Leukemia (2002) 16, 1293–1301  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Expression pattern of HOXB6 in myelomonocytic differentiation and A Giampaolo1, N Felli2, D Diverio3, O Morsilli2, P Samoggia2, M Breccia3, F Lo Coco3, C Peschle2 and UTesta 2

1Department of Clinical Biochemistry, Istituto Superiore di Sanita`, Rome, Italy; 2Department of Hematology and Oncology, Istituto Superiore di Sanita`, Rome, Italy; and 3Department of Cellular Biotechnology and Hematology, University La Sapienza, Rome, Italy

Homeobox encode transcription factors known to be arranged in four gene clusters, named A, B, C and D, mapped important morphogenic regulators during embryonic develop- on 7, 17, 12 and 2, respectively.6 ment. An increasing body of work implies a role for homeobox genes in both hematopoiesis and leukemogenesis. In the Many of the HOX genes have been implicated in the regu- present study we have analyzed the role of the homeobox gene, lation of both normal and leukemic hematopoiesis (reviewed HOXB6, in the program of differentiation of the myeloid cell in Refs 7–10). It seems clear that HOX genes are expressed in lines, NB4 and HL60. HOXB6 expression is transiently induced CD34+ cells and modulated according to stage and lineage- during normal granulocytopoiesis and monocytopoiesis, with specific patterns. Particularly, HOXB genes on the 3′ cluster an initial induction during the early phases of differentiation, + 9,11–14 followed by a blockade of expression at early maturation. The side are highly expressed in primitive CD34 populations enforced expression of HOXB6 in promyelocytic NB4 cells or and are colinearly expressed during differentiation of myeloid, in myeloblastic HL60 cells elicited inhibition of the granulocytic erythroid and lymphoid cells.12,13,15,16 or monocytic maturation, respectively. Furthermore, HOXB6 mutations associated with leukemias are not was frequently expressed (18 out of 49 cases) in AMLs lacking known, but some leukemias are associated with irregular major translocations while it was expressed at very low fre- quency (two out of 47 cases) in AMLs characterized by expression of HOX genes due to translocation or abnormal PML/RAR-␣, AML-1/ETO, CBF␤/MYH11 fusion and rearrange- gene regulation. The HOX 11 gene is deregulated in T-ALL ments of the MLL gene at 11q23. According to these obser- with the t(10;14) translocation,17 while the HOX A9 is vations, we suggest that a regulated pattern of HOXB6 involved in the t(7;11) translocation present in AML classified expression is required for normal granulopoiesis and monocy- as M2–M4 by French–American–British (FAB), that juxtaposes topoiesis. Abnormalities of the HOXB6 expression may contrib- ute to the development of the leukemic phenotype. the complete HOX A9 gene to a gene for nucleoporin 18 Leukemia (2002) 16, 1293–1301. doi:10.1038/sj.leu.2402532 NUP98. Keywords: HoxB6; expression; myelomonopoiesis; differentiation; Concerning HOXB6 expression, we have shown that this leukemias homeobox is expressed in human hematopoietic progenitor cells (HPCs) induced to differentiate into granulocytic lineage, and that a blockade of its expression in HPCs specifically Introduction inhibits granulomonocytic colony formation.13 However, the HOXB6 gene could also be relevant for fetal erythropoiesis, as Hematopoiesis depends on a complex series of cellular events suggested by studies in murine embryos.19 Finally, preliminary which requires both the maintenance of a pool of hematopo- observations based on the analysis of a few acute leukemia ietic stem cells in an undifferentiated state and their matu- patients showed the expression of HOXB6 in some acute ration first to multipotent hemopoietic progenitors and then myeloid leukemias.20 to committed progenitors which first generate a progeny of In this study we have retrovirally transduced the HOXB6 differentiated cells (hemopoietic precursors) and then the gene in promyelocytic NB4 cells and in myeloblastic HL60 mature cellular elements of blood (reviewed in Ref. 1). The and investigated the effects of its enforced and deregulated molecular mechanisms that lead to the generation of a pro- expression, showing an inhibition of the granulocytic and geny of progressively maturing cells starting from pluripotent monocytic maturation, respectively. hemopoietic stem cells are poorly understood and begin only Since products resulting from chromosomal translo- in part to be elucidated.2 cations occurring in the human acute leukemias can interfere The