CTNNA1, CXXC5), No No No 20q12 gain (PTPRT) M45-012 46,XX[20] Normal-like No No Yes M45-013 46,XX[20] Normal-like No No No Leukemia 87 MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 88 differentiation, especially erythrocyte differentiation (Figure 3c). Cluster k2 contained genes associated with cell proliferation and mitosis; it was variably distributed throughout the AML samples, suggesting that the proliferation is not the major factor that distinguishes clusters I and II. Cluster k3 contained immediate early genes. Cluster k4 contained genes associated with differentiation of white blood cells. It was rather downregulated in MYST3-linked AMLs. Cluster k7 was overexpressed in MYST3-linked AMLs and downregulated in the NBM samples. FLT3-ITD FLT3 NPM1 Clusters k5 and k6, overexpressed in sample clusters I and IIa, respectively, contained genes associated with multiple functions and various cell processes. Genes from cluster k5 were not expressed in the NBM samples. We applied a ﬁrst supervised analysis of MYST3-linked AMLs vs others. The analysis of DS yielded 1686 probe sets with a

eukemia; ND, not determined. signiﬁcant expression level among the AML subgroups (Supple- mentary Table 2). The chosen DS signiﬁcance threshold produced less than 55 false positives. The 50 most up- and downregulated probe sets are listed in Table 2. As expected, most genes were part of cluster k7 in the hierarchical clustering. Genes overexpressed in MYST3-linked AMLs compared with other M4/M5 AMLs comprised HOXA9, HOXA10, HOXA11, CEBPA, LMO2 and PTPN6. Pathways upregulated in MYST3- linked AMLs compared with other M4/M5 AMLs included several chemokine signaling pathways and T-cell differentiation (Supplementary Table 3). The CD4 gene was the 50th gene of the signature (Supplementary Table 2). Among the downregulated genes were HOXB3, HOXB5, HOXB6, CCND2,

Normal-likeNormal-like17q12 gainNDNormal-likeNormal-likeNormal-likeNormal-likeNormal-likeNormal-like3q26-ter gain, tri8Trisomy 8Trisomy 8NDNDND No No No D835YCREBBP No No Yes Yes No No ND No No, Yes No No NDCSF3R No No ND D835Y No No No Yes , ND No YesCXXC5 ND No No No ND No No, Yes NPM1 D835H No No No No No, NoSTAT3 No No Yes and No No STAT5B. The classification of the 44 AMLs using the 1,686 genes identified as discriminator between the eight MYST3-linked and the 36 other AMLs is shown in Figure 4a. Before any further conclusion was drawn and because the M5 subtype was predominant (7/8 cases) in our MYST3-linked AMLs, we wondered whether the difference between MYST3- linked AMLs and other M4/M5 AMLs could be due to a difference between M5 and M4 phenotypes. Therefore, we did a second supervised analysis based on M4 vs M5 in 18 normal karyotype AMLs. This analysis identified a 99-probe set with a significant differential expression level between M4 and M5. Only one gene was common with the 1686 probe sets found in the first supervised analysis (data not shown), showing that the difference between MYST3-linked AMLs and other M4/M5 AMLs was not due to the difference between M4 and M5 phenotypes. Finally, we wondered whether the gain of the MYB locus had an influence on MYB mRNA level. We compared MYB expression in AMLs with and without MYB locus gain. Expression was higher in cases with MYB locus gain (Figure 4b). It was also higher in MYST3-linked AMLs without gain, suggesting that MYB expression (due to higher copy- 20,+mar[18]/46,XY[4] ND No No No

À number or another mechanism) participates in the leukemogenesis of MYST3-linked AMLs. Our results show that MYST3-linked AML constitute a rather homogeneous subtype of AML distinct from other M4/M5 AMLs characterized by MYB gain and/or overexpression. 9,+mar[11]/46,XX[9] ND No No Yes ) À Discussion

aCGH shows alteration of the MYB locus in Continued

( MYST3-linked leukemia Array comparative genome hybridization proﬁles showed globally few and variable alterations. The large nonequilibrated

Table 1 No.M45-014 KaryotypeM45-015 46,XX[20] M45-016 46,XX[20] M45-017 46,XX[20] M45-018 46,XX[20] M45-039 46,XX[20] M45-041 46,XX[20] M45-046 46,XX[20] M45-045 46,XX[20] M45-058 46,XX[20] M45-01 46,XX[20] M45-04M45-044 47,XY,+8[1]/46,XY[8] M45-019 47,XX,+8[19]/46,XX[2] 47,XY,+8[20] M45-047 47,XX,+21[6]/46,XX[14] M45-050 46,XX,t(9;11)(p22;q23)[20] M45-051 46,XY,inv(3)(q21q26)[20] M45-0045 46,M45-0052 XY,t(9;22)(q34;q11)[6]/47,idem,+21[17]/46, 46, XY[3] XY,?del(20)(q11q13) or 46,XX, Abbreviations: aCGH, array comparative genome hybridization; AML, acute myeloid leukemia; ITD, internalalterations tandem Fusion duplication; transcripts MLL, mixed lineage l M-BCR-ABL aCGHdetected ND by the karyotypic analysis were also Mutation No No No

