Leukemia (1999) 13, 1675–1690  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu REVIEW

The role of Homeobox genes in normal hematopoiesis and hematological malignancies JW van Oostveen1, JJ Bijl2, FM Raaphorst2, JJM Walboomers2 and CJLM Meijer2

Departments of 1Hematology and 2Pathology, University Hospital, Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

In the last decade it has become clear that homeobox contain- Table 1 Grouping of gene families, coding for transcription fac- ing genes (HOX genes) not only play a significant role in reg- tors, according to their DNA binding motif,244 and their reported func- ulating body formation, but in addition, they are contributing tions to organization and regulation of hematopoiesis. Modern mol- ecular technologies showed that deregulated expression or DNA binding- Gene (family) Function disruption of Hox genes can lead to altered characteristics of domain blood cells or disturbance of blood cell development. In this paper we review the role of HOX proteins in hematopoiesis and leukemogenesis and speculate about their possible target Helix-turn-helix HOX genes embryonic genes and involvement in lymphomagenesis. development, Keywords: transcription factors; homeobox genes; embryogen- organogenesis, esis; hematopoiesis; lymphoma; adhesion hematopoiesis Zinc finger GATA genes/E2A/Pax5 hematopoiesis, endodermal differentiation245 Transcription factors Zinc finger LIM Rbtn2/Lmo2 Embryonic red blood family cells265 Runt AML1(CBFA2) Fetal liver Transcription factors are sequence-specific DNA-binding pro- 266 teins with a variety of functions: some are thought to help fold hematopoiesis Basic helix- SCL gene (tal-1)/c-myc hematopoietic the DNA molecule into distinct domains; others assist in the loop-helix proteins/E2A commitment and initiation of DNA replication, and many control gene tran- differentiation194,246,247 scription. Binding affinity to DNA sequences, the concen- Basic leucine c-fos/c-jun Proliferation248 tration of transcription factors and the presence of cofactors zipper are important determinants in gene activation. Transcription Winged helix forkhead/HNF-3 family embryogenesis, liver factors can be classified according to the three-dimensional organogenesis, tumorigenesis249 structure of their DNA-binding domains. More than 80% of ETS domain ets family Hematopoiesis31,250 all transcription factors are characterized by zinc finger, helix- Rel homology Rel/NF-kappa B family regulation of immune turn-helix, helix-loop-helix, leucine zipper and winged helix domain and inflammatory motifs (Table 1). Moreover, the complexity of transcriptional processes, communication regulation is increased by formation of functional hetero- or 251 homodimers of transcription factors (for instance c-fos and c- between cells 1 HMG-box HMG family (Tcf-1/Lef-1) B and T cell jun ), or by participation in large protein complexes (for development252 instance c-myc). Transcription of a particular gene is an event regulated by a complex network of transcription factors. The HNF-3, hepatocyte nuclear factor; HMG, high mobility group; HOX, total set of transcription factors present in a cell – induced by homeobox; the Table is modified from Clevers HC, Grosschedl R. extracellular signals – provides a specific genetic imprint that Transcriptional control of lymphoid development: lessons from results in a distinct response. Transcription factors have a gene targeting. Immunol Today 1996; 17: 336–343. regulatory role in proliferation and in all kinds of differen- tiation processes, like embryogenesis, organogenesis, and also hematopoiesis (Table 1).2–9 ensure its own survival by proliferation before differentiating into all lineages of blood cells. This balance of proliferation and differentiation is highly dynamic, and pluripotent stem Hematopoiesis cells and progenitor cells are produced in relation to physio- logical stresses of the organism (such as bleeding, or Specialized blood cells of all lineages originate from the pluri- infection). The intriguing question arises how this process of potent stem cell by hematopoiesis. Blood cells have a limited blood cell development is controlled, and which genes are life-span, and new cells are generated continuously through- involved in this complex process. out life. The ancestral pluripotent stem cell must therefore Hematopoiesis during embryogenesis

