Original Article

Leukemia (2009) 23, 85–94; doi:10.1038/leu.2008.257; published online 25 September 2008

Acute Leukemias

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 Adélaïde1, 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, C Pérot12, S Olschwang1, F Bertucci1, M Chaffanet1, M Lessard2, M-J Mozziconacci1 and D Birnbaum1 on behalf of the Groupe Francophone de Cytogénétique Hématologique (GFCH)

  1. 1Département d'Oncologie Moléculaire, Centre de Recherche en Cancérologie de Marseille, Institut Paoli-Calmettes, Marseille, France
  2. 2Laboratoire d'Hématologie, CHU de Hautepierre, Strasbourg, France
  3. 3Laboratoire d'Hématologie, Hôpital Cardiologique-Haut Lévêque, Bordeaux, France
  4. 4Laboratoire de Cytogénétique, CHU du Bocage, Dijon, France
  5. 5Laboratoire d'Hématologie, CHU Lyon Sud, Lyon, France
  6. 6Département de Génétique, Centre Henri Becquerel, Rouen, France
  7. 7Centre de Génétique Médicale, Ghent, Belgique
  8. 8Laboratoire de Génétique, CHU d'Angers, Angers, France
  9. 9Service de Génétique, Hôpital Maison Blanche, Reims, France
  10. 10Laboratoire d'Hématologie, Hôpital Nord, Saint-Etienne, France
  11. 11Laboratoire de Cytogénétique Hématologique, Hôpital Pitié-Salpêtrière, Paris, France
  12. 12Laboratoire de Cytogénétique Onco-Hématologique, Hôpital Saint-Antoine, Paris, France

Correspondence: Dr D Birnbaum, Centre de Recherche en Cancérologie de Marseille, UMR891 Inserm, 27 Bd. Leï Roure, 13009 Marseille, France. E-mail: daniel.birnbaum@inserm.fr

13These authors contributed equally to this work.

Received 6 May 2008; Revised 1 August 2008; Accepted 21 August 2008; Published online 25 September 2008.



The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.


array-CGH, acute myeloid leukemia, gene expression profiling, MYB, MYST3, t(8;16)



The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia (French-American-British classification (FAB) AML-M4, M5a and M5b). It fuses two genes encoding histone acetyltransferases (HAT), MYST3 (also called MOZ) located at 8p11 to CREBBP (also called CBP) located at 16p13.1, 2, 3 Variant translocations involve other HAT-encoding genes, such as EP300 at 22q13,4 MYST4 at 10q22, NCOA2 at 8q135 or NCOA3 at 20q12.6 MYST3-linked acute myeloid leukemias (AMLs) share specific features, such as frequent extramedullary involvement, disseminated intravascular coagulation, erythrophagocytosis and a poor prognosis.7 They are often secondary, therapy-related AMLs.8, 9 Because of its rarity, the molecular biology of the MYST3-linked AMLs remains poorly understood. A study of three cases using DNA microarrays has shown that they have a distinct gene expression profile compared with other AMLs.10

Here, we have established the genome and/or gene expression profiles of a series of 61 M4/M5 AMLs including MYST3-linked AMLs and variants by using array comparative genome hybridization (aCGH) and DNA microarrays, respectively. We show that MYST3-linked AMLs have specific gene alterations and gene expression profiles within the M4/M5 group.


Patients and methods


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 Organization12 criteria, the panel comprised 16 M4, 44 M5, and one case of AML with no more data available. Some cases have been previously reported in detail, namely M45-1, M45-3, M45-4, M45-5, M45-7, M45-11, M45-13, M45-16, M45-19, M45-20, M45-21, M45-23, M45-26,7 M45-052,13 M45-031,14 M45-0326 M45-062 and M45-063.4 Three BM samples were used as normal controls (normal bone marrow (NBM)). They were collected from breast cancer patients without micrometastases.

The aCGH study was carried out on 52 of the 61 samples, including 18 MYST3-linked AMLs, 16 AMLs with normal karyotype, nine AMLs with 11q23 (mixed lineage leukemia (MLL)) abnormalities, four AMLs with inv(16; CBF-beta-MYH11 fusion), one AML with t(8;21) (RUNX1-RUNX1T1 aka AML1-ETO fusion) and four AMLs with trisomy 8.