Leukemia MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 89

Figure 1 Examples of genomic profiles of AML cases. Genomic profiles were established with CGH analytics software (Agilent Technologies) for patients suffering from AML associated with a normal karyotype (NK AML) or MLL or MYST3 rearrangements (MLL-linked or MYST3-linked AML, respectively), (a) The aCGH profile of chromosome 11 in case M45-010 associated with MLL rearrangement shows copy-number gain of the 11q23-qter region. In regions including MLL and 11q24.2-qter, the gain level was sufficiently high (log2 ratio 41) to be considered as a gene or regional amplification, respectively. (b) Comparison of chromosome 12 aCGH profiles of cases M45-010 and M45-1 associated with MLL and MYST3 rearrangements, respectively, shows copy-number losses of the 12p arm spanning a common region. Case M45-10 lost the genomic interval (chr12:9,133,033-23,305,796) with a copy-number transition targeting the A2M gene at the telomeric part. Case M45-1 lost the genomic interval (chr12:10,358,563-32,223,529) with a copy-number transition targeting from centromere to telomere, KLRD1 and BICD1, respectively (hg17 human genome mapping; build 35 from NCBI, May 2004 version). (c) The aCGH profile of chromosome 13 in case M45-26 shows various copy-number aberrations in 13q13-qter region. (d) Comparison of chromosome 3 aCGH profiles of cases M45-01 and M45-7 shows copy-number gains of the 3q26-qter region. Among the cases with limited genomic alterations, case M45-011 profile (e) shows a small loss including CXXC5 gene on 5q31, whereas a small deleted region is characterized at 4q24 spanning the paralogous gene CXXC4 in case M45-26 (f). aCGH, array comparative genome hybridization; AML, acute myeloid leukemia; MLL, mixed lineage leukemia.

detected by aCGH. Many localized alterations were further fused to MLL in AML with t(10;11)(q22;q23).19,20 The MLL detected by aCGH, usually in a non-recurrent manner. protein itself (aka CXXC7) has a CXXC domain.21 The cysteine- However, the presence of paralogous genes (CXXC4 and rich CXXC domain binds Zinc and DNA, and recruits the CXXC5) in small regions of loss could suggest that these genes transcription corepressor C-terminal binding protein 1 (CTBP1). play a role in leukemogenesis. Another paralog, CXXC6,is It is possible that these genes are eliminated because they

Leukemia MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 90

Figure 2 Alteration of the MYB locus detected by array comparative genomic hybridization. For each AML case, the regional genomic proﬁles were established, with CGH analytics software (Agilent Technologies), within the genomic interval (133.6–137.4 Mb) of chromosome 6 (hg17 human genome mapping; build 35 from National Center for Biotechnology Information (NCBI), May 2004 version). Color proﬁles correspond to different cases. Seven cases showed MYB locus (arrow) copy-number gain (six local; one regional, M45-4). (a and b) chromosome views of two cases, one (a) with regional q22–25 gain (M45-4) the other (b) with local q23 gain centered on MYB (M45-19), (c) zoom on the gained MYB region; one case with no gain (M45-032) is shown to visualize the normal baseline. aCGH, array comparative genome hybridization; AML, acute myeloid leukemia.

Figure 3 Global gene expression proﬁling of M4/M5 acute myeloid leukemias. (a) Hierarchical clustering of 47 samples and 15 293 genes/ESTs based on mRNA expression levels. Each row represents a gene and each column represents a sample. The expression level of each gene in a single sample is relative to its median abundance across all the 44 samples and is depicted according to a color scale shown at the bottom. Red and green indicate expression levels above and below the median, respectively. The magnitude of deviation from the median is represented by the color saturation. The dendrogram of samples (above matrix) represents overall similarities in gene expression proﬁles and is zoomed in (b). Colored bars to the right indicate the locations of gene clusters of interest that are zoomed in (c). (b) Dendrogram of samples. Top, dendrogram: two large groups of samples (designated I and II) are distinguished by clustering. Two subclusters (a and b) were recognized in branch II. Down, some relevant features of samples are represented according to a color ladder (unavailable, oblique feature): karyotype of samples (white, normal karyotype; red, MYST3-linked; orange, inv(16); dark blue, MLL-linked; dark green, trisomy 8. In light green are the three normal bone marrow samples), and outcome status (white, patient alive; black, patient dead). (c) Expanded view of selected gene clusters (colors are as in panel a). Some genes included in these clusters are indicated and referenced by their Human Genome Organization (HUGO) abbreviation as used in ‘Entrez Gene’. EST, expressed sequence tag; MLL, mixed lineage leukemia.