process of hemopoietic differentiation seems to be with regulated expression of genes that control normal hema- orchestrated at the molecular level by a series of transcription topoiesis,21 including HOX genes, it seemed of interest to factors which act according to a hierarchic pattern and follow- evaluate the expression pattern of HOXB6 in acute myeloid ing differentiation-specific pathways.2 Among these transcrip- tion factors a relevant role seems to be played by homeobox leukemias. HOXB6 mRNA was found to be expressed at vari- (HOX) genes. able levels in about 35% of acute myeloid leukemias classi- Products of HOX genes are localized in the nucleus and fied as M1, M2, M4, M5 and M6 by FAB. By contrast, almost represent a major class of transcription factors controlling all AMLs (45 out of 47) characterized by fusion genes underly- ␣ cellular proliferation/differentiation during early embryonic ing most common translocations (PML/RAR- , AML-1/ETO, ␤ development.3 Homeogenes, originally described in Droso- CBF /MYH11 and rearrangements of the MLL gene) showed phila melanogaster, are organized in gene clusters evol- HOXB6 expression to be absent. utionarly conserved.4,5 In humans the homeobox genes are According to these observations we suggest that a regulated pattern of HOXB6 expression is required for normal gran- ulopoiesis and monocytopoiesis. Abnormalities of HOXB6 expression in acute leukemia may contribute to the develop- Correspondence: UTesta, Department of Hematology-Oncology, Isti- ment of the leukemic phenotype. tuto Superiere di Sanita`, Viale Regina Elena, 299 Rome, 00161 Italy Received 12 September 2001; accepted 20 February 2002 HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1294 Materials and methods Reverse-transcription polymerase chain reaction (RT- PCR) analysis Cloning of HOXB6 gene Total RNA was extracted by the standard guanidium thiocyan- ␮ Total RNA was extracted from human embryonal carcinoma ate-CsCl in the presence of 12 gofEscherichia coli rRNA or 28 cell line NT2/D1 induced to differentiate by all-trans retinoic by the guanidium thyocyanate/acid phenol method tech- acid (ATRA)10−5 m. The RNA extraction was performed by the nique. standard guanidinium thiocianate-CsCl technique. Two RNA was reverse-transcribed (Moloney murine leukemia micrograms of total RNA were reverse transcribed (RT) with virus; BRL, Gaithersburg, MD, USA) with oligo (dT) (BRL) as oligo (dT) as the primer. An aliquot of RT-RNA was amplified a primer. The products were equalized by amplification of the using pfu DNA polymerase (Stratagene, La Jolla, CA, USA) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or and the following primers: forward-primer CCCAATCTCGGAT human ribosomal protein S26 genes. cDNA from NT2/D1 ter- ATACTAC, reverse-primer CTCGGGTGGGGGAGCCAGGA.22 atocarcinoma cells induced to differentiate for 14 days with −5 29 The PCR reaction mix was then directly ligated into the pCR- ATRA 10 m was normalized together with normal and leu- Script Amp SK(+) (Stratagene) and used for bacterial transform- kemic cell cDNAs: teratocarcinoma RT-RNA was used as ation. A vector possessing the correct insert (674 bp) was ident- positive control in each PCR. ified and sequenced to confirm identity with published To evaluate HOXB6 expression, an aliquot of cDNA ෂ sequence of HOXB6.22 ( 30 ng) was amplified within the linear range by 35 PCR cycles (the cycle number was chosen on the basis of dose– response curve for the NT2/D1 templates and analysis of ali- quots of PCR reactions removed at different PCR cycles) using Retroviral generation and infection of NB4 and HL60 the following primers: forward-primer CTTTGCCACTTC cell lines CTCCTATTAC, reverse-primer CCAGACATACACACGTTAC CAG. This amplification generated a 318 bp fragment of Briefly, the HOXB6 cDNA region encompassing the complete human HOXB6 gene.22 PCR products were verified by South- coding sequence was subcloned at the BAMHI site of the ern blotting utilizing an internal oligomer as a probe. 23 PINCO retrovirus. Viral supernatant was produced by transi- Evaluation of mRNA levels was performed by densitometric ent transfection of Phoenix packaging cells with retroviral analysis (Instant Imager, Packard, Canberra, Australia). construct as described in Ref. 23. NB4 and HL60 cell line infection was obtained by three cycles of centrifugation of the cells in viral supernatant at 1800 r.p.m. for 40 min in the Cell surface marker and morphology analysis of HPC ␮ presence of 4 g/ml of polybrene. cultures