Correspondence: JW van Oostveen, Department of Hematology, Uni- The anatomical site of hematopoiesis changes during early life versity Hospital Vrije Universiteit (VU), De Boelelaan 1117 Amster- 10 11 dam, PO Box 7057, NL-1007 MB Amsterdam, The Netherlands; Fax: phases (reviewed by Zon and Auerbach ). During 31 20 4442601 embryogenesis, early hematopoiesis occurs in the yolk sac, Received 30 December 1998; accepted 12 July 1999 starting in humans at day 15 and continuing for 6 weeks. Review JW van Oostveen et al 1676 Hematopoietic stem cells – derived from the ventral meso- early hematopoietic progenitor cells into the lymphoid lin- derm – and vascular progenitors migrate to the yolk sac to eage.43,44 Several other genes specifically involved in the lym- form blood cell islands.12 These islands consist of an endoder- phopoiesis have been found, such as Pax5/BSAP, E2A, Oct-2, mal layer (supporting growth), a central core of hematopoietic PU.1, Ets-1 and Tcf-1.45–53 Of these transcription factors both stem cells, and an endothelial layer that surrounds the blood Pax5 and Oct-2 genes belong to the HOX class II genes, which islands.13 Although progenitors of most blood cells are contain a homeobox in addition to another characteristic present, these stem cells predominantly differentiate into sequence (see below). erythroid cells and occasionally into monocytes and megakar- yocytes.14–16 They are primarily important for embryonic hem- atopoiesis17 and die during further development of the Development of the lymphoid lineage embryo. In the embryo itself, an additional population of hem- atopoietic stem cells is present in the dorsal mesentery (also The stem cell first differentiates into a lymphoid and a myeloid called the aorta gonad mesonephros region, or AGM). This progenitor cell. All lymphocytes are derived from a common population initiates definitive hematopoiesis (including that of lymphoid progenitor cell. Differentiation is accompanied by the lymphoid lineage), first in fetal liver and spleen, and sub- changes in morphology and immunophenotype, eg loss of sequently in bone marrow (BM).17 BM remains the primary stem cell marker CD34 during differentiation and induction hematopoietic organ in adult life. Developmentally controlled of lineage-specific markers such as CD19 and CD22. Lympho- changes in location of blood cell development requires coor- cytes are responsible for the body’s response to foreign anti- dinate regulatory decisions in cell fate and reorganization of gen. As the number of potential antigens is enormous, this cells. requires a large pool of lymphocytes, each displaying speci- ficity for a single antigen. Antigen responses are divided into the humoral immune response, mediated by B cells, and the Transcription factors in hematopoiesis cellular immune response, mediated by T cells. Stimulated by specific cytokines like IL–7 and Flt3 ligand, Hematopoiesis includes many decisions in lineage commit- B cells initially develop in the BM. Their maturation is ment that depend on coordinate expression of lineage-specific accompanied by rearrangement of immunoglobulin (Ig) genes genes. This development program is governed by transcription encoding their membrane-associated antigen receptor. This factors. Blood cell development is particularly complex produces a large population of B cells each expressing their because it occurs at different body sites, partly determined by unique Ig molecules. Immature B cells with a self-reactive developmental stage. Fetal hematopoiesis occurs in the liver, receptor are negatively selected before they leave the BM. whereas in adults it is initiated in BM. Lymphoid cells reach After migration and homing to lymph nodes, follicular B cells their final maturation stage in peripheral lymphoid organs, that recognize the appropriate antigen on follicular dendritic such as lymphnodes, spleen and extranodal lymphoid tissues. cells continue to mature in the germinal center reaction.54 The microenvironment of these specific body sites has a pro- During this process cells with an antigen-specific Ig receptor found influence on the differentiation program.18,19 Therefore, are selected to survive,55 and will further differentiate into genes that can specify positional information, such as homeo- plasma cells and memory B cells. Cells without antigen speci- box (HOX) genes, might be strongly related with the control ficity, or expressing a self-specific antigen receptor, undergo of hematopoiesis.20 programmed cell death (apoptosis). Expression studies have revealed a number of transcription T cell development occurs primarily in a separate organ, factor genes that are active in specific cell lineages and differen- the thymus. This gland is formed from the third pharyngeal tiation stages during hematopoiesis. Their function was clarified pouch and consists of well-defined cortical and medullar in vitro by transfection studies, and in vivo by knock-out and zones. Immature prothymocytes enter the thymus, attracted by transgenic mice experiments. Disruption of transcription factor chemotactic factors. Here, they interact with stromal cells that genes often resulted in a block of terminal differentiation of produce lineage-specific cytokines such as IL-7, stem cell fac- particular hematological cell types, disclosing their role in a tor (SCF), Flt3 ligand and tumor growth factor (TGF). This particular lineage. Homeobox genes were originally identified drives the T cells into further differentiation.56 T cells acquire as master control genes during embryonic development, but antigen receptor diversity by rearrangement of T cell receptor they affect various steps in hematopoiesis as well.21–32 These (TCR) genes,54 followed by selection for T cells reacting with genes, the class I homeobox genes, are the subject of this non-self antigens.57 This process generates various types of T- review. Although knock-out mice have been generated for sev- helper cells (CD4 positive) and cytotoxic T cells (CD8 eral genes of the HOX gene family, defects in hematopoiesis positive).58,59 Commitment and differentiation of cells to the of such knock-out mice have only recently been described for lymphoid lineage is under precise genetic control, and there the HOXA9 gene.33 HOXA9 knock-out mice exhibit disturbed is evidence that especially the HOXC cluster plays a role in differentiation of myeloid and megakaryocytic cells. Early T cell this regulatory process.26,27 development is severely affected and associated with decreased intracellular expression of CD3 and the TCR ␤-chain, while at the cell surface expression of IL-7R and E cadherin are Development of non-lymphoid lineages reduced.34 Members of the GATA gene family appear crucial for development of various cell types, like GATA-1 for erythroid All non-lymphoid blood cells are derived from a common cells,35–38 and GATA-3 for T-cells.39 By contrast, GATA-2 plays myeloid progenitor cell. Progenitors differentiate along parti- a role in differentiation of all hemopoietic lineages, suggesting