Leukemia Table 2 List of top 50 differentially expressed genes

Probe set ID Symbol Description Ref Seq transcript ID Cytoband Status Camos et al.10

203279_at EDEM1 ER degradation enhancer, mannosidase a-like 1 NM_014674 chr3p26 2 Overexpression in MYST3-linked subgroup a 213153_at SETD1B SET domain containing 1B XM_037523 chr12q24 31 Overexpression in MYST3-linked subgroup 213150_at HOXA10 Homeobox A10 NM_018951 chr7p15-p14 Overexpression in MYST3-linked subgroup 206589_at GFI1 Growth factor independent 1 NM_005263 chr1p22 Overexpression in MYST3-linked subgroup 227002_at FAM78A Family with sequence similarity 78, member A NM_033387 chr9q34 Overexpression in MYST3-linked subgroup 213147_at HOXA10 Homeobox A10 NM_018951 chr7p15-p14 Overexpression in MYST3-linked subgroup 219463_at C20orf103 Chromosome 20 open reading frame 103 NM_012261 chr20p12 Overexpression in MYST3-linked subgroup a 222622_at MGC4692 Hypothetical protein MGC4692 FFOverexpression in MYST3-linked subgroup 226691_at KIAA1856 KIAA1856 protein XM_376567 chr7p22 1 Overexpression in MYST3-linked subgroup 206580_s_at EFEMP2 EGF-containing ﬁbulin-like extracellular matrix protein 2 NM_016938 chr11q13 Overexpression in MYST3-linked subgroup 229257_at KIAA1856 KIAA1856 protein XM_376567 chr7p22 1 Overexpression in MYST3-linked subgroup 208072_s_at DGKD Diacylglycerol kinase, d130 kDa NM_003648 chr2q37 1 Overexpression in MYST3-linked subgroup 238107_at EST CDNA FLJ43318 ﬁs, clone NT2RI2018311 FFOverexpression in MYST3-linked subgroup 1558685_a_at LOC158960 Hypothetical protein BC009467 F chrXq28 Overexpression in MYST3-linked subgroup 220097_s_at TMEM104 Transmembrane protein 104 NM_017728 chr17q25 1 Overexpression in MYST3-linked subgroup 209262_s_at NR2F6 Nuclear receptor subfamily 2, group F, member 6 NM_005234 chr19p13 1 Overexpression in MYST3-linked subgroup 226939_at CPEB2 Cytoplasmic polyadenylation element-binding protein 2 NM_182485 chr4p15 33 Overexpression in MYST3-linked subgroup 209229_s_at SAPS1 SAPS domain family, member 1 NM_014931 chr19q13 42 Overexpression in MYST3-linked subgroup 205743_at STAC SH3 and cysteine rich domain NM_003149 chr3p22 3 Overexpression in MYST3-linked subgroup 203546_at IPO13 Importin 13 NM_014652 chr1p34 1 Overexpression in MYST3-linked subgroup 206687_s_at PTPN6 Protein tyrosine phosphatase, nonreceptor type 6 NM_002831 chr12p13 Overexpression in MYST3-linked subgroup 208915_s_at GGA2 Golgi-associated, g-adaptin ear-containing, ARF-binding protein 2 NM_015044 chr16p12 Overexpression in MYST3-linked subgroup 204249_s_at LMO2 LIM domain only 2 (rhombotin-like 1) NM_005574 chr11p13 Overexpression in MYST3-linked subgroup 222955_s_at FAM45B Family with sequence similarity 45, member B NM_018472 chrXq25 Overexpression in MYST3-linked subgroup 225377_at C9orf86 Chromosome 9 open reading frame 86 NM_024718 chr9q34 3 Overexpression in MYST3-linked subgroup

203853_s_at GAB2 GRB2-associated binding protein 2 NM_012296 chr11q14 1 Underexpression in MYST3-linked subgroup a Murati A MYB 227882_at FKRP Fukutin-related protein NM_001039885 chr19q13 32 Underexpression in MYST3-linked subgroup