A total of 44 M4/M5 cases with good quality RNA were selected for global gene expression profile analysis, including eight MYST3-linked AML and 36 other samples comprising 18 cases with normal karyotype, six with 11q23 abnormalities, four with inv(16), two with trisomy 8 and six with various karyotypes. Of the 61 cases analyzed by aCGH and/or gene expression profile, mutations of nucleophosmin 1 (NPM1) were found in 11 cases (mostly in AMLs with normal karyotype, as expected), and an internal tandem duplication and mutation of the FLT3 gene in four and six cases, respectively (Table 1).

Nucleic acids extraction

Bone marrow aspirates or peripheral blood samples were collected from 61 patients at the time of diagnosis. Blasts and mononuclear cells were purified after density gradient centrifugation of BM aspiration or whole blood (cases M45-3, M45-4, M45-11, M45-16, M45-010, M45-035, M45-036 and M45-037), and processed immediately or cryoconserved at -80 °C. High-quality total RNA and DNA was extracted by the Allprep DNA/RNA isolation kit (Qiagen, Germany) from blood or BM blast cells. RNA quality and purity were assessed with Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). NBM samples were processed similarly.

Array comparative genomic hybridization

Genomic imbalances were analyzed by aCGH using 244K CGH Microarrays (Hu-244A, Agilent Technologies, Massy, France) following a previously described protocol.15 Scanning was performed with Agilent Autofocus Dynamic Scanner (G2565BA, Agilent Technologies). Data analysis was performed as described previously15 and visualized with CGH analytics 3.4 software (Agilent Technologies). Extraction data (log2 ratio) was performed from CGH analytics, whereas normalized and filtered log2 ratio were obtained from 'feature extraction' software (Agilent Technologies). Data generated by probes mapped to X and Y chromosomes were eliminated. The final data set contained 225 388 unique probes covering 22 509 genes and intergenic part following the hg17 human genome mapping. Copy number changes were characterized as reported previously.15 Results were displayed using TreeView program. Identification of copy-number variations was based on a 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 Fisher's exact test and false discovery rate was applied to correct the hypothesis of multiple testing.

Gene expression profiling

We used Affymetrix U133 Plus 2.0 human oligonucleotide microarrays. Preparation of cRNA, hybridizations, washes and detection were performed as recommended by the supplier and as described previously.16 Data were analyzed by the Robust multichip average method in R using the Bioconductor (http://www.bioconductor.org/) and associated package17 as described previously.16 Before analysis, a first filtering process removed the genes, from the data set, with low and poorly measured expression as defined by an expression value inferior to 100 U in all 44 samples retaining 26 376 genes/expressed sequence tags (ESTs). A second filter, based on the intensity of s.d., was applied to exclude genes showing low expression variation across the analyses. Standard deviation was calculated on log2 transformed data, in which the lowest values were first floored to a minimal value of 100 U, that is, the background intensity, retaining 15 618 genes/ESTs with s.d. superior to 0.4. An unsupervised analysis was performed on 44 samples on 15 618 probe sets. Before hierarchical clustering, filtered data were log2 transformed and submitted to the cluster program using the median-centered data on genes, Pearson correlation as similarity metric and centroid linkage clustering. Results were displayed using TreeView program. To identify and rank genes discriminating eight cases of MYST3-linked and 36 M4/M5 AMLs, a supervised analysis was applied to the 26 376 genes/ESTs. A discriminating score (DS) was calculated for each gene. DS=(M1-M2)/(S1-S2) where M1 and S1, respectively, represent mean and s.d. of expression levels of the gene in MYST3-linked subgroup, and M2 and S2 in M4/M5 AMLs subgroup. Confidence levels were estimated by 100 random permutations of samples.

Functional processes and pathways were identified by using Ingenuity software (Ingenuity Systems, Redwood City, CA, USA).



aCGH profiling

Using genome-wide, high-density arrays, we established the 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 profile was said 'normal-like' because no alteration was detected. A second type of profiles showed gains or losses visible on the karyotype and affecting large regions of the genome, such as trisomy 8, gains of 11q23-qter (Figure 1a), deletions of 12p (including the CDKN1B locus; Figure 1b), rearrangement of chromosome 13 (Figure 1c), gain of 3q26-qter (Figure 1d) or 6q22–25 (case 4). Finally, few profiles showed rare and limited gains or losses that affected few or single genes, such as deletions encompassing CTNNA1 and CXXC5 at 5q31 (case 011, normal karyotype, Figure 1e), reminiscent of a 5q-syndrome,18 CXXC4 at 4q24 (case 23, MYST3, Figure 1f), IKZF1/Ikaros at 7p12 (case 055) or a cluster of chemokines at 17q12 (case 010). Among these alterations, a gain of MYB at 6q23 was detected in seven MYST3-linked AML samples (Figure 2). In six cases, only MYB and its neighbor gene AHI1 were gained, whereas in the seventh case (4) the MYB gain was included in a larger gained region also detected by karyotype.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

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 >1) 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.