cular lineages to mature blood cells, under influence of a that this gene is required for the expansion of the early progeni- multilineage stimulating factor (IL-3) and lineage-restricted tor cell pool.40–42 Lack of the Ikaros gene resulted in the com- growth factors (eg IL-5, Epo (erythropoietin) and GM-CSFs plete lack of lymphoid cells (including early lymphoid (granulocyte–macrophage colony-stimulating factors).60 This progenitors), suggesting that Ikaros affects differentiation of is accompanied with downregulation of the progenitor marker Review JW van Oostveen et al 1677 CD34 and induction of lineage-specific markers such as CD15 first discovered homeobox gene of Drosophila, called (granulocyte), CD14 and CD68 (monocyte/microphage), Antennapedia. The class II, or diverged homeobox genes, CD41 and CD42 (megakaryocyte) and H-antigen, which exhibit Ͻ50% homology to the Antennapedia gene. Class II forms the backbone of the ABO blood group proteins on homeobox genes are grouped in several families, based on erythrocytes.61 Cells also undergo morphological changes, the presence of additional conserved sequences, such as the which are most dramatic for erythroid and megakaryocytic paired box in genes of the Pax family and the POU box in development. This results in denucleated erythrocytes and the POU domain family (including members as Oct-1, 2, 3 platelets, respectively.62 Commitment and differentiation of and 4 and Pit-1).81 In this review we only discuss the homeo- myeloid cells is carefully controlled. Genes of the HOX cluster box containing class I gene cluster. are involved in this process: HOXA for the myeloid lineage, Human class I genes are called HOX genes in the new HOXB for the erythroid lineage. Recent studies suggest nomenclature.82,83 HOX are organized in clusters, and human that HOXC genes might also be involved in myeloid HOX elements can partly be aligned with those in Drosphila differentiation.32 (Figure 2). Drosophila homeobox genes encompass the antennapedia-complex (ANT-C) and bithorax-complex (BX- C),84 located on one chromosome. The 39 human HOX genes Homeobox genes are organized in four clusters (HOXA, -B, -C and -D), located 83,85–87 General aspects of homeobox genes on chromosomes 7, 17, 12 and 2, respectively. Each cluster contains a minimum of nine HOX genes. HOX genes Homeobox genes are characterized by the presence of a con- belonging to different clusters but with highly homologous served 183 bp sequence, the homeobox (Figure 1) first disco- homeodomains, can be positionally aligned on the chromo- vered in genes of the fruit-fly Drosophila melanogaster.63 some. This arranges HOX genes in 13 paralogue groups; for Nuclear magnetic resonance and crystallographic studies instance, paralogue group one consists of HOXA1, HOXB1 showed that the three-dimensional structure of the homeobox and HOXD1 (Figure 2). The homology between the homeodo- is a helix-turn-helix motif, consisting of four helices.64–68 This mains of HOX genes belonging to the same paralogue group motif recognizes DNA sequences having core sequences rang- is higher than between those of one cluster, suggesting that ing from TGAT to TAAT69,70 to TTAT and TTAC. Regions out- first a tandem duplication and subsequently a duplication of 80,88–90 side the homeobox in the variable domain provide binding the complete cluster occurred during evolution. The specificity to homeoproteins for specific DNA sequences. As genomic organization of HOX genes and cis-regulatory explained below in the process of binding to DNA, homeo- sequences suggest that processes involving enhancer sharing proteins appear to display cooperative binding to other form the basis for highly orchestrated expression of HOX regulatory proteins. genes, necessary for the guidance of sequential steps in the 91–93 Embryonic expression studies and analysis of homeotic differentiation pathways. mutants revealed that homeobox genes specify cell identity and positioning in the developing embryo.19,71–74 Some mutations in homeobox genes resulted in the duplication of HOX gene expression intact morphological structures to anterior or posterior located segments in Drosophila. For example, the Drosophila home- Each of the four clusters covers a distance of approximately 81 otic mutant called Antennapedia resulted in formation of an 150–200 kb on the chromosome. Each single gene has a additional pair of legs on the head, instead of antennae.75–78 polyadenylation site and is thought to have its own promoter. Ј The identification of homeobox genes in many other species Normally, at the 5 end of the homeobox gene, one or more indicates that homeobox genes provide an evolutionary introns are present. HOXC4, HOXC5 and HOXC6 are parti- conserved elementary regulatory mechanism for major cular because they are also under control of a master pro- 94,95 differentiation programs.78–80 moter. It is not known whether transcription from the common promoter or the individual promoters is regular for these genes. Organization of homeobox genes at the chromosomal Most HOX genes give rise to more than one RNA transcript, level generated by the use of alternative splice sites or by differen- tial promoter use, as has been shown for HOXC6 and Homeobox genes are categorized in two large groups. The HOXA9.96–98 Alternative splice variants were described that Ͼ class I homeobox genes are highly homologous ( 80%) to the are functionally equivalent.99 The distinct tissue distribution of alternative transcripts of several HOX genes implies that there are also alternative transcripts that might encode func- tionally different proteins. However, it is not known whether all alternative transcripts encode functional proteins.97,98,100 Several mechanisms have been identified that regulate expression of HOX genes. The identification of TAAT Figure 1 Structure of the homeobox protein. Homeobox proteins sequences and retinoic acid response elements in upstream are composed of several structural units, including a variable region, regions allows the binding and activation or repression by which determines a protein’s specific activity, a small connective homeoproteins101–106 (autoregulation) and activation of hinge region (IYPWM) and a homeodomain of 60 amino acid expression by retinoic acid.107–116 Early HOX gene expression sequence, encoded by the homeobox, that is similar in all these pro- in Drosophila is dependent on segmentation genes of the pair- teins. The aminoterminal region of the proteins often begin with rule (activators) and gap (repressor) families. During later MSSLYYXN and the carboxyterminal domain tends to be acidic, indi- cating that it might act as a transcriptional domain. The homeodomain stages of development, the maintenance of HOX expression consists of four alpha helices (1–4), one of which (black) recognizes is guided by the polycomb (Pc-G) and trithorax (Trx-G) and binds to a specific DNA sequence in the target genes. genes.117–122 Pc-G genes repress HOX-gene transcription, Review JW van Oostveen et al 1678