222731_at ZDHHC2 Zinc ﬁnger, DHHC-type containing 2 NM_016353 chr8p21 3-p22 Underexpression in MYST3-linked subgroup leukemias myeloid acute MYST3-linked in gain a

213222_at PLCB1 Phospholipase C, b1 (phosphoinositide specific) NM_015192 chr20p12 Underexpression in MYST3-linked subgroup al et 222730_s_at ZDHHC2 Zinc-finger, DHHC-type containing 2 NM_016353 chr8p21 3-p22 Underexpression in MYST3-linked subgroup 228622_s_at DNAJC4 DnaJ (Hsp40) homolog, subfamily C, member 4 NM_005528 chr11q13 Underexpression in MYST3-linked subgroup 226142_at GLIPR1 GLI pathogenesis-related 1 (glioma) NM_006851 chr12q21 2 Underexpression in MYST3-linked subgroup 238759_at KIAA1212 KIAA1212 NM_018084 chr2p16 1 Underexpression in MYST3-linked subgroup a 205686_s_at CD86 CD86 molecule NM_006889 chr3q21 Underexpression in MYST3-linked subgroup a 213511_s_at MTMR1 Myotubularin-related protein 1 NM_003828 chrXq28 Underexpression in MYST3-linked subgroup 212959_s_at GNPTAB N-acetylglucosamine-1-phosphate transferase, a- and b-subunits NM_024312 chr12q23 2 Underexpression in MYST3-linked subgroup 217865_at RNF130 Ring-finger protein 130 NM_018434 chr5q35 3 Underexpression in MYST3-linked subgroup 206782_s_at DNAJC4 DnaJ (Hsp40) homolog, subfamily C, member 4 NM_005528 chr11q13 Underexpression in MYST3-linked subgroup 208826_x_at HINT1 Histidine triad nucleotide-binding-protein 1 NM_005340 chr5q31 2 Underexpression in MYST3-linked subgroup a 240572_s_at LOC374443 CLR pseudogene NR_002814 chr12p13 31 Underexpression in MYST3-linked subgroup 224797_at ARRDC3 Arrestin domain containing 3 NM_020801 chr5q14 3 Underexpression in MYST3-linked subgroup 201136_at PLP2 Proteolipid protein 2 (colonic epithelium enriched) NM_002668 chrXp11 23 Underexpression in MYST3-linked subgroup 216095_x_at MTMR1 Myotubularin-related protein 1 NM_003828 chrXq28 Underexpression in MYST3-linked subgroup 207721_x_at HINT1 Histidine triad nucleotide-binding protein 1 NM_005340 chr5q31 2 Underexpression in MYST3-linked subgroup a 200093_s_at HINT1 Histidine triad nucleotide-binding protein 1 NM_005340 chr5q31 2 Underexpression in MYST3-linked subgroup a 1555961_a_at HINT1 Histidine triad nucleotide-binding protein 1 NM_005340 chr5q31 2 Underexpression in MYST3-linked subgroup a 210895_s_at CD86 CD86 molecule NM_006889 chr3q21 Underexpression in MYST3-linked subgroup a 223000_s_at F11R F11 receptor NM_016946 chr1q21 2-q21 3 Underexpression in MYST3-linked subgroup a 218836_at RPP21 Ribonuclease P 21 kDa subunit NM_024839 chr6p21 33 Underexpression in MYST3-linked subgroup 229498_at EST MRNA; cDNA DKFZp779M2422 (from clone DKFZp779M2422) FFUnderexpression in MYST3-linked subgroup Abbreviations: CLR, clear; EGF, epidermal growth factor; ER, endoplasmic reticulum; EST, expressed sequence tag; LIM, Lin11, Isl-1, Mec-3; SET, suppressor of variegation 3–9, enhancer of zeste, trithorax. Probe sets are ordered according to decreasing DS (discriminating score). Leukemia 91 MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 92

Figure 4 Supervised classification of MYST3-linked AMLS vs other M4/M5 AMLs. (a) Classification of 44 AMLs using the 1686 genes identified as discriminator between the eight MYST3-linked and 36 other AMLs. Top, karyotype as in Figure 3. Genes are ordered from top to bottom by their decreasing DS. AML samples are ordered from left to right according to the decreasing correlation coefficient of their expression profile with the median profile of the MYST3-linked (bottom). Solid orange line, threshold 0 that separates the two classes of samples, predicted MYST3-linked class (left of the line) from the others (right of the line). To the right is indicated, by horizontal lines, the position of the genes in common with the gene expression signature published by Camos et al.10 (MYST3 GES). (b) Box plots of the expression level of the MYB gene relative to its observed genomic aberration. According to the genomic status of MYB (that is, gain and nongain) and the karyotype, AMLs were classified into three groups. Median and range are indicated. AML, acute myeloid leukemia; DS, discriminating score.