To evaluate the differentiation of HPCs along the E and M Hematopoietic growth factors and cell culture lineages the cells were stained with the following monoclonal antibodies labeled either with FITC or PE: anti-CD34, anti- Recombinant human growth factors were obtained from stan- CD15, anti-CD14 (Pharmingen, San Diego, CA, USA). Fluor- dard commercial sources.24,25 Iscove’s medium was freshly escent cells were analyzed with a flow cytometer (FACS Scan, prepared weekly. Becton Dickinson, San Diego, CA, USA). Cell lines HL60 and NB4 were maintained in RPMI 1640 For cell morphology analysis cytospin preparations of cells medium supplemented with 10% heat-inactivated fetal calf were performed at different days of culture and stained with serum (FCS). May–Gru¨nwald-Giemsa. At least 200 cells were counted and classified according to their differentiation/maturation stage. Adult peripheral blood (PB) HPC purification and clonogenic assay Cell surface and morphology analysis of NB4 and HL60 cells HPCs were purified from PB buffy coat according to the method previously reported24 and subsequently modified as To evaluate the granulocytic differentiation capacities of both described.26 NB4 cells transduced with the empty vector containing only the reporter gene GFP (GFP NB4) or transduced with the HOXB6 (HOXB6 NB4), these cells were grown in vitro for 5– HPC granulopoietic (G) and monocytopoietic (M) 7 days in the presence of different doses of ATRA (from 10−9 liquid suspension culture to 5 × 10−7 m) and then analyzed for both cell surface markers and cell morphology. For analysis of cell surface membrane Step IIIP HPCs were seeded (5 × 104 cells per ml) and grown antigens the cells were labeled with the following PE-labeled in liquid FCS− medium supplemented in G cultures with low monoclonal antibodies: anti-CD11a, anti-CD11b, anti- amounts of IL-3 (1 U/ml) and GM-CSF (0.1 ng/ml) and a satu- CD11c, anti-CD15, anti-CD18 and anti-CD54 (all from rating amount of G-CSF (10 ng/ml) as described;26 in M cul- Pharmingen). Fluorescent cells were analyzed as above and tures with Flt3 ligand (100 ng/ml), IL-6 (10 ng/ml) and M-CSF the results are expressed in terms of both the percentage of (10 ng/ml) as described.27 Cells were incubated in a fully positive cells and mean fluorescent intensity, expressed in

humidified atmosphere of 5% CO2/5% O2/90% N2 and were arbitrary units. periodically counted and analyzed for cell morphology, mem- To evaluate the monocytic differentiation capacities of brane phenotype and HOXB6 . HL60 cells transduced either with the empty vector (GFP

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1295 HL60) or with the HOXB6 gene (HOXB6 HL60), these cells were grown for 4 days in the presence of 1␣-25OH-VitD3 (250 ␮g/ml) and then analyzed for cell surface markers and cell morphology. For analysis of cell surface membrane anti- gens the cells were labeled with the following PE-labeled monoclonal antibodies: anti-CD11a, anti-CD11b, anti- CD11c, anti-CD18, anti-CD14 and anti-CD54 (all from Pharmingen). Fluorescent cells were analyzed as above.

Leukemic patient samples

Cryopreserved cell samples from 96 patients with acute myeloid leukemia, gathered from a single institution, were studied and subclassified according to FAB nomenclature. The original samples contained a range of 20 to 500 × 106 cells each and Ͼ80% blastic infiltration. Leukocytes were isolated by Ficoll–Hypaque density centrifugation, lysed in guanidine and freezed in liquid nitrogen. RNA was isolated by the guani- dium thyocyanate/acid phenol method.28 All the samples were analyzed for the presence of PML/RAR-␣, AML-1/ETO and CBF-␤/MYH11 fusion transcripts according to the primers, protocols and criteria of the Euro- pean BIOMED1 Concerted Action for standardization of mini- mal residual disease (MRD) studies in acute leukemia.30 All assays were carried out using appropriate positive (cDNAs from the NB4, KASUMI 1 and ME-1 cell lines) and negative + controls (all reagents H2O). Controls as well as samples were amplified in parallel with GAPDH primers, as internal control. Figure 1 RT-PCR analysis of HOXB6 mRNA in purified HPCs dif- MLL rearrangements were evaluated by Southern blot analysis ferentiating along the granulocytic (a) or monocytic (b) lineages. HOXB6 mRNA levels were investigated also in circulating neutrophils using 10 ␮g of high molecular weight DNA electrophoresed, 32 (G) and monocytes (Mo), as well as in the positive control, ie ATRA- blotted and hybridized with the appropriate P-labeled induced teratocarcinoma cells (C+). probe.31 Enforced expression of HOXB6 in NB4 promyelocytic cells impaired granulocytic maturation Statistical analysis Given this pattern of HOXB6 expression during the normal granulocytic and monocytic differentiation it seemed of inter- Patient characteristics and complete remission rates were compared using the chi-square test. Overall survival time was calculated from the rate of diagnosis until death or at last fol- low-up examinations. Survival curves were estimated using the product-limit method of Kaplan–Meier and were compared using the log-rank square.