Figure 2 Comparative organization of HOX complexes. The alignments of the Drosophila and human complexes and chromosomal localiza- tions are shown. Gray intensities indicate similar homeodomain sequences. The order of the genes along the chromosome is as shown, with the order roughly corresponding to where along the anterior–posterior body axis gene is expressed or, in the case of mammals, the anterior- most limit of expression. Categories 1–13 are called ‘paralogue’ groups and are indicative of homeodomain sequence similarity. Black ovals indicate the lack of a paralogue from a cluster. The assignment of the Drosophila homeobox genes Scr (Sex comb reduced), Ant (Antennapedia), Ubx (Ultrabithorax) and abd-A (Abdominal-A) to any particular paralogue group is uncertain. Paralogue group 10–13 do not have homologues in the Drosophila homeobox gene clusters. The direction of transcription from each gene is indicated by an arrow. Note that genes early expressed in the embryo are more sensitive to retinoic acid (RA) than later expressed genes. Modified from Manak and Scott.243

while Trx-G genes contribute to strong expression.123–125 Fol- genital malformations demonstrated the involvement of HOX lowing their discovery in Drosophila, Pc-G genes have also genes mutations in these syndromes.145–147 Furthermore, stud- been identified in mammals, including humans, where they ies with knock-out mice revealed the involvement of HOX11 appear to have a similar role.125–131 The mixed lineage-, or in the spleen,148 Pax1 and Hoxa-3 in the developing thy- myeloid-lymphoid, leukemia gene (MLL) is the only known mus149–150 while Hoxa3, -b3 and -d3 mutant mice show spe- human Trx-G gene.132,133 Human Pc-G genes are differentially cific disturbance of thymus, parathyroid and thyroid develop- transcribed during development of CD34+ into CD34− BM ment.151 Similarly, mice expressing a Hlx homeobox progenitor cells,131 but other than that little else is known transgene exhibit severe disturbance of both T and B cell about their role in hematopoiesis. development.152 Thus, HOX genes not only play a role in development of the embryo, but they also contribute to regu- lation of hematopoiesis. Importantly, this holds true for both HOX gene expression in embryogenesis and fetus and adult. In the following sections, we shall turn our organogenesis attention to the significance of HOX genes for control of hem- atopoiesis, and their involvement in oncogenesis. Furthermore The physical order of the genes is also reflected by the temporal HOX genes might be involved in different types of human sequence of their activation during embryogenesis and organo- solid tumors,142,143,153–155 however, these malignancies are genesis.134–136 The expression order along the anterior–pos- beyond the scope of this review. terior axis of the embryo is colinear with the 3Ј–5Ј organization of HOX genes on the chromosomes (Figure 2).3,4,76,137–139 Moreover, transcripts of a given HOX gene can usually not be Homeobox genes in hematopoiesis detected in the developing embryo until the product of its 3Ј neighbor is present. This so-called temporal colinearity may HOX gene expression in hematopoietic cell lines occur only in cells with high mitotic rates, resulting in a strong linkage between patterning and growth control and an uni- Analysis of human and mouse hematopoietic cell lines directional pattern process.140 The expression domains of HOX revealed that some HOX genes were only expressed in cell genes in vertebrates have marked anterior boundaries, but less lines of one hematopoietic lineage, while other HOX genes sharp posterior boundaries, and this results in extensive overlap were expressed in cell lines of several lineages.42–44,156–162 of expression domains in the posterior regions. This implies that HOX genes that are expressed in cell lines of all lineages are the nature of body structures which grow out from individual HOXC8, HOXA7 and HOXB7. HOX genes showing a lineage- segments apparently depends on the specific combination of restricted expression are HOXA10 (myelomonocytic cell HOX products.141 lines),158 and HOXC4 (predominantly expressed in lymphoid Involvement of HOX genes in the formation of organs is cell lines).45 Furthermore, expression of all HOXB genes was reflected by their expression in kidney, colon, liver, spleen, mainly associated with cell lines of the erythroid lineage.42,43 skin, spinal cord and hematopoietic cells in human adults.142–144 Except for HOXB1, which is not expressed in the hematopo- In addition, recent studies of human synpolydactyly and con- ietic compartment, and HOXB13, which has been identified Review JW van Oostveen et al 1679 only recently, no expression pattern in other hematopoietic bone marrow cells than in more primitive populations. Some cell lines is yet known of HOXB genes.163 All, but one164 study of these genes (like ENX-1, M31, HP1HS␣ and HP1HS␥) appear reported that genes of the HOXD cluster are not expressed in the earliest clonogenic precursors, whereas other Pc-G in the hematopoietic compartment. Taken together, no clear genes (M32, M33, Mel-18 and Mph1/Rae3) were only detect- coordinate expression of clustered members of HOX genes able at later stages of differentiation. Interestingly, the ninth was found in particular hematopoietic cell lineages, but known human Pc-G gene, Bmi-1, was detected at low fre- instead HOX genes appear expressed in a complex pattern of quencies in mature bone marrow cells but was preferentially lineage- and non-lineage restriction. expressed in primitive long-term culture-initiating cells with high proliferative potential. Progressive upregulation of Pc-G gene expression possibly induces progressive formation of het- Functional analysis of HOX gene expression in hematopoietic erochromatin, ultimately resulting in 3Ј→5Ј closure of the cell lines: Several groups made use of the capability of HOX clusters. some hematopoietic cell lines to differentiate after induction with an appropriate stimulus. Erythroid-myeloid cell lines transfected with a HOXB6 antisense construct, showed an Functional analysis of HOX gene expression in normal hema- increase of erythroid features. Transfection with a HOXB6 topoiesis: New techniques, including retrovirus-mediated sense construct resulted in the loss of erythroid features, indi- transfection of HOX genes in mouse BM and the generation cating its involvement in erythropoiesis.165 Also, retroviral of knock-out mice, have made it possible to monitor the effect gene transfer of HOXB4 in embryonic stem cells enhanced of modulated HOX expression in vivo. Both overexpression their erythropoietic potential.166 HOXB7 is temporarily acti- and blocking of expression of one individual HOX gene in vated in promyelocytic cell line HL60 after induction with Vit bone marrow cells has an enormous effect on development