encode transcriptional repressors of HOX genes.22 Our only MYST3-linked AMLs with other types of AMLs and not only with AML1-ETO (eight twenty one) sample showed a loss of the M4/M5 cases as in our study. Interestingly, 143 of our 1686 IKZF1 gene on 19p13, which encodes the zinc-finger protein probe sets (98 unique genes) were common with this published Ikaros. Ikaros recruits CTBP1 and represses the NOTCH path- list, including CEBPA, GGA2, HOXA10, PRL, PTPN6, STK11 way-associated HES1 gene,23 which is located in 3q28, a region and RET (see Figure 4a and Supplementary Table 2). that we found gained in two samples. Frequent loss of the IKZF1 is observed in BCR-ABL-linked B-ALLs (B-cell acute lymphoid leukemias (ALLs)).24,25 Our result shows that this alteration is not MYB, HOX and MYST3-linked AMLs specific of B-ALL, but may also occur in AML. MYB was not included in any of the identified k gene clusters. Our results suggest that, although M4/M5 AMLs show few This suggests that MYB-induced effect on transcription is not alterations that seem highly variable, they might actually be sufficient to be individualized in M4/5 AMLs, perhaps because linked to only few common pathways of leukemogenesis. other pathways in non-MYST3-linked AMLs lead to the same The most noticeable, because both recurrent and restricted to effects. Similarly, FLT3 and NPM1 alterations were almost the MYST3-linked AMLs, were the alterations of the MYB and equally distributed among clusters I, IIa and IIb, and had no AHI1 (Abelson helper integration site 1) loci. Because MYB has major impact on sample or gene clustering. MYB is a nuclear been the focus of similar observations in T-cell ALL (T-ALL),26,27 transcription regulator essential in multiple steps and lineages of we surmised that MYB rather than AHI1 is involved in MYST3- hematopoiesis. Constitutive overexpression of MYB or of its linked AMLs. This was confirmed by gene expression analysis: altered viral counterpart, v-MYB, transforms myelomonocytic MYB but not AHI1 mRNA expression was consistently elevated cells in vitro,28,29 and causes maturation block in monocyte– in MYST3-linked AMLs compared with other M4/M5 AMLs; macrophage differentiation,30 and induces acute monoblastic except for two samples with gain, AHI1 mRNA level was close leukemia in chickens. MYB is required for T-cell development.31 to background in all AML samples including six samples with Translocations with juxtaposition to the TCRB locus and short gain (not shown). The level of the MYB gain is compatible with somatic duplications of the MYB locus are involved in child- locus duplication, as observed in T-ALL.26,27 hood T-ALL.26,27 Major interacting partners of MYB are CEBP factors and CREBBP and EP300 HATs.32–35 Two sites of MYB interaction have been described on CREBBP; they are preserved Gene expression profiling shows that MYST3-linked in the MYST3–CREBBP fusion protein, and thus the MYB/MYST– leukemias constitute a homogeneous and proliferative CREBBP transcription complex can function in t(8;16) myelo- subtype monocytic cells. Interestingly, CREBBP expression was Hierarchical clustering showed that most MYST3-linked AMLs downregulated in MYST3-linked samples, indicating a major were grouped together and could thus constitute a rather perturbation of the normal CREBBP function by gene alteration, homogeneous subgroup of AMLs. This could be due to their gene expression and MYB partner increase. In contrast, CEBPA relatively specific gene alterations (MYST3-CREBBP fusion and expression was upregulated. The disruption of the balance MYB gain). This is in agreement with their clinical features between CCAAT/enhancer binding protein (CEBP) and CREB including poor prognosis.7 However, no significant difference in binding protein (CREBBP) factors might play a role in t(8;16)- survival could be found between clusters I and II. associated leukemogenesis. A gene expression signature of three cases of MYST3-linked HOXA9 seems to be a central player in leukemogenesis and AMLs has been reported.10 It was obtained by comparing its direct or indirect alterations may define a subgroup