Results

HOXB6 expression during normal granulocytic and monocytic differentiation

Using specific unilineage cell culture systems, allowing the selective differentiation/maturation of purified HPCs along either the granulocytic or monocytic lineages,25–27 we have evaluated HOXB6 mRNA expression by RT-PCR during the different stages of granulocytopoiesis and monocytopoiesis. HOXB6 mRNA was only scarcely expressed in quiescent HPCs and its expression markedly increased during the initial stages of both granulocytic and monocytic differentiation, with a peak of expression around day 5 of culture (Figure 1). At later stages of differentiation/maturation, HOXB6 mRNA Figure 2 Molecular map of PINCO/HOXB6 retroviral construct. The two long terminal repeats (LTR), the Epstein–Barr virus nuclear progressively declined, being undetectable or detectable only antigen-1 (EBNA-1) gene and the gene encoding for the enhanced at very low levels in mature neutrophils and monocytes, green fluorescence protein (GFP), controlled by a cytomegalovirus respectively. promoter (CMV) are shown. The cloning sites (BamHI) are indicated.

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1296 est to evaluate a possible role of this gene in the control of We evaluated the capacity of GFP-NB4 as well as HOXB6- granulo–monocytic differentiation. NB4 cells to undergo granulocytic differentiation using ATRA To evaluate this possible role we transduced with a retrovi- as a chemical inducer of neutrophilic maturation. GFP-NB4 ral vector expressing the HOXB6 gene (Figure 2) a leukemic cells exhibited morphological and phenotypical changes (a cell line capable of differentiating to granulocytes, such as marked enhancement of CD54, CD11b and CD18 expression) NB4 cells. The analogous vector containing GFP alone was typically associated with induction of granulocytic matu- used as a control (GFP). ration, while these changes were only scarcely observed in NB4 cell line did not display HOXB6 mRNA expression HOXB6 expressing NB4 cells (Figure 3b). detectable at the level of sensitivity of RT-PCR and RNAse ATRA dose–response studies showed that at all doses of the protection techniques (data not shown). In contrast, NB4 cells chemical inducer HOXB6-NB4 cells displayed a reduced transduced with HOXB6 gene under the control of a retroviral granulocytic maturation as compared to those observed in promoter constitutively expressed HOXB6 mRNA, at a level GFP-NB4 cells, as evidenced by analysis of the levels of CD54 which remained constant during the differentiation ATRA and CD18 achieved after treatment with different doses of induced (Figure 3a). ATRA (Figure 4).

Figure 3 Expression of the granulocytic differentiation markers in NB4 cells transduced either with PINCO empty vector (GFP) or with PINCO/HOXB6 (HOXB6) expressing vector and treated with ATRA (5 × 10−7 m). (a) HOXB6 transduced NB4 cells were analyzed for HOXB6 mRNA expression at various time point after ATRA induction. (b) Expression analysis of CD54, CD11b and CD18 differentiation markers in GFP and HOXB6-NB4 cells. Mean ± s.e. of three independent experiments is shown.

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1297 the large majority of HL60 cells (wt or transduced with the empty vector) at consistently high levels; however, the CD14 antigen was induced only on about 50% of HOXB6 cells and only at low levels (Figure 5c).

HOXB6 expression in AML

Given these effects of HOXB6 on granulocytic and monocytic maturation it seemed of interest to evaluate HOXB6 expression on leukemic blasts derived from AML patients. Ninety-six cases of AML, subdivided according to the FAB classification, were screened for HOXB6 expression. Interestingly, a consistent proportion (18/49) of AMLs lack- ing major translocations were found to be HOX B6+ (ranging from 22 to 60% of AMLs classified as M1, M2, M4 M5 and M6 by FAB) (Table 1); the cases expressing the highest levels of HOXB6 mRNA were found among M2 and M4 leukem- ias (Figure 6). By contrast, almost all AMLs (45/47) characterized by fusion genes underlying most commons translocation showed HOX B6 expression to be absent (Table 2). In particular, leukemic blasts from 15 cases of M3 PML/RAR-␣+ were constantly HOXB6 negative (Table 2); similarly, HOXB6 expression was undetectable in 12 cases of AML with t(8;21), AML-1/ETO+, in 10/11 cases of AML with inversion of 16, CBF␤/MYH11+, and in eight out of nine AML with MLL rearrangements. The frequency of HOXB6 expression was sig- nificantly (P Ͻ 0.001) different in the two AML groups with or without major translocations. M3 leukemic blasts tested negative for HOXB6 expression even after in vitro treatment with ATRA, which downregulates PML/RAR-␣ expression and induces granulocytic maturation of these cells (data not shown). Clinical outcome according to HOXB6 expression was investigated in the group of 49 patients with normal karyo- Figure 4 Dose–response analysis of the effects of increasing con- centrations of ATRA on CD54 and CD18 expression in GFP and type. HOXB6-NB4 cells, analyzed at day 7. A representative experiment Patients with positive HOXB6 expression and patients with is shown. negative HOXB6 expression had essentially identical com- plete remission rates of 66.6% and 62.7% (P = 0.43). There was a trend towards longer median survival in patients lacking HOXB6 expression: median survival 26 months (range 1–86) vs median survival of 15 months (range 1–84); however, this Constitutive HOXB6 expression in HL-60 cells difference did not achieve statistical significance (P = 0.24). inhibited monocytic maturation