D3 or TPA leading to monocytic differentiation. By contrast, of specific cell pools. For example, mouse BM cells, over- HL60 cells induced to differentiate into the granulocytic expressing HOXB8 as a result of retroviral transfection, lineage lack HOXB7 expression. This was subsequently had enhanced capacity to form IL-3-dependent cell lines.174 confirmed by inhibition of colony formation derived from Furthermore, an increase was noted in self-renewal of early GM-CSF-stimulated bone marrow (BM) in an antisense clonogenic cells, concomitant with expansion of progenitor assay.167,168 Finally, Blatt et al169 reported that overexpression cells and a propensity to evolve into leukemia. of HOXB8 (Hox2.4) inhibited the myeloid differentiation of Sauvageau et al175 recently demonstrated that over- IL-6 inducible mouse cells. Thus, modulation of HOX gene expression of HOXB4 in normal mouse BM results in an expression in hematopoietic cell lines results in differences in increase of the most primitive cell populations, without devel- lineage commitment and maturation of these cells. opment into leukemia or other malignancies. Serial transplan- tations of bone marrow cells, initially transfected with HOXB4, revealed a 50-fold higher number of totipotent hema- HOX gene expression in normal hematopoietic bone topoietic stem cells in primary and secondary recipients, com- marrow cells pared with mice bearing a control vector expressing neomycin resistance alone. After retroviral gene transfer in embryonic Analysis of unfractionated normal human BM cells using RNase stem cells, HOXB4 was shown to influence the early progeni- protection assays demonstrated expression of HOXB2, HOXB6 tors with mixed erythroid/myeloid features and not the pro- and HOXA10,43,158 indicating that HOX expression in cell lines genitors of later stages, specific for the erythroid, granulocytic is not solely due to the transformed state. Furthermore, RT-PCR or monocytic lineage.176 Also, overexpression of the human analysis on small purified subpopulations of CD34+ hematopo- paralogue HOXC4 in human hematopoietic progenitor cells, ietic progenitor cells showed that the number of HOX genes resulted in progenitor cell expansion, without changing their expressed was the highest for the HOXA cluster, and decreased capacity to differentiate properly (Corte, personal for genes of the HOXB and HOXC clusters. No expression of communication). These results suggest that paralogue mem- the HOXD cluster genes was found.170–173 Not only the number bers HOXB4 and HOXC4 may have a similar function during of HOXA genes, but also the expression level of these genes the same time span in hematopoiesis. was higher than that of HOXB genes in enriched CD34+ subpo- Another gene investigated in this model is HOXA10, the pulations, including the self-renewing stem cell.172 expression of which is restricted to myeloid cells. Mice over- In addition, it appeared from two studies that HOX genes expressing HOXA10 in their bone marrow cells have are colinearly expressed during differentiation of myeloid and increased numbers of stem cells and myeloid progenitor erythroid cells.172,173 Downregulation of the 3Ј end located cells.177 Additionally, the number of megakaryocytic and genes (eg HOXB3) was observed in more mature fractions, primitive blast colonies was increased in the recipient ani- including the committed progenitor cells, while genes located mals. By contrast, HOXA10-overexpressing marrow cells did at the 5Ј end (eg HOXA10) seem to be more actively not contribute to the B cell population, suggesting that expressed throughout the CD34+ compartment. Their tran- HOXA10 switch-off is required for B cell differentiation. scription decreases only when cells progress to mature CD34− Moreover, the HOXA10-transfected animals were extremely stages.172 Induction of human progenitors into erythroid and susceptible to a myeloproliferative disorder that resembled granulocytic lineages in vitro revealed a 3Ј to 5Ј end wave of acute myeloblastic leukemia. HOXB gene expression colinear with differentiation,173 similar An alternative approach to functional analysis of HOX to the expression order in the embryonic development genes is to reduce or knock-out their normal expression. Using (Figure 2). homologous recombination in embryonic stem cells, mice Expression of HOX regulatory genes, like Pc-G genes, dur- with a defect in HOXA9 were created.178 Mutants were heal- ing human hematopoiesis remains relatively unexplored. Les- thy and fertile, but had reduced numbers of peripheral blood sard and colleagues131 demonstrated that the expression level granulocytes and lymphocytes. Lymphoid tissues, spleen and of eight out of nine Pc-G genes is much higher in more mature thymus were smaller, and the number of myeloid and B cells Review JW van Oostveen et al 1680 was reduced. By contrast, no significant effect on pluripotent required. Recently, the mechanism by which HOX11 inter- stem cells seemed to be observed.52 However, more recent feres in the regulation of the cell cycle was shown by Kawabe data of the same investigators (ASH 1998) indicate a major et al.193 By using a two hybrid system they found that HOX11 stem cell defect in these animals. These results indicated that can interact with the protein phosphatases PP2A and PP1, HOXA9 activates differentiation of stem cells, myeloid pro- resulting in disruption of the G2/M cell cycle checkpoint. genitors and, in contrast to HOXA10, lymphoid progenitor The third translocation, t(1;19)(q23;p13.3), produces a cells. chimeric fusion protein that links the amino-terminal trans- An antisense oligodeoxynucleotides (ASOs) approach has activating domain of E2A to the carboxy-terminal DNA bind- been used to diminish HOX gene function in vivo as well. ing domain of the diverged class II homeobox containing Blocking expression of HOXA5 with ASOs had a dual effect regulatory gene, PBX1.194–196 The translocation correlates with on growth of BM cells. Firstly, myeloid colony forming was 25% of pre-B ALL.197 Five alternative splice variants of E2A- inhibited, and secondly, the erythroid progenitor population PBX1 have been identified. Protein products were shown to was expanded.179 In contrast to HOXA5, an ASO blockade of possess various transforming potentials.198 Whether PBX1 several HOXB and C genes led to inhibition of erythroid col- alone has transforming potential is unknown, but PBX proteins ony formation.20,180 Some pitfalls using the antisense ASOs can form DNA-binding complexes with HOX proteins.199 treatment must be considered, however. First, only a 50% These complexes are thought to form the basis for autoregu- reduction of mRNA levels is generally achieved, and an effect lation and HOX-induced cellular transformation.199,200 of HOX inhibition may not necessarily be observed. Secondly, Inordinate HOX gene expression as a result of abnormal ASOs could bind non-specifically and affect the function of regulation has been observed in leukemic cells compared other genes. with their normal counterparts.181,201 For example, the diverged class II homeobox containing gene HLX (formerly known as HB24) is strongly expressed in activated lympho- Expression of HOX genes in hematological malignancies cytes and hematopoietic progenitor cells.202,203 In samples of ALL and AML, however, very high levels of mRNA transcripts It will not be surprising that genes which are involved in regu- were found, in contrast to the normal levels detected in CML lation of differentiation and proliferation can contribute to the material (which represent more mature cells).204 Transient malignant transformation of cells, because their deregulated expression of HLX into a subpopulation of hematopoietic pro- expression could lead to defective control of cell growth. genitors resulted in impairment of differentiation into mature HOX genes not only control the differentiation and prolifer- hematopoietic cells, suggesting that downregulation of HLX is ation of embryonic cells, but their expression in adult necessary for normal