Full figure and legend (282K)

Figure 2.
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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.

Full figure and legend (121K)

Expression profiling

To determine how the presence or absence of genome 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 three NBM samples. After filtering, 15 618 genes/ESTs were retained. Before hierarchical clustering, data were log2 transformed and submitted to the cluster program using the median-centered data on genes, Pearson correlation as similarity metric and centroid linkage clustering. Results are displayed in Figure 3 using the TreeView program. Two main clusters of samples (I and II) were distinguished in the dendrogram. Two subclusters (a and b) were recognized in branch II. Subcluster IIa contained six MYST3-linked AMLs, suggesting that this type of AML is rather homogeneous. The other cases clustering with MYST3-linked AMLs in IIa were AMLs with normal karyotype. MLL-linked AMLs were distributed within different subclusters. Three CBF AMLs were grouped together in cluster I. The three NBM samples clustered together in cluster IIb.

Figure 3.
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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.

Full figure and legend (261K)

On the basis of QT clustering method and visual inspection, seven clusters of genes (k1–k7) were identified (Figure 3a). Gene cluster k1, overexpressed in the NBM samples (Figure 3b) and in a subcluster of cluster I, contained genes associated with 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. 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 significant expression level among the AML subgroups (Supplementary 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, CREBBP, CSF3R, CXXC5, NPM1, STAT3 and 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.

Figure 4.
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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.

Full figure and legend (155K)

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-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.



aCGH shows alteration of the MYB locus in MYST3-linked leukemia

Array comparative genome hybridization profiles showed globally few and variable alterations. The large nonequilibrated alterations detected by the karyotypic analysis were also detected by aCGH. Many localized alterations were further detected by aCGH, usually in a non-recurrent manner. However, the presence of paralogous genes (CXXC4 and CXXC5) in small regions of loss could suggest that these genes play a role in leukemogenesis. Another paralog, CXXC6, is fused to MLL in AML with t(10;11)(q22;q23).19, 20 The MLL protein itself (aka CXXC7) has a CXXC domain.21 The cysteine-rich CXXC domain binds Zinc and DNA, and recruits the transcription corepressor C-terminal binding protein 1 (CTBP1). It is possible that these genes are eliminated because they encode transcriptional repressors of HOX genes.22 Our only AML1-ETO (eight twenty one) sample showed a loss of the IKZF1 gene on 19p13, which encodes the zinc-finger protein Ikaros. Ikaros recruits CTBP1 and represses the NOTCH pathway-associated HES1 gene,23 which is located in 3q28, a region 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 specific of B-ALL, but may also occur in AML.

Our results suggest that, although M4/M5 AMLs show few alterations that seem highly variable, they might actually be linked to only few common pathways of leukemogenesis.

The most noticeable, because both recurrent and restricted to the MYST3-linked AMLs, were the alterations of the MYB and AHI1 (Abelson helper integration site 1) loci. Because MYB has been the focus of similar observations in T-cell ALL (T-ALL),26, 27 we surmised that MYB rather than AHI1 is involved in MYST3-linked AMLs. This was confirmed by gene expression analysis: MYB but not AHI1 mRNA expression was consistently elevated in MYST3-linked AMLs compared with other M4/M5 AMLs; except for two samples with gain, AHI1 mRNA level was close to background in all AML samples including six samples with gain (not shown). The level of the MYB gain is compatible with locus duplication, as observed in T-ALL.26, 27

Gene expression profiling shows that MYST3-linked leukemias constitute a homogeneous and proliferative subtype

Hierarchical clustering showed that most MYST3-linked AMLs were grouped together and could thus constitute a rather homogeneous subgroup of AMLs. This could be due to their relatively specific gene alterations (MYST3-CREBBP fusion and MYB gain). This is in agreement with their clinical features including poor prognosis.7 However, no significant difference in survival could be found between clusters I and II.