Since the pattern of HOXB6 expression during normal mono- Discussion cytopoiesis strictly resembles that observed during granulopo- iesis, it seemed of interest to evaluate whether a constitutive HOXB6 expression in HL-60 could impair the capacity of In recent years, several members of the homeobox gene family these cells to undergo monocytic maturation in response to have been identified as important regulators of hemopoietic treatment with 1␣25OH-VitD3. Wild-type HL-60 cells did not cell differentiation. The expression patterns of these genes express HOXB6 mRNA at detectable levels by RT-PCR and have been extensively studied in cell lines and, to a less RNAse protection (data not shown), while a high and consti- extent, in normal hemopoietic cells (reviewed in Refs 9 and tutive HOXB6 expression was observed in HL-60 cells trans- 10). Several of the HOX genes exhibited a temporal pattern duced with HOXB6 gene and such expression remained of expression, with a peak associated with the early stages of unmodified after incubation of the cells with 1␣25OH-VitD3, HPC differentiation and downregulation of several HOX genes a chemical inducer of monocytic maturation (Figure 5a). After being associated with later differentiation.12,13 4 days of treatment with this inducer wild type (wt) as well as HOXB6 is a prototype of these HOX genes, its expression HL-60 cells transduced with the empty vector (GFP) being restricted to the early phases of HPC differentiation (Ref underwent monocytic maturation according to morphological 13 and the present paper), with downmodulation at and phenotypical criteria. However, these changes were gre- intermediate/late stages of maturation. atly inhibited in HL60 cells expressing the HOXB6 gene Given this peculiar pattern of expression it seemed of inter- (Figure 5b). The analysis of the kinetics of induction of CD14, est to evaluate the role of this HOX gene in granulocytopoiesis a membrane antigen specifically expressed in monocytes, and in monocytopoiesis. Enforced expression of HOXB6 in showed that this antigen was induced by 1␣25OH-VitD3 on NB4 and HL60 cell lines, which do not express HOXB6, elic-

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1298

Figure 5 Effect of HOXB6 enforced expression in HL60 cells on the monocytic maturation induced by 1␣25OH-VitD3 (250 ␮g/ml). (a) HOXB6 transduced HL60 cells were analyzed for HOXB6 mRNA expression during monocytic differentiation. (b) Left: analysis of the expression of CD11b, CD18, CD11a and CD14 in GFP and HOXB6 expressing HL60 cells treated for 4 days in the presence of 1␣25OH-VitD3. Right: percentage of cells identified as mature monocytes in cells grown as above. Mean ± s.e. of three independent experiments is shown. (c) Kinetic analysis of the expression of CD14 in wild-type, GFP and HOXB6-HL60 cells: the data are reported in terms of both the percentage of positive cells (left) and of the mean fluorescence intensity (right). A representative experiment is shown.

Table 1 HOXB6 expression in acute myeloid leukemic samples of HOXB6 on granulocytic and monocytic maturation lacking major translocations suggests that a silencing of this HOX gene is required for the normal development of granulopoiesis and monocytopoiesis. Sample FAB HOXB6-positive sample No. HOXB6-positive It is of interest that the effects of HOXB6 are different from No. frequency those previously reported for HOXB7 in the HL60 cellular sys- tem. In fact, overexpression of HOXB7 in these cells elicited 1 M0 — 0/1 + + an inhibition of ATRA-induced granulocytic maturation, but 2–10 M1 2( ); 3( ) 2/9 ␣ 11–23 M2 11(++++); 12(++); 13(+); 14(+) 4/13 did not affect the monocytic maturation induced by 1 25OH- 24–33 M4 24(++); 25(+); 26(+); 27(±); 28(±) 5/10 VitD3.32 However, at variance with HOXB6, HOXB7 34–44 M5 34(+); 35(+); 36(+); 37(±) 4/11 expression was induced during VitD3-driven maturation of 45–49 M6 45(+); 46(±); 47(±) 3/5 HL60 cells. Previous studies on HOXB6 have suggested a possible role 18/49 of this gene in the control of erythropoiesis. Thus, transfection of the K562 erythroid cell line with a HOXB6 antisense con- Expression was detected by RT-PCR analysis as shown in Figure 6. struct induced an increase of erythroid features, while an Bands were quantitated by densitometric scanning of autoradio- opposite phenomenon was observed when the cells were graphs, using the value (+) when the band was similar to the posi- 11 tive control (ATRA-induced NT2/D1 teratocarcinoma cells). See transfected with a HOXB6 sense. Furthermore, it was shown also Figure 6 and Materials and methods section. that during embryonic/fetal murine development HOXB6 expression is closely associated with the regulation of the erythropoietic system.19 In the present study, we show that a ited a marked inhibition of granulocytic and monocytic matu- regulated pattern of HOXB6 expression is also required for ration, respectively. The inhibition of ATRA-induced granulo- normal granulocytic and monocytic differentiation. cytic maturation of NB4 cells is particularly interesting, as There is growing evidence indicating that HOX retinoic acid is known to regulate homeobox gene expression, play a role in leukemic transformation (reviewed in Ref. 33): including HOXB6, in other cell types.29 This inhibitory effect (1) in AMLs, several HOXA, HOXB and HOXC genes are

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1299

Figure 6 RT-PCR analysis of HOXB6 mRNA in leukemic blasts derived from AMLs lacking major translocations. Only positive cases are shown, see also Table 1. C+ indicates ATRA-induced teratocarcinoma cells used as positive control. The expression pattern of HOXB6 gene was confirmed in at least two other experiments.

Table 2 HOXB6 expression in acute myeloid leukemic samples kemic blasts in that it was similarly expressed in different sub- ␣ ␤ characterized by PML/RAR- , AML-1/ETO, CBF /MYH11 fusion and types of AML, including M1, M2, M4, M5 and M6. rearrangements of the MLL gene at 11q23 Furthermore, with the exception of only two cases, HOXB6 expression was found to be absent in AMLs positive for the Sample No. Molecular HOX B6-positive HOX B6-positive defect sample No. frequency expression of leukemogenetic fusion proteins (such as PML/RAR-␣, MLI-1/ETO, CBF␤/MYH11 and MLL gene 1–15 PML/RAR␣ — 0/15 rearrangements). These AMLs account for about 40% of total 16–27 AML-1/ETO — 0/12 AMLs and for the large majority (Ͼ90%) of AMLs with known 28–38 CBF␤/MYH11 28(±) 1/11 oncogenetic molecular defects.21 39–47 MLL/SOUTH 39(+) 1/9 Since it was previously reported that HOXC6, paralogue to HOXB6, was expressed in AML-M3,34 we analyzed the 2/47 expression of this gene in all M3 blasts. We detected HOXC6 expression in three out 15 cases (data not shown). Expression was detected by RT-PCR analysis. Bands were quantit- In particular, M3 leukemias were constantly found to be ated by densitometric analysis (see also legend to Table 1). negative for HOXB6 expression, which remained absent fol- lowing ATRA treatment. Reasons underlying this finding are at present unclear to us. Nevertheless, it is of interest that also in two previous studies HOXA935 and HOXA1036 were found to be expressed in all types of AMLs, with the constant excep- expressed, while in lymphoid malignancies, HOXC genes are tion of M3 leukemias. Based on those observations and on the preferentially expressed; (2) in rare cases of leukemia HOX results of the present study, it is tempting to suggest that HOX genes are mutated, such as the HOXA9 gene fused together gene expression is suppressed in M3 leukemias probably with the nucleoporin gene as the consequence of the t(7;11) through an aberrant signaling mediated by the PML/RAR-␣.37 translocation.18 HOX gene expression in several cell types is stimulated fol- This is the first study which reports the pattern of HOXB6 lowing activation of retinoic acid receptors;29 thus, it may be expression in a large number of AMLs. We observed HOXB6 hypothesized that an aberrant signaling through the RAR-␣ expression in about 35% of AMLs lacking major translo- fused with PML suppresses the stimulatory effect of normal cations, with some cases of M2 and M4 leukemia expressing RARs on HOX gene expression. particularly high levels of HOXB6 mRNA (see Table 1 and Whatever mechanism is involved in HOXB6 expression in Figure 6). The expression pattern of HOXB6 in AMLs does not AMLs, this phenomenon does not seem to be related to seem to be related to the differentiation potential of the leu- clinical outcome.