differentiation of hematopoietic progeni- organs142,143,181,182 also suggests a function in the mainte- tors. That this gene also has oncogenic transformation nance of the differentiation state of cells. Aberrant expression capacity was proven by the fact that HLX-transfected Jurkat of these genes might be a result of DNA amplification, translo- cells induced tumor formation when injected into nude cated genes, mutations or deregulated gene expression. mice.205 Another example of leukemic transformation through deregulated HOX gene expression is presented by HOXA9. Recent studies have shown that co-activation of the Hoxa9 Leukemias: HOX gene mutations associated with leukem- and Meis1 genes is potentially sufficient for transformation ias are not known, but some leukemias are associated with and co-transfection of these genes in mouse hematopoietic irregular expression of HOX genes due to translocation or cells rapidly leads to development of AML.206,207 abnormal gene regulation. Translocations have been reported There is scarce evidence that trx-G and Pc-G genes are for class I and II homeobox genes. Class I gene HOXA9 was directly involved in oncogenesis by HOX proteins. The MLL found to be involved in translocation t(7;11)(p15;p15) present (Hrx) trithorax gene is implicated in mixed lineage (myeloid- in AML FAB M2-M4, linking the complete HOXA9 gene to a lymphoid) leukemia.208–210 Knocking out the Bmi-1 Pc-G gene gene for nucleoporin NUP98.183,184 Diverged, class II homeo- in mice specifically affects expression of HOXC5 and box gene HOX11 is juxtaposed to the J␦1 region of the TCR HOXC6,211 while expression of Bmi-1 as a transgene results delta gene in the translocation t(10;14)(q24;q11). This translo- in a high incidence of lymphomas.212 This is most likely cation has been found in 5–7% of T-ALL.185–187 related to downregulation of the p16/INKA4 gene, an inhibitor A variant translocation t(7;10)(q35;q24) fuses HOX11 to the of the cell cycle.213 TCR␤ gene on chromosome 7.188 Since the coding region of The expression of HOXC genes and in particular of the HOX11 is not affected by the translocation,189 deregulated HOXC4 gene,26 seems to be associated with commitment to HOX11 expression is thought to be a key event in the devel- the lymphoid lineage. We focussed on the expression of opment of leukemias. The amino- and carboxyl termini of HOXC4, HOXC5 and HOXC6 since these three genes have a HOX11, however, seem dispensable for transformation.190 common promoter, and produce one mRNA molecule that is The transforming capacity of HOX11 was demonstrated in spliced in separate transcripts. The expression pattern of these vitro by transfection of HOX11 into NIH 3T3 mouse cells. 3Ј end cluster-C genes were studied by RT-PCR in normal HOX11 can transactivate various mammalian and yeast pro- hematopoiesis and hematopoietic leukemias. Acute B- and T- moters,191 suggesting that the oncogenic activity of HOX11 lymphatic leukemias showed HOXC4 and -C6 expression but might not be restricted to T cell leukemias. Retroviral lacked HOXC5 expression like normal lymphoid cells.27 In transduction of HOX11 in mouse BM cells, enriched for the myeloid lineage, the expression patterns of HOXC4 and progenitors, yielded cell lines at high frequency consisting of -C6 were largely similar in neoplastic cells of AML.32 How- immature cell lines of the myeloid lineage.192 However, these ever, HOXC5 gene transcripts were poorly expressed in these cells are IL-3-dependent and showed no leukemogenicity, neoplastic cells and in normal myeloid cells. Surprisingly, suggesting that HOX11 promotes proliferation and blocks ter- none of the three genes were expressed in CML. Expression minal differentiation of hematopoietic cells.192 For further pro- of all three genes could generally be detected in immature gression to malignancy secondary events (eg mutations) are AMLs (FAB M1–M4 and M6). In mature (normal) myeloid cells Review JW van Oostveen et al 1681 the expression of these genes seemed to be downregulated,32 contain the t(2;5) translocation and normally have an agreeing with the results of studies reported in the literature. intermediate prognosis. Mature myeloid leukemias localized in tissue simultaneously Given the involvement of HOX genes in hematopoiesis, expressed HOXC4, HOXC5 and HOXC6 genes, in contrast to organogenesis of hematopoietic organs, and pathogenesis of leukemic myeloid cells in the same stage of maturity. These tumors and leukemias, investigation of HOX gene expression findings are suggestive for a relationship between HOXC gene in lymphomas seemed warranted. expression and the presence of adhesion molecules at the sur- We studied the expression pattern of the 3Ј end cluster-C face of these cells (see below). genes HOXC4, HOXC5 and HOXC6 in lymphoid hematopo- In conclusion, if HOX regulatory genes are of central impor- iesis to see whether the expression pattern of HOXC genes in tance in normal hematopoietic development, then disruption NHL, the neoplastic equivalents of normal lymphoid cells, is of this pathway might be a common feature in human altered compared to normal lymphoid cells. Lymphoid B and leukemias.207 T cell lines, arrested at different stages of maturity, early ALL, and healthy lymphoid cells from peripheral blood and hyp- erplastic tonsillar tissue were used as a model for normal Non-Hodgkin’s lymphomas: To explain our interest in the lymphoid development. Semiquantitative RT-PCR demon- relationship between HOXC genes and non-Hodgkin’s lym- strated increased expression of HOXC4 and HOXC6 with pro- phomas (NHL), we would like to briefly discuss the develop- gressive maturation of both B- and T-lymphocytes. In contrast ment of the present conceptual definition of NHL. to HOXC4 and HOXC6 gene expression in neoplastic and NHL form a heterogeneous group of malignant lymphoprol- normal lymphoid cells, we detected expression of HOXC5 iferations. They can be roughly divided into B and T cell lym- only in lymphoma-derived cell lines and some B cell lym- phomas depending on lineage markers, like Ig- or TCR-gene phomas.27 These findings suggested a role for HOXC5 in lym- rearrangements, and markers expressed on the cell surface. In phomagenesis. the past, several classifications have been used to describe To distinguish HOXC gene expression in normal cells from these various types of lymphomas. The updated Kiel classi- that in lymphoma cells, we used non-radioactive RNA in situ fication is nowadays widely used in Europe. The concept of hybridization (RISH) to determine HOXC transcription at the this classification was that various types of non-Hodgkin’s single cell level. We further analyzed expression of HOXC4, lymphomas are thought to represent malignant counterparts of HOXC5 and HOXC6 in histologic subtypes of primary lymphoid cells. They are arrested at various stages of normal cutaneous lymphomas, in benign cutaneous lymphoid lesions differentiation, while maintaining their proliferating and hyperplastic lymphoid organs. HOXC4 and HOXC6 were activity.214 The Kiel classification was mainly based on nodal detected by RT-PCR and RISH in almost all samples of primary lymphomas. Extranodal lymphomas, such as mycosis fungo- cutaneous NHL, benign lymphoid lesions and hyperplastic ides and maltomas that display unique clinicopathological lymphoid organs.223 However, expression of HOXC5 was features, did not fit in this concept. Present insights in the only detectable by RISH in primary cutaneous anaplastic large biology of lymphocytes, in particular the recirculation of lym- T cell lymphomas. Semiquantitative RT-PCR revealed phocytes through the body and homing of lymphocytes to spe- expression of HOXC5 in some other subtypes of cutaneous B- cific body sites, increased our understanding of extranodal and T-NHL, but this was much weaker than in primary lymphomas.215,216 The recirculation pattern of lymphocytes is cutaneous anaplastic large T cell lymphomas. Since we could specific for particular body compartments, meaning that there not induce HOXC5 expression in