A gene expression signature of three cases of MYST3-linked AMLs has been reported.10 It was obtained by comparing MYST3-linked AMLs with other types of AMLs and not only with M4/M5 cases as in our study. Interestingly, 143 of our 1686 probe sets (98 unique genes) were common with this published list, including CEBPA, GGA2, HOXA10, PRL, PTPN6, STK11 and RET (see Figure 4a and Supplementary Table 2).

MYB, HOX and MYST3-linked AMLs

MYB was not included in any of the identified k gene clusters. This suggests that MYB-induced effect on transcription is not sufficient to be individualized in M4/5 AMLs, perhaps because other pathways in non-MYST3-linked AMLs lead to the same effects. Similarly, FLT3 and NPM1 alterations were almost equally distributed among clusters I, IIa and IIb, and had no major impact on sample or gene clustering. MYB is a nuclear transcription regulator essential in multiple steps and lineages of hematopoiesis. Constitutive overexpression of MYB or of its altered viral counterpart, v-MYB, transforms myelomonocytic cells in vitro,28, 29 and causes maturation block in monocyte–macrophage differentiation,30 and induces acute monoblastic leukemia in chickens. MYB is required for T-cell development.31 Translocations with juxtaposition to the TCRB locus and short somatic duplications of the MYB locus are involved in childhood T-ALL.26, 27 Major interacting partners of MYB are CEBP factors and CREBBP and EP300 HATs.32, 33, 34, 35 Two sites of MYB interaction have been described on CREBBP; they are preserved in the MYST3–CREBBP fusion protein, and thus the MYB/MYST–CREBBP transcription complex can function in t(8;16) myelomonocytic cells. Interestingly, CREBBP expression was downregulated in MYST3-linked samples, indicating a major perturbation of the normal CREBBP function by gene alteration, gene expression and MYB partner increase. In contrast, CEBPA expression was upregulated. The disruption of the balance between CCAAT/enhancer binding protein (CEBP) and CREB binding protein (CREBBP) factors might play a role in t(8;16)-associated leukemogenesis.

HOXA9 seems to be a central player in leukemogenesis and its direct or indirect alterations may define a subgroup of leukemias originating in progenitors having acquired self-renewal. The MYB gene is a downstream target of HOXA-mediated transformation.36 Constitutive expression of HOXA9 in primary murine marrow immortalizes a late myelomonocytic progenitor preventing it from executing terminal differentiation.37 HOXA9 is also fused to NUP98 in acute leukemia with t(7;11).38 Finally, there is a direct link between MLL alterations and HOXA9 expression.39, 40, 41, 42, 43, 44 As predicted, upregulation of HOXA genes, especially HOXA9, suggests that increased self-renewal and stem cell features are prominent in MYST3-linked AMLs (see Argiropoulos and Humphries for review45). This is also the case of AMLs with NPM1 mutation, and it is noticeable that three cases clustering with MYST3-linked AMLs in cluster IIa showed NPM1 mutation. It is worth to note that, like MYB alteration, HOXA upregulation is a prominent feature of T-ALL.

The HOXA10, HOXA11, LMO2, PTPN6 and GFI1 genes were also upregulated in MYST3-linked AMLs compared to the other M4/M5 AMLs. PTPN6, also called SHP1, is a phosphatase that interacts with HOXA10.46 The growth factor independent 1 transcription repressor (GFI1) transcription factor inhibits PU1/SPI1 differentiation effect. In contrast, HOXB genes were downregulated. The reason for this opposite variation of HOXA and HOXB genes is unknown. Perhaps HOXB genes are more associated with stem cell expansion than self-renewal.

In conclusion, our results show gain of MYB, overexpression of HOXA genes and CD4 in MYST3-linked AMLs. They suggest a community with T-ALL,26, 27, 47 which could be related to the existence of the recently identified common macrophage-T progenitor.48, 49 Both MYST3-linked AMLs and T-ALLs could originate from this common progenitor; variation in the dosage of regulatory factors could further induce leukemia in either the T-lymphoid or myelomonocytic lineage. The regulation of early myelomonocytic differentiation, which is controled by a complex network of factors,50 is affected at several levels in t(8;16) leukemia including the formation of chimeric HATs, amplification of MYB and overexpression of HOXA genes. Combined upregulation of HOXA9 expression and CREBBP and MYB loci alteration and dysregulation suggest that perturbation of MYB/CREBBP factor complex may be a major event in MYST3-linked AMLs.



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This study was supported by Inserm, Institut Paoli-Calmettes and grants from Association pour la Recherche sur le Cancer (2007; AM).

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