Leukemia MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 93 of leukemias originating in progenitors having acquired 5 Murati A, Ade´laide J, Mozziconacci MJ, Popovici C, Carbuccia N, self-renewal. The MYB gene is a downstream target of HOXA- Letessier A et al. Variant MYST4-CBP gene fusion in a t(10;16) mediated transformation.36 Constitutive expression of HOXA9 acute myeloid leukaemia. Br J Haematol 2004; 125: 601–604. in primary murine marrow immortalizes a late myelomonocytic 6 Esteyries S, Perot C, Ade´laide J, Imbert M, Lagarde A, Pautas C et al. NCOA3, a new fusion partner for MOZ/MYST3 in M5 acute progenitor preventing it from executing terminal differentia- myeloid leukemia. Leukemia 2008; 22: 663–665. tion.37 HOXA9 is also fused to NUP98 in acute leukemia with 38 7 Gervais C, Murati A, Helias C, Struski S, Eischen A, Lippert E et al. t(7;11). Finally, there is a direct link between MLL alterations Acute Myeloid Leukemia with 8p11 (MYST3) translocation: 39–44 and HOXA9 expression. As predicted, upregulation of cytologic, cytogenetic and molecular studies of 30 cases. HOXA genes, especially HOXA9, suggests that increased self- Leukemia 2008; 22: 1567–1575. renewal and stem cell features are prominent in MYST3-linked 8 Tasaka T, Matsuhashi Y, Uehara E, Tamura T, Kakazu N, Abe T AMLs (see Argiropoulos and Humphries for review45). This is et al. Secondary acute monocytic leukemia with a translocation also the case of AMLs with NPM1 mutation, and it is noticeable t(8;16)(p11;p13): case report and review of the literature. Leuk Lymphoma 2004; 45: 621–625. that three cases clustering with MYST3-linked AMLs in cluster 9 Troke PJ, Kindle KB, Collins HM, Heery DM. MOZ fusion proteins IIa showed NPM1 mutation. It is worth to note that, like MYB in acute myeloid leukaemia. Biochem Soc Symp 2006; 73: 23–39. alteration, HOXA upregulation is a prominent feature of T-ALL. 10 Camos M, Esteve J, Jares P, Colomer D, Rozman M, Villamor N The HOXA10, HOXA11, LMO2, PTPN6 and GFI1 genes were et al. Gene expression profiling of acute myeloid leukemia with also upregulated in MYST3-linked AMLs compared to the other translocation t(8;16)(p11;p13) and MYST3-CREBBP rearrangement M4/M5 AMLs. PTPN6, also called SHP1, is a phosphatase that reveals a distinctive signature with a specific pattern of HOX gene interacts with HOXA10.46 The growth factor independent 1 expression. Cancer Res 2006; 66: 6947–6954. transcription repressor (GFI1) transcription factor inhibits 11 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposals for the classification of the acute PU1/SPI1 differentiation effect. In contrast, HOXB genes were leukaemias. French-American-British (FAB) co-operative group. downregulated. The reason for this opposite variation of HOXA Br J Haematol 1976; 33: 451–458. and HOXB genes is unknown. Perhaps HOXB genes are more 12 Jaffe ES, Harris NL, Stein H, Vardiman JW. World Health associated with stem cell expansion than self-renewal. Organization Classification of Tumors: Pathology and Genetics In conclusion, our results show gain of MYB, overexpression of Tumor of Haematopoietic and Lymphoid Tissues. IARC Press: of HOXA genes and CD4 in MYST3-linked AMLs. They suggest Lyon, 2001, p 352. a community with T-ALL,26,27,47 which could be related to the 13 Chaffanet M, Mozziconacci MJ, Fernandez F, Sainty D, Lafage- existence of the recently identified common macrophage-T Pochitaloff M, Birnbaum D et al. A case of inv(8)(p11q24) associated with acute myeloid leukemia involves the MOZ and progenitor.48,49 Both MYST3-linked AMLs and T-ALLs could CBP genes in a masked t(8;16). Genes Chromosomes Cancer 1999; originate from this common progenitor; variation in the dosage 26: 161–165. of