PBL stimulated with ␣- are subpopulations of lymphocytes that home specifically to CD3/CD28 mAbs in the presence of IL-2, HOXC5 might act lymph nodes, to the MALT (mucosa-associated lymphoid as a putative oncogen in the genesis of cutaneous anaplastic tissue) or other body compartments. This homing is mediated large T cell lymphomas.223 by specific adhesion molecules, which react with site-specific Keeping in mind that HOX genes provide cells with pos- adhesion molecules on the endothelial cells of high endo- itional as well as developmental information, different histol- thelial venules (HEV), named vascular adressins or tissue pos- ogic subtypes of B- and T-NHL were analyzed for expression ition markers. For example, lymphocytes expressing of HOXC4, HOXC5 and HOXC6. Expression of HOXC4 and cutaneous lymphocyte antigen CLA (the proposed skin hom- HOXC6 could be detected by RISH in the majority of lym- ing receptor217,218), interact with E-selectin on the superficial phomas. Interestingly, HOXC6 could not be detected in dermal vessels.219 Those expressing the ␣4␤7 integrin, serving lymphnode-based B-CLL. HOXC5 gene expression appeared as the homing receptor for mucosal sites, interacts with MAd- associated with primary cutaneous anaplastic large T cell lym- CAM-1, present on the HEV of the Peyer’s patch.220,221 This phomas and B cell lymphomas with plasmocytic differen- compartmentalization of the immune system is accompanied tiation from the oro-digestive tract.30 Thus, HOXC5 showed a with different functions and the existence of distinct effector type- and site-restricted expression pattern in NHL and may immune cell populations. be involved in formation of these lymphomas. Extending the concept of the ‘neoplastic equivalent’ with this aspect of tissue-restricted lymphocytes, we can now define NHL as neoplastic counterparts of functional recirculat- ing tissue restricted lymphocytes activated after antigen stimu- Target genes of homeoproteins lation.222 From this point of view it is conceivable that lym- phomas may arise with similar histologic features, but with Although expression patterns of many class I HOX genes have different clinical behavior, different translocations, different been described in embryogenesis and hematopoiesis, the tar- adhesion molecule expression pattern, or differences in the gets of the majority of their protein products have yet to be presence of viral sequences (such as EBV or HTLV-1). Thus, identified. Genes that have been shown to be controlled by anaplastic lymphomas arising in the skin express CLA, lack homeoproteins encode adhesion molecules, transcription the t(2;5) translocation and have a good prognosis. By factors (in particular the HOX genes themselves) and growth contrast, nodal anaplastic lymphomas do not express CLA, factors. Review JW van Oostveen et al 1682 Modulation of adhesion molecule expression Modulation of transcription factor expression Another group of target genes of the homeoproteins are genes By modulation of HOX gene expression in vitro it was coding for transcription factors (Table 3). The most obvious observed that homeoproteins are able to influence the transcription factors under regulation of homeoproteins are adhesive capacity of myeloid leukemia cells.224 Adhesion the homeobox genes themselves. Moreover, coordinate molecules are categorized in the following families: the expression of HOX genes in embryogenesis is achieved in part immunoglobulin (Ig) gene superfamily, cadherins, integrins, by cross-talk between the various members of the HOX selectins and the CD44 family. Analysis of adhesion molecule complexes.7,72,134 For several HOX genes auto- and/or cross- profile of cells, overexpressing HOX genes, revealed that regulatory interactions within the homeotic network have members from different adhesion molecule families were been described by genetic analysis and in transfection affected. Examples are given in Table 2. assays.234–237 For example, human HOXD9 (formerly HOX4C) Cotransfection experiments demonstrated that the is transactivated by the HOXD9 protein itself and by HOXD10 expression of N-CAM, a member of the Ig gene superfamily, (formerly HOX4D) protein by cotransfection. Conversely, is involved in the development of the neural network in mouse Hoxd-8 (formerly Hox-4.3) represses activation of embryogenesis. This protein, present on natural killer (NK) HOXD9.106 Furthermore, HOXC5 can be transactivated by cells, could be activated by increased levels of Xenopus HOX genes from the same cluster (HOXC6), as well as from HoxC6 and HoxB9.225,226 In contrast, HoxB8 had an inhibi- the HOXD cluster (HOXD9 and HOXD10).102 In vivo experi- tory effect on the expression of N-CAM, indicating that tan- ments with Drosophila and mice indicated that a cofactor is demly arranged genes, such as HOXB8 and HOXB9, act necessary for autoregulation of Hoxb-1.101 These experiments together to control one gene by differential regulation.225 In demonstrate that the HOX cross-talk region (HCR) can be addition, a study of HOX gene expression by the human meta- occupied simultaneously by several protein molecules and static melanoma cell line Me 655/2 demonstrated that mela- allows various combinations of HOX proteins to interact. noma clones could be classified into two populations: one Although no data are present on cross- or auto-regulatory group expressed HOX-C genes, which correlated with low events in hematopoietic cells, these regulatory circuits may be level expression of ICAM and absent expression of VLA-2, active in hematopoiesis as well. -5 and -6. By contrast, high level expression of VLA-2, -5 and In addition to regulation of homeobox gene expression by -6, and ICAM coincided with silence of the HOX-C locus.227 homeoproteins, gene expression of other transcription factors, In addition to these class I genes, class II homeobox genes involved in organogenesis and proliferation, have also been (Phox2 and Cux) also showed cross-talk by either activating shown to be under control of homeoproteins (Table 3). Con- (Phox2) or inactivating (Cux) N-CAM expression.228 Further- cerning hematopoiesis, it is of interest that knock-out mice for more, decreased expression of ICAM-1 (CD54) was found in HOXA3 have a reduced expression of the Pax1 gene, suggest- myeloid FDC-P1 cells after enforced expression of the mouse ing that Pax1 is a target of HOXA3. Moreover, these mutants homeobox genes Hlx.229 In the same study, the expression of were athymic, which is in agreement with the phenotype of CD44 also decreased as a result of the Hlx overexpression.229 Pax1 mutant mice.150 Since Pax1 mutants also had reduced CD44 is an integral membrane protein, which is widely dis- + + + numbers of CD4 /CD8 immature and CD4 mature thymo- tributed over hematopoietic and non-hematopoietic cells. In cytes, Pax1 is also involved in the maturation of thymo- addition to important adhesion events necessary for migration, cytes.149 Moreover, the gene encoding transcription factor T cell activation230 and probably lymphopoiesis231,232 within Thy-1, involved in the maturation of thymocytes, was dis- the bone marrow, a variant of CD44 was shown to have meta- closed as a target of Pax1. The expression order from HOXA3, static potential.233 Pax1 to Thy-1 in the development of the thymus and thymo- Integrins can also be modulated by HOX genes. For cytes indicates an early function as master control genes for instance, increased expression of ␣IIb␤3, a surface fibronectin HOX genes in organogenesis and lymphopoiesis. receptor, was induced by HOXD3 in the erythroleukemia cell lines HEL and K562.164 No example of HOX-mediated regu- Modulation of expression of other targets of lation of selectins are known. All adhesion molecules have homeoproteins important functions in embryogenesis, morphogenesis, lin- eage commitment, hematopoiesis and cell migration, stressing Other targets of homeoproteins, not belonging to adhesion the importance of homeoproteins in these cellular events. molecules or transcription factors are mentioned in Table 4.

Table 2 Genes of adhesion molecules as targets of homeoproteins

Adhesion Species HOX Function Reference molecules

N-CAM human/xenopus HOXC6/HOXB8/HOXB9 neural development 226, 253, 254 mice Pax6/Pax8/Phox2/Cux ICAM-1 mice Hlx embryogenesis 229 CD44 mice Hlx embryogenesis 229 ␣IIb␤3 human HOXD3 embryogenesis 164 L-CAM human HOXD9/HNF-1 liver development 255 Cytotactin mice Pax6/Pax8/evx-1 (counter)adhesion, ordering tissues 239, 254 E-cadherin mice HOXA9 Cell adhesion 34 mgl-1 mice HOXC8 putative adhesion 256 Connectin drosophila Ubx cell adhesion 257

CAM, cell adhesion molecule; Ubx, Ultrabithorax. Review JW van Oostveen et al 1683 Table 3 Genes of transcription factors as targets of homeoproteins

Target gene Species Regulatory homeoprotein Function Reference

HOXD9 human HOXD9/HOXD10/HOXD8 embryogenesis, limb development 106 HOXC5 human HOXC6/HOXD9/HOXD10 embryogenesis 102 Pax1 mice HOXA3 thymus development 150 TTF-1 human/mice HOXB3 expression of thyroglobulin and thyroperoxidase 258 Thy-1 mice Pax1 maturation of thymocytes 149 c-fos mice Pax1/Pax6/Pax8 proliferation 254 fkh human/drosophila HOXA5/Scr liver development 259, 260 salm drosophila Ant transcription factor 261 fkh, forkhead; SCR, sex comb reduced; TTF-1, thyroid transcription factor; Ant, Antennapedia; Ubx, Ultrabithorax.

Table 4 Genes encoding other proteins as targets of homeoproteins

Other genes Species Regulatory homeoprotein Function Reference

␤-APP Mice HOXC8 cellulary regulatory processes 262 CD19 Mice Pax5 (BSAP) B cell development 48 Thyroglobulin/thyroperoxidase Mice Pax8 thyroid development 263 Decapentaplegic Drosophila Ubx growth factor (TGF␤) 264

␤-APP, ␤-amyloid precursor protein (involved in Alzheimer disease and Down syndrome); Ubx, Ultrabithorax.

Interestingly, the Pax5 protein (BSAP, B-cell lineage-specific embryogenesis, starting with the 3Ј genes and progressing to activator protein), is able to induce the expression of B cell 5Ј genes of the HOX cluster. This ‘wave’ of HOX gene membrane protein CD19.34 BSAP is essential for B cell expression has been found in CD34+ BM cells173 and T development34 and deregulated BSAP expression has been cells238 after activation, suggesting a temporal axis of HOX implicated in formation of large-cell lymphomas and some gene expression in hematopoiesis. Moreover, most mature follicular lymphomas.39 The finding that many target genes of CD34+ subpopulations showed a preference for expression of homeoproteins are involved in hematopoiesis emphasizes 5Ј end genes compared to expression of more 3Ј end genes their regulatory role in this process. Furthermore, deregulated in more immature fractions.172 HOX gene expression might have profound effects on prolifer- Concerning positional information, we have considered a ation and differentiation of a cell, and promote neoplasia. role of homeobox genes in homing of lymphocytes to specific body sites by regulation of adhesion molecules, which would give these cells a positional imprint. Immature blood cells lay Epilogue ordered in the three-dimensional structure of the BM, inter- acting with a stromal meshwork through adhesion molecules In this part we will discuss two major aspects which have not and cytokines.19 Progression of maturation is accompanied been dealt with in the previous sections: general aspects of with migration within the BM, requiring downregulation of HOX gene expression in hematopoiesis, and the significance adhesion molecules determined by the given BM localization, of HOXC5 in NHL. in combination with expression of other adhesion molecules. Thus, blood cell differentiation is tightly associated with a spe- cific position and a spatial axis, that might be coordinated by General aspects of HOX gene expression in hematopoiesis HOX genes. However, the release of hematopoietic cells from BM to peripheral blood requires downregulation or shedding of Our study on HOXC4, HOXC5 and HOXC6 gene expression in myeloid and lymphoid hematopoietic compartments adhesion molecules. Since it seems possible that adhesion revealed a lineage- and stage-restricted expression pattern of molecule expression can be controlled by homeoproteins, downregulation of the expression of (particular) HOX genes in these genes. Other groups showed commitment of some HOX + genes to particular lineages or stages of hematopoiesis. An mature BM populations is also necessary. Compared to CD34 immature BM cells HOXA10 is only found in CD34− mature interesting question is whether there are parallels between the + − function of HOX genes in the formation of the embryonic cells. Indeed, transition from the CD34 to the CD34 com- body plan and their regulatory role in hematopoiesis. In other partment was accompanied with the downregulation of words, does the homeotic network regulate the process of HOXA10 gene expression.201 This indicates that HOXA10 is + blood cell maturation by providing positional and temporal temporarily expressed during maturation of immature CD34 + information? Secondly, are HOX genes expressed in a spatial BM cells to mature CD34 BM cells. and temporal manner during hematopoiesis? When mature BM cells are released in the peripheral blood Stage- and lineage-restricted expression patterns of HOX stream, adhesion molecules are required for the homing of genes in hematopoiesis indicates that their transcription is lymphocytes to lymphoid organs and extranodal lymphoid coordinated in time. Indeed, HOX gene expression during tissues. Therefore, we suggest that a second ‘wave’ of HOX hematopoiesis appears to occur in a pattern similar to that in gene expression is activated, reinducing (other) adhesion mol- Review JW van Oostveen et al 1684 ecules. This view is supported by our observation of HOXC4 pendent functions: the C-terminus represses immediate–early and HOXC6 expression in mature lymphocytes. gene expression via CArG elements. Mol Cell Biol 1990; 10: 4243–4255. 2 Driever W, Nu¨sslein-Volhard C. The bicoid protein determines Significance of HOXC5 expression in non-Hodgkin’s position in the Drosophila embryo in a concentration-dependent manner. 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