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1300 This striking difference among different types of AMLs sug- involvement in human cancer. Leuk Lymphoma 1995; 16:209– gests that HOXB6 expression in AMLs could be specifically 215. associated with leukemias whose molecular defects remain to 18 Borrow J, Shearman AM, Stanton VPjr, Becher R, Collins T, Willi- ams AJ, Dube I, Katz F, Kwong YL, Morris C, Ohyashiki K, Toyama be determined. K, Rowley J, Housman DE. The t (7;11) (p15; p15) translocation in acute myeloid leukemia fuses the genes for nucleoporin NUP 98 and class I homeoprotein HOX A9. Nat Genet 1996; 12: Acknowledgements 159–167. 19 Zimmermann F, Rich IN. Mammalian homeobox B6 expression can be correlated with erythropoietin production sites and erythro- The authors are indebted to Dr Alessandra Care` for critical poiesis during development, but not with hematopoietic or non- reading of the manuscript and helpful suggestions. We thank hematopoietic stem cell populations. Blood 1997; 89: 2723- Giuseppe Loreto and Alfonso Zito for photographic assistance. 2735. This work was supported in part by grant ‘Homeobox genes 20 Ohnishi K, Tobita T, Sinjo K, Takeshita A, Ohno R. Modulation in adult hematopoiesis’ from Istituto Superiore di Sanita` of homeobox B6 and B9 genes expression in human leukemia cell lines during myelo-monocytic differentiation. Leuk Lymphoma and by grants from AIRC, MURST 70% and Ministero della 1998; 31: 599–608. Sanita`. 21 Look AT. Oncogenic transcription factors in the human acute leu- kemias. Science 1997; 278: 1059–1064. 22 Shen WF, Detmer K, Simonitch-Eason, TA, Lawrence HJ, Largman References C. Alternative splicing of the HOX 2.2 homeobox gene in human hematopoietic cells and murine embryonic and adult tissues. Nucleic Acids Res 1991; 19: 539–545. 1 Weissman IL. Stem cells: units of development, units of regener- 23 Grignani F, Kinsella T, Mencarelli A, Valtieri M, Riganelli D, Grig- ation, and units in evolution. Cell 2000; 100: 157–168. nani F, Lanfrancone L, Peschle C, Nolan GP, Pelicci PG. High 2 Fuchs E, Segre JA. Stem cells: a new lease on life. Cell 2000; 100: efficiency gene transfer and selection of human hematopoietic 143–155. progenitor cells with a hybrid EBV/retroviral vector expressing the 3 Krumlauf R. Cell 1994; 78: 191–201. green fluorescence protein. Cancer Res 1998; 58: 14–19. 4 Deschamps J, Meijlink F. Mammalian homeobox genes in normal 24 Gabbianelli M, Sargiacomo M, Pelosi E, Testa U, Isacchi G, development and neoplasia. Crit Rev Oncol 1992; 3: 117–173. Peschle C. ‘Pure’ human hematopoietic progenitors: permissive 5 Mavilio F. Regulation of homeo-box containing genes by morpho- action of basic fibroblast growth factor. Science 1990; 249: gens. Eur J Biochem 1993; 212: 273–288. 1561–1564. 6 Scott MP. Vertebrate homeobox genes nomenclature. Cell 1992; 25 Labbaye C, Valtieri M, Testa U, Giampaolo A, Meccia E, Sterpetti 71: 551–553. P, Parolini I, Pelosi E, Bulgarini D, Cayre Y, Peschle C. Retinoic 7 Lawrence HJ, Largman C. Homeobox genes in normal hematopo- acid downmodulates erythroid differentiation and GATA-1 iesis and leukemia. Blood 1992; 80: 2445–2453. expression in purified adult progenitor culture. Blood 1994; 83: 8 Giampaolo A, Pelosi E, Valtieri M, Montesoro E, Sterpetti P, Sam- oggia P, Camagna A, Mastroberardino G, Gabbianelli M, Testa U, 651–656. Peschle C. HOXB gene expression and function in differentiating 26 Labbaye C, Valtieri M, Barberi T, Meccia E, Masella B, Pelosi E, purified hematopoietic progenitors. Stem Cells 1995; 13 Condorelli G, Testa U, Peschle C. Differential expression and (Suppl. 1): 90–105. functional role of GATA-2, NF-E2 and GATA-1 in normal adult 9 Magli MC, Largman C, Lawrence HJ. Effects of HOX homeobox hematopoiesis. J Clin Invest 1995; 95: 2346–2358. genes in blood cell differentiation. J Cell Physiol 1997; 173: 27 Gabbianelli M, Pelosi E, Montesoro E, Testa U, Peschle C. Multi- 168–177. level effects of FLT3 ligand on human hematopoiesis: expansion 10 Van Oostveen JW, Raaphorst FM, Walboomers JM, Mejer CJLM. of putative stem cells and proliferation of granulo–monocytic The role of homeobox genes in normal hematopoiesis and hema- progenitors/monocytic precursors. Blood 1995; 86: 1661–1670. tological malignancies. Leukemia 1999; 19: 1675–1690. 28 Chomczynski P, Sacchi N. Single-step method of RNA isolation by 11 Shen WF, Detmer K, Mathews CHE, Hack FM, Morgan DA, Larg- acid guanidinium thiocyanate-phenol-chloroform extraction. Anal man C, Lawrence HJ. Modulation of homeobox gene expression Biochem 1987; 162: 156–159. alters the phenotype of human hematopoietic cell lines. EMBO J 29 Simeone A, Acampora D, Arcioni L, Andrews PW, Boncinelli E, 1992; 11: 983–989. Mavilio F. Sequential activation of HOX2 homeobox genes by reti- 12 Sauvageau G, Landsorp PM, Eaves CJ, Hogge DE, Dragowska WH, noic acid in human embryonal carcinoma cells. Nature 1990; Reid DS, Largman C, Lawrence H.J, Humphries RK. Differential 346: 763–766. expression of homeobox genes in functionally distinct CD34+ sub- 30 van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, populations of human bone marrow cells. Proc Natl Acad Sci USA Saglio G, Gottardi E, Rambaldi A, Dotti G, Griesinger F, Parreira 1994; 91: 12223–12227. A, Gameiro P, Diaz MG, Malec M, Langerak AW, San Miguel JF, 13 Giampaolo A, Sterpetti P, Bulgarini D, Samoggia P, Pelosi E, Valti- Biondi A. Standardized RT-PCR analysis of fusion gene transcripts eri M, Peschle C. Key functional role and lineage-specific from chromosome aberrations in acute leukemia for detection of expression of selected HOXB genes in purified hematopoietic pro- minimal residual disease. Leukemia 1999; 13: 1901–1928. genitor differentiation. Blood 1994; 84: 3637–3647. 31 Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, 14 Sauvageau G, Thorsteindottir U, Eaves CJ, Lawrence HJ, Largman Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sand- C, Lansdorp PM, Humphreis RK. Overexpression of HOXB4 in berg AA. Rearrangement of the MLL gene in acute lymphoblastic hematopoietic cells causes the selective expansion of more primi- and acute myeloid leukemias with 11q23 chromosomal translo- tive populations in vitro and in vivo. Genes Dev 1995; 9: cations. N Engl J Med 1993; 329: 909–914. 1753–1765. 32 Lill MC, Fuller JF, Herzig R, Crooks GM, Gasson JC. The role of 15 Care` A, Testa U, Bassani A, Tritarelli E, Montesoro E, Samoggia the homeobox gene HOX B7, in human myelomonocytic differen- P, Cianetti L, Peschle C. Coordinate expression and proliferative tiation. Blood 1995; 85: 692–697. role of HOXB genes in activated adult T lymphocytes. Mol Cell 33 Buske C, Humphries RK. Homeobox genes in leukemogenesis. Int Biol 1994; 14: 4872–4877. J Hematol 2000; 71: 301–308. 16 Quaranta MT, Petrini M, Tritarelli E, Samoggia P, Care` A, Bottero 34 Bijl JJ, van Oostveen JW, Walboomers JMM, Brink ATP, Vos W, L, Testa U, Peschle C. HOXB cluster genes in activated natural Ossenkoppele GJ, Meijer CJLM. Differentiation and cell-type- killer lymphocytes: expression from 3′ to 5′ cluster side and pro- restricted expression of HOXC4, HOXC5 and HOXC6 in myeloid liferative function. J Immunol 1996; 157: 2462–2469. leukemias and normal myeloid cells. Leukemia 1998; 12: 17 Lichthy BD, Ackland-Snow J, Noble L, Kamel-Reid S, Dube ID. 1724–1732. Dysregulation of HOX 11 by chromosome translocations in T-cell 35 Lawrence HJ, Rozenfeld S, Cruz C, Matsukuma K, Kwong A, acute lymphoblastic leukemia a paradigm for homeobox gene Komuves L, Buchberg AM, Largman C. Frequent co-expression of

Leukemia HOXB6 expression in myelomonocytic differentiation and AML A Giampaolo et al 1301 the HOXA9 and MEIS1 homeobox genes in human myeloid leuke- 37 Grignani F, Fagioli M, Alacaly M, Longo L, Pandolfi PP, Donti E, mias. Leukemia 1999; 13: 1993–1999. Biondi A, Lo Coco F, Grignani F, Pelicci PG. Acute promyelocytic 36 Lawrence HJ, Sauvageau G, Ahmadi A, Lau T, Lopez AR, LeBeau leukemia: from genetics to treatment. Blood 1994; 83: 10–25. MM, Largman C. Stage and lineage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells. Exp Hematol 1995; 23: 1160–1166.

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