regulatory factors could further induce leukemia in either the 14 Murati A, Ade´laide J, Popovici C, Mozziconacci MJ, Arnoulet C, T-lymphoid or myelomonocytic lineage. The regulation of early Lafage-Pochitaloff M et al. A further case of acute myelomonocytic myelomonocytic differentiation, which is controled by a leukemia with inv(8) chromosomal rearrangement and MOZ- complex network of factors,50 is affected at several levels in NCOA2 gene fusion. Int J Mol Med 2003; 12: 423–428. t(8;16) leukemia including the formation of chimeric HATs, 15 Ade´laide J, Finetti P, Bekhouche I, Repellini L, Geneix J, amplification of MYB and overexpression of HOXA genes. Sircoulomb F et al. Integrated profiling of basal and luminal breast cancers. Cancer Res 2007; 67: 11565–11575. Combined upregulation of HOXA9 expression and CREBBP and 16 Bertucci F, Finetti P, Cervera N, Charafe-Jauffret E, Mamessier E, MYB loci alteration and dysregulation suggest that perturbation Ade´laide J et al. Gene expression profiling shows medullary breast of MYB/CREBBP factor complex may be a major event in cancer is a subgroup of basal breast cancers. Cancer Res 2006; 66: MYST3-linked AMLs. 4636–4644. 17 Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U et al. Exploration, normalization, and summaries of high Acknowledgements density oligonucleotide array probe level data. Biostatistics 2003; 4: 249–264. This study was supported by Inserm, Institut Paoli-Calmettes 18 Liu TX, Becker MW, Jelinek J, Wu WS, Deng M, Mikhalkevich N and grants from Association pour la Recherche sur le Cancer et al. Chromosome 5q deletion and epigenetic suppression of the (2007; AM). gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation. Nat Med 2007; 13: 78–83. 19 Lorsbach RB, Moore J, Mathew S, Raimondi SC, Mukatira ST, Downing JR. TET1, a member of a novel protein family, is fused to References MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia 2003; 17: 637–641. 20 Ono R, Taki T, Taketani T, Taniwaki M, Kobayashi H, Hayashi Y. 1 Aguiar RC, Chase A, Coulthard S, Macdonald DH, Carapeti M, LCX, leukemia-associated protein with a CXXC domain, is fused to Reiter A et al. Abnormalities of chromosome band 8p11 in MLL in acute myeloid leukemia with trilineage dysplasia having leukemia: two clinical syndromes can be distinguished on the basis t(10;11)(q22;q23). Cancer Res 2002; 62: 4075–4080. of MOZ involvement. Blood 1997; 90: 3130–3135. 2 Borrow J, Stanton Jr VP, Andresen JM, Becher R, Behm FG, 21 Allen MD, Grummitt CG, Hilcenko C, Min SY, Tonkin LM, Chaganti RS et al. The translocation t(8;16)(p11;p13) of acute Johnson CM et al. Solution structure of the nonmethyl-CpG- myeloid leukaemia fuses a putative acetyltransferase to the binding CXXC domain of the leukaemia-associated MLL histone CREB-binding protein. Nat Genet 1996; 14: 33–41. methyltransferase. EMBO J 2006; 25: 4503–4512. 3 Giles RH, Dauwerse JG, Higgins C, Petrij F, Wessels JW, 22 Sakamoto Y, Watanabe S, Ichimura T, Kawasuji M, Koseki H, Baba Beverstock GC et al. Detection of CBP rearrangements in acute H et al. Overlapping roles of the methylated DNA-binding protein myelogenous leukemia with t(8;16). Leukemia 1997; 11: MBD1 and polycomb group proteins in transcriptional repression 2087–2096. of HOXA genes and heterochromatin foci formation. J Biol Chem 4 Chaffanet M, Gressin L, Preudhomme C, Soenen-Cornu V, 2007; 282: 16391–16400. Birnbaum D, Pe´busque MJ. MOZ is fused to p300 in an acute 23 Kathrein KL, Chari S, Winandy S. Ikaros directly represses the monocytic leukemia with t(8;22). Genes Chromosomes Cancer notch target gene Hes1 in a leukemia T cell line: implications for 2000; 28: 138–144. CD4 regulation. J Biol Chem 2008; 283: 10476–10484.

Leukemia MYB gain in MYST3-linked acute myeloid leukemias A Murati et al 94 24 Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, phils or macrophages, independent of enforced meis expression. Dalton JD et al. Genome-wide analysis of genetic alterations in Mol Cell Biol 2000; 20: 3274–3285. acute lymphoblastic leukaemia. Nature 2007; 446: 758–764. 38 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, 25 Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. Toyama K et al. Fusion of the nucleoporin gene NUP98 to HOXA9 BCR-ABL1 lymphoblastic leukaemia is characterized by the by the chromosome translocation t(7;11)(p15;p15) in human deletion of Ikaros. Nature 2008; 453: 110–114. myeloid leukaemia. Nat Genet 1996; 12: 154–158. 26 Clappier E, Cuccuini W, Kalota A, Crinquette A, Cayuela JM, Dik 39 Ayton PM, Cleary ML. Transformation of myeloid progenitors by WA et al. The C-MYB locus is involved in chromosomal MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev translocation and genomic duplications in human T-cell acute 2003; 17: 2298–2307. leukemia (T-ALL), the translocation defining a new T-ALL subtype 40 Dorrance AM, Liu S, Yuan W, Becknell B, Arnoczky KJ, Guimond in very young children. Blood 2007; 110: 1251–1261. M et al. Mll partial tandem duplication induces aberrant Hox 27 Lahortiga I, De Keersmaecker K, Van Vlierberghe P, Graux C, expression in vivo via specific epigenetic alterations. J Clin Invest Cauwelier B, Lambert F et al. Duplication of the MYB oncogene 2006; 116: 2707–2716. in T cell acute lymphoblastic leukemia. Nat Genet 2007; 39: 41 Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. 593–595. Expression of HOX genes, HOX cofactors, and MLL in pheno- 28 Dvorakova M, Kralova J, Karafiat V, Bartunek P, Dvorak M. An ex typically and functionally defined subpopulations of leukemic and vivo model to study v-Myb-induced leukemogenicity. Blood Cells normal human hematopoietic cells. Leukemia 1999; 13: 687–698. Mol Dis 2001; 27: 437–445. 42 Krivtsov AV, Armstrong SA. MLL translocations, histone modifica- 29 Fu SL, Lipsick JS. Constitutive expression of full-length c-Myb tions and leukaemia stem-cell development. Nat Rev Cancer 2007; transforms avian cells characteristic of both the monocytic and 7: 823–833. granulocytic lineages. Cell Growth Differ 1997; 8: 35–45. 43 Milne TA, Martin ME, Brock HW, Slany RK, Hess JL. Leukemo- 30 Yanagisawa H, Nagasawa T, Kuramochi S, Abe T, Ikawa Y, genic MLL fusion proteins bind across a broad region of the Hox a9 Todokoro K. Constitutive expression of exogenous c-myb gene locus, promoting transcription and multiple histone modifications. causes maturation block in monocyte-macrophage differentiation. Cancer Res 2005; 65: 11367–11374. Biochim Biophys Acta 1991; 1088: 380–384. 44 Zeisig BB, Milne T, Garcia-Cuellar MP, Schreiner S, Martin ME, 31 Lieu YK, Kumar A, Pajerowski AG, Rogers TJ, Reddy EP. Requirement of c-myb in T cell development and in mature T Fuchs U et al. Hoxa9 and Meis1 are key targets for MLL-ENL- cell function. Proc Natl Acad Sci USA 2004; 101: 14853–14858. mediated cellular immortalization. Mol Cell Biol 2004; 24: 32 Fung SM, Ramsay G, Katzen AL. MYB and CBP: physiological 617–628. relevance of a biochemical interaction. Mech Dev 2003; 120: 45 Argiropoulos B, Humphries RK. Hox genes in hematopoiesis and 711–720. leukemogenesis. Oncogene 2007; 26: 6766–6776. 33 Oelgeschlager M, Janknecht R, Krieg J, Schreek S, Luscher B. 46 Eklund EA, Goldenberg I, Lu Y, Andrejic J, Kakar R. SHP1 protein- Interaction of the co-activator CBP with Myb proteins: effects on tyrosine phosphatase regulates HoxA10 DNA binding and Myb-specific transactivation and on the cooperativity with NF-M. transcriptional repression activity in undifferentiated myeloid cells. EMBO J 1996; 15: 2771–2780. J Biol Chem 2002; 277: 36878–36888. 34 Ramsay RG. c-Myb a stem-progenitor cell regulator in multiple 47 Dik WA, Brahim W, Braun C, Asnafi V, Dastugue N, Bernard OA tissue compartments. Growth Factors 2005; 23: 253–261. et al. CALM-AF10+ T-ALL expression profiles are characterized by 35 Sandberg ML, Sutton SE, Pletcher MT, Wiltshire T, Tarantino LM, overexpression of HOXA and BMI1 oncogenes. Leukemia 2005; Hogenesch JB et al. c-Myb and p300 regulate hematopoietic stem 19: 1948–1957. cell proliferation and differentiation. Dev Cell 2005; 8: 153–166. 48 Bell JJ, Bhandoola A. The earliest thymic progenitors for T cells 36 Hess JL, Bittner CB, Zeisig DT, Bach C, Fuchs U, Borkhardt A et al. possess myeloid lineage potential. Nature 2008; 452: 764–767. c-Myb is an essential downstream target for homeobox-mediated 49 Wada H, Masuda K, Satoh R, Kakugawa K, Ikawa T, Katsura Y transformation of hematopoietic cells. Blood 2006; 108: 297–304. et al. Adult T-cell progenitors retain myeloid potential. Nature 37 Calvo KR, Sykes DB, Pasillas M, Kamps MP. Hoxa9 immortalizes a 2008; 452: 768–772. granulocyte-macrophage colony-stimulating factor-dependent 50 Friedman AD. Transcriptional regulation of granulocyte and promyelocyte capable of biphenotypic differentiation to neutro- monocyte development. Oncogene 2002; 21: 3377–3390.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia