(2013) 27, 1000–1008 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

REVIEW The role of HOX in normal hematopoiesis and acute leukemia

RA Alharbi1,2, R Pettengell3, HS Pandha1 and R Morgan1

The (HOX) genes are a highly conserved family of homeodomain-containing transcription factors that specify cell identity in early development and, subsequently, in a number of adult processes including hematopoiesis. The dysregulation of HOX genes is associated with a number of malignancies including (AML) and acute lymphoid leukemia (ALL), where they have been shown to support the immortalization of leukemic cells both as chimeric partners in fusion genes and when overexpressed in their wild-type form. This review covers our current understanding of the role of HOX genes in normal hematopoiesis, AML and ALL, with particular emphasis on the similarities and differences of HOX function in these contexts, their hematopoietic downstream targets and implications for therapy.

Leukemia (2013) 27, 1000–1008; doi:10.1038/leu.2012.356 Keywords: hematopoiesis; human HOX; murine Hox; AML; ALL

INTRODUCTION enhance proliferation signal transduction pathways and induce Acute myeloid leukemia (AML) is a heterogeneous group of the proliferation of hematopoietic stem cells (HSCs) or precursors genetically and phenotypically aggressive disorders where the and usually affect kinase signaling pathways, such as FLT3, KIT, differentiation of hematopoietic progenitor cells is blocked, NRAS/KRAS and JAK/STAT . Class I mutations take place increasing their self-renewal ability and disturbing the normal late and cause disease progression. Class II mutations target regulation of proliferation.1,2 In the UK, AML is the most frequent hematopoietic genes leading to a block in acute leukemia in adults, accounting for 77% of cases. The median myeloid differentiation and conferring the self-renewal ability of age at presentation is 69 years and the male: female ratio is about hematopoietic precursors. These mutations take place early and 5–7 5 : 4. The disease is commonly classified by either the French– initiate the AML disease. One of the most affected and mutated American–British system, or that described by the World Health transcription factors are homeobox (HOX) genes. Organization. The former is based on morphology and maturation stage, and classifies AML into eight groups (M0–M7). The latter is HOX genes also based on morphology, but also includes immunophenotyp- The HOX genes are a family of homeodomain-containing ing, genetics and clinical manifestations, and classifies AML into transcription factors,8 initially characterized in as four main groups: AML with recurrent genetic abnormalities, AML master regulators of trunk and tail development during with myelodysplasia-related changes, therapy-related AML and embryogenesis.9 Duplication of the original cluster MDS, or AML that does not fit into any of these groups. Non- has given rise to 39 genes in , separated into four random chromosomal alterations, such as balanced translocations, clusters known as A, B, C and D.9–11 These clusters are located on monosomies, trisomies, inversion and deletions have been found four different , HOXA (7p15), HOXB (17q21), HOXC in the leukemic cells of almost 55% of AML patients, and until (12q13) and HOXD (2q31).12 Ancestors of the original gene in each recently they were considered to be the most crucial prognostic of the clusters are known as paralogs, and generally they show a factors for complete remission, likelihood of relapse and overall 3,4 high degree of sequence similarity as well as functional survival. redundancy (Figure 1).13 Recent advances in molecular diagnosis have resulted in both The arrangement of HOX genes into clusters allows for gene alterations and the dysregulation of specific genes becoming sharing, which enables a precise spatial and temporal increasingly important as prognostic elements in AML. This has coordination of expression during development. The relative helped to clarify the numerous heterogeneities of AML subsets, 5 regulatory also varies between HOX genes, giving particularly AML subsets showing normal karyotype AML , and rise to what is often referred to as a ‘HOX code’,14 and resulting furthered understanding of the molecular mechanisms of in the following distinctive criteria: (1) Temporal distribution. The leukemogenesis. expression of HOX genes starts from the 30 end of the cluster and proceeds stepwise towards the 50 end. (2) Spatial distribution. The 30 most member of the cluster is expressed with a more anterior Genetic changes in AML limit than the next member and each subsequent member has a The origin of AML is associated with two distinct genetic changes, more posterior limit of expression resulting in an overlapping referred to as Class I and Class II. Class I consists of mutations that series of expression domains. (3) Posterior prevalence. In each

1Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK; 2Faculty of Applied Medical Sciences, Albaha University, Albaha, KSA and 3St. George’s, University of London, London, UK. Correspondence: Dr R Morgan, Oncology, University of Surrey, Leggett Building, Guildford, Surrey GU2 7WG, UK. E-mail: [email protected] Received 8 May 2012; revised 19 November 2012; accepted 21 November 2012; accepted article preview online 5 December 2012; advance online publication, 8 January 2013 The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1001 Complex (Ant-C) Bithorax Complex (BX-C)

Drosophilia Iab Pb Dfd Scr Antp Ubx Abd A Abd B (HOM-C)

Chromosome number

HOX A 1324 5 6 79 10 11 12 13 7p15

HOX B 1234569 78 13 17q21 Mammalian (HOX-C) HOX C 4135 68910111212q13

HOX D 34 89 10 11 12 13 2q31

3` 5` Figure 1. A schematic structure of clustered HOX genes. The 39 HOX genes are located on four different chromosomes. of human HOX genes HOX-C to Drosophila HOM-C genes is shown by colors. Blank squares show missing genes. individual cluster, the function of the posterior gene products is considered differentiated (BM) cells.26,28 The analysis dominant over the more anterior genes.13 of HOX in human multipotent stem cells and T-cell precursors showed that HOXA genes are prominently expressed during T-cell development, in particular the Abd-HOXA HOX cofactors genes including HOXA7-HOXA11, with only HOXB3 and HOXC3 DNA-binding site studies suggest that HOX have expressed from the other HOX clusters.29 relatively limited selectivity and specificity, and they need cofactor interactions in order to increase both.15–17 The most important HOX cofactors are the three loop extension proteins, Gain of function studies which comprise the pre-B-cell leukemia (PBX) and myeloid The function of HOX genes in normal hematopoiesis has been ectropic insertion site (MEIS) families.16,17 These cofactors have 18 widely studied using gene expression analysis and knockin or crucial roles in development and hematopoiesis. For example, knockout studies in HSCs and early hematopoietic progenitors Pbx1 null mice die during the embryonic stage as a result of severe 19 (Table 1). Generally the overexpression of a HOX gene leads to an hematopoietic defects, and Meis1-deficient mice fail to generate expansion of stem and progenitor cell populations together with a , exhibit severe hemorrhaging and likewise die 20 block on differentiation. Notable examples of this include the during the embryonic stage. In zebrafish, Meis1 and Pbx overexpression of murine Hoxb6, which resulted in the expansion contribute to the production of erythropoietic cells and the 21 of murine HSCs and myeloid precursors, together with the inhibi- inhibition of myelopoiesis. Generally, Hox proteins 1–10 bind to 44 22 23 tion of and lymphopoiesis, and overexpression Pbx1, whereas Hox proteins 9–13 bind to Meis1. of murine Hoxb3 that resulted in several hematological abnormali- ties, such as a block of B- and T-cell differentiation as well as a delay in myeloid precursor proliferation.37 Overexpression of HOX GENES IN HEMATOPOIESIS human HOXC4 resulted in expansion of early and committed HOX genes are expressed in HSCs and progenitors in a manner myeloid and erythroid progenitors,47 and knockin of human reminiscent of their expression in early development, with lineage HOXA5 caused an increase in the number of myeloid progenitors and differentiation stage-restricted patterns. Thus, for example, and blocked erythroid differentiation.30,31 Likewise, overexpres- HOXB3, HOXB4 and HOXA9 are highly expressed in uncommitted sion of HOXA10 in human cord blood or fetal liver CD34 þ hematopoietic cells, whereas HOXB8 and HOXA10 are expressed in hematopoietic progenitors resulted in a significant production of myeloid committed cells. The different HOX clusters also have blast cells and myelopoiesis concomitant with a complete block of specific patterns of lineage-restricted expression, whereby HOXA erythroid differentiation and a severe reduction in B-cell develop- genes are expressed in myeloid cells, HOXB genes in erythroid ment.36 Other HOX genes are required for the maintenance of cells and HOXC genes in lymphoid cells. Intriguingly, the HOXD progenitor or stem cell status and promote their proliferation, genes are not expressed in hematopoiesis despite having similar especially HOXA9 and HOXB4. The former is the most preferentially regulatory regions to the other clusters.24–26 Different mammalian expressed HOX gene in human CD34 þ HSCs and early hemato- CD34 þ cell subpopulations express at least 22 of the 39 HOX poietic progenitors and is subsequently downregulated during genes.27 Anterior ‘30 end’ HOX genes (HOX1-6) are highly differentiation. Murine Hoxa9 overexpression enhances HSC expressed in the most primitive HSCs. Subsequently, the anterior expansion and myeloid progenitor proliferation and, with a long HOX genes are downregulated and posterior ‘50 end’ HOX latency, leads to leukemia.33,34 In contrast to myeloid progenitors, genes are expressed during commitment.28 HOX genes are Hoxa9 overexpression resulted in a partial inhibition of pre-B-cell highly expressed in the most primitive HSCs and progenitors, differentiation, but did not affect T-cell development.34 Hoxb4 is while their expression is almost absent in CD34 À cells, which are also highly expressed in HSCs and downregulated during

& 2013 Macmillan Publishers Limited Leukemia (2013) 1000 – 1008 The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1002 Table 1. HOX gene studies

HOX gene Gain of function Loss of function Species References

HOXA5 mMyeloid progenitors and block erythroid mErythroid progenitors and kmyelomonocytic Human 30,31 differentiation. cells. Hoxa7 k MEP, reticulocytosis and . Mouse 32 Hoxa9 mHSCs expansion and myeloid progenitor kkCMP, GMP, CLP, lymphoid precursors, Mouse 32–35 proliferation. Block erythroid differentiation repopulating ability andkspleen cellularity andkpre-B-cell differentiation. and size. HOXA10 mmBlast cells and myelopoiesis, kB-cell Human 36 differentiation and block erythroid differentiation. Hoxb3 Block B and T-cell differentiation and a delay kkB-cell progenitors and BM cellularity. Mouse 37,38 in myeloid precursor proliferation. HOXB4/ Hoxb4 mm HSCs expansion. kk Hematopoietic organs cellularity and Human/mouse 39–42 size,kHSCs and HPs andmHoxb genes. Hoxb3/b4 kk HSCs and HPs. Mouse 43 Hoxb6 mHSCs expansion and myeloid precursor mEarly erythroid progenitors. Mouse 44,45 proliferation.kErythropoiesis and lymphopoiesis. Hoxc3 kErythroid progenitors. Mouse 46 HOXC4 mEarly and committed myeloid and erythroid Human 47 progenitors. Hoxc8 kkErythroid, and macrophage Mouse 48 colony formation potential. Abbreviations: CLP, common lymphoid precursors; CMP, common myeloid progenitors; GMP, granulocyte/monocyte precursors; HOX, homeobox; HP, Hematopoietic progenitor; HSCs, hematopoietic stem cells; MEP, megakaryocytic/erythroid progenitors.

differentiation.26,28 Its overexpression in murine and human cell all Hoxa genes, except Hoxa13, and upregulation of Hoxc4, Hoxc9 lines results in a remarkable expansion of HSCs in vivo and in vitro and Hoxc11, also suggesting functional redundancy and complex without resulting in leukemia or lineage disturbances.39,40 Indeed, genetic interactions between Hox genes. the self-renewal ability of Hoxb4-transduced HSCs is 20–50-fold An exception to the general prevalence of functional redun- greater than untreated cells, and can be increased still further by dancy amongt the HOX gene family is HOXA9, the most highly knocking down Pbx1.49,50 expressed HOX family member in HSCs. Hoxa9 À / À mice showed significant deficiencies in myeloid and lymphoid cells concomitant with a significant defect in repopulating ability. These deficiencies Loss of function studies include common myeloid progenitors, granulocyte/monocyte In addition to the knockin and overexpression approaches precursors, common lymphoid precursors and lymphoid precur- described above, knockdown and deletion studies in murine sors (pro- and pre-B cells, pro-T cells).35 There is also a corres- models and cell lines have also been used to evaluate the role of ponding reduction in spleen cellularity and size. Notably, HOX genes in hematopoiesis. However, owing to the functional compared with the entire cluster b deficient fetal liver HSCs, redundancy of HOX genes, the results of knockdown assays are Hoxa9 À / À fetal liver HSCs exhibited a more dramatic defect in sometimes difficult to interpret and do not always reflect the repopulating ability,35,42 and HSCs from Hoxa9/b3/b4 null mice findings of studies where the gene has been overexpressed. For had the same repopulating ability as those from Hoxa9 null example, it has been found that Hoxb4 null mice exhibit a mice.35 Gene knockdown studies have revealed that some addi- significant reduction in size and cellularity of hematopoietic tional HOX genes are also essential in normal hematopoiesis. organs, such as spleen and liver, and a slight decrease in HSCs and Knockdown of HOXA5 led to an increase in erythroid progenitors hematopoietic progenitor number without a significant distur- 30,31 41,42 and a reduction in the number of myelomonocytic cells, and bance of lineage commitment. Likewise, Hoxb3 null mice Hoxa7 null mice showed a reduction in megakaryocytic/erythroid display a notable reduction in B-cell progenitors, and a reduction progenitors as well as reticulocytosis and thrombocytopenia.32 in BM cellularity, but no significant reduction in B-cell numbers in 38 À / À Knockout of Hoxb6 resulted in an increase in early erythroid the spleen. Hoxb3/b4 mice have a greater reduction in HSCs progenitors in murine BM and fetal liver cells.45 Likewise, and hematopoietic progenitors, yet no difference in hemato- À / À 43 Hoxc3 mice showed a reduction in late erythroid pro- poietic cell commitment. Using quantitative PCR, it has been 46 þ genitors without affecting the hemoglobinization size, and demonstrated that fetal liver cells (c-Kit )ofHoxb4 null mice Hoxc8 deficient mice showed a significant reduction in erythroid, expressed other Hoxb genes to a significantly higher level than granulocyte and macrophage colony formation potential.48 control cells.42 Bijl and colleagues34 found that an individual loss of Hoxb4 or even the complete loss of the Hoxb cluster (b1-b9) did not affect Upstream regulators of HOX genes the ability of murine fetal liver HSCs to self-renew. The repopu- Knockout models of HOX gene upstream regulators have helped lation and differentiation potential were retained, compared with to define their role in normal hematopoiesis. Regulators include wild-type control cells. Thus, the Hoxb cluster may not be transcriptional activators such as mixed lineage, myeloid lymphoid necessary for hematopoiesis, with members of the other Hox leukemia (MLL) and a family of caudal-type HOX transcription clusters presumably having largely duplicate roles. Analysis of Hox factors (CDX1, CDX2 and CDX4). The existence of HOX genes in gene expression in fetal liver cells (c-Kit þ Hoxb1-Hoxb9 À / À ), clusters makes them particularly sensitive to changes in chromo- revealed genetic interactions between members of the Hoxa, somal organization, and repressors of HOX transcription include b and c clusters, whereby these cells exhibited downregulation of genes that mediate this process, most notably members of the

Leukemia (2013) 1000 – 1008 & 2013 Macmillan Publishers Limited The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1003 polycomb group.51 These regulators have crucial roles in normal unfavorable prognosis of AML,64 and it also regulates its own development and hematopoiesis through the regulation of HOX cofactor, Meis1, through binding to Meis1 upstream regulator genes. A number of studies demonstrated that Mll-deficient genes cerb1 and .65 More proliferative genes have been embryonic bodies and Mll conditional knockout mice showed a recently identified as downstream targets for Hoxa9 including dramatic reduction in HSCs and hematopoietic progenitors. In Camk2d, Cdk6, Erg, Etv6, Flt3, Foxp1, Gfi1, Kit, Lck, Lmo2, Myb and addition, these embryonic bodies and the mice exhibited greatly Sox4.66 In the same study, it was shown that Hoxa9 downregulates reduced expression of a number of Hox genes including Hoxa7, differentiation and inflammation genes including Ifit1, Tlr4, Ccl3, Hoxa9, Hoxa10 and other Hoxb and Hoxc genes.52,53 Likewise, Ccl4, Csf2rb, Ifngr1, Runx1, Cd28 and Cd33. The fusion compound-deficient zebrafish and murine ESCs showed 98 (NUP98-HOXA9) has been found to stimulate dysregulation of the embryonic hematopoietic progenitors as the proliferation of HSCs by activating the expression of other well as impaired expression of Hox genes.54,55 However, it has HOX genes including HOXA9, HOXA7, MEIS1 and PBX3. It also been shown that Cdxs are not essential for normal hematopoiesis upregulates a number of leukemogenic transcription factors in adult mice. For example, Cdx4-deficient mice showed minimal including EVI1 and MEF2C, and tyrosine kinases hematopoietic defects, though it is highly expressed in wild-type including FLT3 and KIT.68 myeloid progenitor cells.56 In addition, human CDX2 is not It is also of note that there are both overlapping opposing expressed in normal HSCs, or in myeloid, B-cell, or T-cell functions between the closely related HOXA9 and HOXA10 progenitors.57–59 transcription factors. For example, in a similar manner to Hoxa9, Hoxa10 activates the expression of proliferative genes that result in myeloid progenitor expansion such as Itgb3, Hif, Tgfb2 and Fgf2 HOX downstream target genes in hematopoietic cells by direct binding to their promoters.69,70,81,82 HOXA10 also The mechanism by which HOX genes regulate hematopoiesis activates the transcription of anti-apoptotic genes such as is not yet fully understood. However, -wide analyses DUSP4, which encodes mitogen-activated protein phosphatase 2. after experimentally induced changes in HOX gene expression Mitogen-activated protein kinase 2 in turn prevents cell death by have identified some potential downstream targets. Amongt downregulating JNK.71 Hoxa10 decreases erythroid differentiation these are the HOX genes themselves, some of which have and megakaryopoiesis by activating Hoxa5 and inactivating Gata1, been shown to auto-activate their own expression or to cross- respectively,69 and it also induces Cdx4 expression in myeloid regulate their neighbors, or their cofactors. HOXA9, HOXA10 and cells.72 HOXB4 are the most comprehensively studied genes in this Unlike HOXA9, HOXA10 can also exert anti-proliferative respect because of their key roles in normal hematopoiesis and effects. For example, in cooperation with its trimeric cofactors, leukemia. It is particularly noteworthy that HOXA9 positively HOXA10 induces P21 transcription leading to cell cycle arrest regulates the transcription of other HOX genes including HOXA7 and differentiation.73 It also represses CYBB transcription,74 and HOXA10 and its cofactors PBX3 and MEIS1.60 A summary of thereby acting in an opposing manner to HOXA9. In a fusion HOX downstream target genes is presented in Table 2. form with Nup98, Hoxa10 activates more than 400 genes As described above, HOXA9 is a key regulator of hematopoiesis including the self-renewal genes Flt3, Prnp, Hlf, Jag2.75 and behaves as an in leukemia. It is therefore Many HOXB4 target genes have also been identified in three unsurprising that it activates the transcription of genes known recent studies.76,83,84 Overexpression of Hoxb4 resulted in to regulate cell proliferation and survival. For example, Hoxa9 transcriptional upregulation of Meis1, Dntt, Hlf, Sox4 and Runx2, directly activates the Pim1 gene, the product of which enhances while it downregulated the transcription of lymphoid specific proliferation by activating c-Myb, and also exerts an anti-apoptotic genes, such as B220, and myeloid-specific genes, such as Hmbs.76 effect by phosphorylating and inactivating the BAD protein.61,80 Some HOXB4 targets seem to vary in a context-dependent C-Myb has also been identified as an indirect transcriptional manner, for example it has been found to downregulate the target of Hoxa9-Meis1 that mediates transformation in Mll-Enl transcription of C- in the HL-60 cell line leading to cell (Eleven nineteen leukemia).67 Other HOXA9 targets include the differentiation,77 while it activates c-Myc transcription in murine oncogene ID2, which is upregulated, and BIM, which encodes an BM cells.78 As with HOXA9, Hoxb4 activates the transcription of its apoptotic factor and is downregulated.62 HOXA9 also activates the neighboring genes, Hoxb2, Hoxb3 and Hoxb5.41 Hoxb4 also CYBB gene, which encodes Gp91phox (a phagocyte respiratory activates the AP-1 complex members Fra-1 and Jun-B, which burst oxidase protein), and is expressed in differentiated myeloid leads in turn to an increase in the level of cyclin-D1 and a decrease cells.63 In mice, Hoxa9 has been shown to directly activate the in the level of c-Fos transcription, thereby increasing the transcription of the Flt3 gene, which is associated with an proliferation capacity of HSCs.79

Table 2. A summary of mammalian HOX target genes

HOX protein Targets of transcriptional activation Targets of transcriptional Species References repression

HOXA9/Hoxa9 Pim1, ID2, CYBB, HOXA7, HOXA10, PBX3 and MEIS1/ Flt3, Cerb1, Pknox1, BIM/ Itfi1, Tlr4, Ccl3, Ccl4, Human/mouse 60–66 Camk2d, Cdk6, Erg, Etv6, Foxp1, Gfi1, Kit, Lck, Lmo2, Myb and Sox4. Csf2rb, Ifngr1, Runx1, Cd28, and Cd33. Hoxa9-Meis1 c-Myb. Mouse 67 NUP98-HOXA9 HOXA7, HOXA9, MEIS1, PBX3, EVI1, MEF2C, FLT3 and KIT. Human 68 HOXA10/ P21 and DUSP4/ Itgb3, Hlf, Tgfb2, Fgf2, Hoxa5, and Cdx4. CYBB/Gata1. Human/mouse 69–74 Hoxa10 Nup98-Hoxa10 Flt3, Prnp, Hlf and Jag2. Mouse 75 HOXB4/Hoxb4 Meis1, Dntt, Hlf, Sox4, Runx2, and C-myc/ Hoxb2, Hoxb3, Hoxb5, B220 and HMBS/ C-MYC. Human/mouse 41,76–79 Fra-1 and Jun-B. Abbreviations: HOX, homeobox; MEIS, myeloid ectropic insertion site; NUP98, nucleoporin 98.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1000 – 1008 The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1004 THE ROLE OF HOX GENES IN ACUTE LEUKEMIA precursor cells.101 Conversely, a number of studies demonstrated Numerous studies have now shown that HOX genes can promote that the expression of HOXA genes is not crucial for MLL the development of AML by forming chimeric fusions with other leukemogenesis, yet their expression affects disease . genes, but more recent work has also shown that their miss- For instance, Hoxa7 and Hoxa9 influence AML latency and expression, in particular their overexpression, is also important in phenotype; yet they are not essential to initiate Mll-Gas7- 32 the formation of malignancy. mediated leukemogenesis. Furthermore, suppression of HOXA9 expression in cells with a chimeric MLL-AF9 gene reduces the HOX fusion proteins survival of leukemic cells and changes the disease phenotype, but it does not affect AML initiation.60 One of the most frequent fusion partners for HOX genes is NUP98, The dysregulation of another regulator of HOX genes, the CDX a member of the nuclear pore family (Figure 2). It is localized in gene family, has also been shown to drive the development of the nuclear membrane and functions as a selective transporter for AML. CDX2 is expressed in the majority of AML cases (90%), but RNA and proteins between the nucleus and cytoplasm. NUP98- not in normal adult hematopoiesis, and Cdx2-elevated expression HOX fusion proteins have been reported in AML and other leads to AML with only a short latency period.57 In contrast, the . In AML, NUP98-HOXA9 is associated with a t(7;11) 85,86 closely related CDX4 gene is expressed in 25% of AML cases, and (p15;p15) translocation. There are eight other HOX genes that 87,88 in normal adult hematopoiesis, and Cdx4 overexpression in can be fused with NUP98, including HOXA11 and HOXA13, 89,90 91 murine whole BM results in AML but only with a long latency HOXD11 and HOXD13, and HOXC13. Thus, only the 50 end period.102 This latency can be accelerated in mice through members of each HOX complex have been documented to be cooperation of Meis1 which results in the overexpression of a fused with NUP98 in AML. However, Hoxb3 has been shown to be 92 number of Hox genes including Hoxa6, Hoxa7, Hoxa9, Hoxb4, a potential leukemogenic partner with Nup98, suggesting that Hoxb8 and Hoxc6.103 Cdx2 expression alone is sufficient to drive the ability to be a fusion NUP98 partner is not limited to the 50 end the upregulation of a related set of HOX genes,58 demonstrating HOX genes. the importance of the Cdx family in the dysregulation of Hox Nup98-Hox fusion proteins result in AML with a long latency, genes during AML. around 11–12 months. However, this latency can be reduced to The dysregulation of HOX gene expression is also associated two months by co-overexpression of the Hox cofactor Meis1 and 93–95 with the nucleophosmin 1 (NPM1) . NPM1 is a chaperone the receptor Flt3. FLT3 has an essential role in protein that shuttles between the nucleus and cytoplasm, the regulation of early hematopoietic progenitors growth, and although its predominant localization is in the nucleus.104 NPM1 causes increased and uncontrolled self-renewal of these cells 96 has a crucial role in several biological processes, such as ribosome through a FLT3 -independent pathway. biogenesis, genomic stability and cell cycle progression. In adult AML, NPM1 mutation is the most common genetic aberration, HOX overexpression in AML reported in about 35% of all adult AML and approximately in 45– HOX genes may also be indirectly involved in AML through 55% of normal karyotype AML.105–107 In pediatric AML, NPM1 chromosomal rearrangements that involve their upstream reg- mutations are significantly less common, occurring in 8–10% of ulators, such as MLL. MLL fusion proteins constitute about 10% of cases, and in about a quarter of normal karyotype cases.108,109 The therapy-related AML and 3% of de novo AML.97 There are more relocation of NPM1 into the cytoplasm (NPMc þ ) occurs only in than 64 translocation partner genes that fuse with the MLL AML.110 This relocation causes upregulation of a number of HOX N-terminus.98 Normally, Mll positively regulates the transcription genes, some of which are similar to those seen in AML initiated by of Hox genes by maintaining their expression through direct a MLL chimeric gene fusion, while some are distinct. Thus for binding to a proximal region.99 MLL fusion proteins example HOXA4, HOXA6, HOXA7, HOXA9, HOXB9 and MEIS1 are activate HOX gene transcription more efficiently than MLL alone,97 overexpressed in both contexts, while HOXB2, HOXB3, HOXB5, especially the 50 end members of the HOXA cluster, together with HOXB6 and HOXD4 are upregulated in NPMc þ AML only.111 It has their co-activator MEIS1. As a consequence, myeloid differentiation been reported that activation of a humanized Npm1 allele led to is blocked and proliferation is enhanced. Consistent with this overexpression of Hoxa5, Hoxa7, Hoxa9 and Hoxa10, induction of proposed mechanism, it has been reported that MLL-AF9, like HSC self-renewal and the expansion of myelopoiesis.112 The exact NUP98-HOXA9, leads to a block in erythroid/myeloid maturation mechanism of the association between the NPM1 mutation and and to erythroid hyperplasia,100 and the Mll-Enl the upregulation of HOX genes is still unclear. A possible requires Hoxa7 and Hoxa9 for efficient immortalization of myeloid explanation is that NPM1 directly disturbs the expression of HOX

a HOX NH2 MD PM H HD COOH

b NUP98

NH2 FG GLEBS FG RBD COOH

c NUP98-HOX

NH2 FG GLEBS FG PM H HD COOH

Figure 2. Structures of AbdB HOX, NUP98 and the predictive fusion protein NUP98-HOX. (a) A general structure of AbdB HOX (9–13) proteins that have been reported to fuse with NUP98. (b) Structure of normal NUP98 protein. (c) Structure of predictive NUP98-HOX fusion protein. This fusion eliminates MD and RBD from AbdB and NUP98, respectively. The arrows represent the breakpoints. MD: MEIS domain, PM: Pbx motif, H: hexapeptide, HD: homeodomain, FG: phenylalanine-glycine, GLEBS: gle2p-binding-like motif, RBD: RNA-binding domain.

Leukemia (2013) 1000 – 1008 & 2013 Macmillan Publishers Limited The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1005 genes, or alternatively, that NPM1 mutations arrest the ACKNOWLEDGEMENTS differentiation of early hematopoietic progenitors in which HOX RA Alharbi gratefully acknowledges the support of Albaha University. expression is upregulated.104

HOX gene dysregulation in acute lymphoid leukemia REFERENCES The dysregulation of HOX genes has also been reported in 1 Frohling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malig- acute lymphoid leukemia (ALL) including both B- and T-precursor nancies: pathogenetic and clinical implications. J Clin Oncol 2005; 23: ALL (B-ALL and T-ALL), especially when MLL translocations are 6285–6295. involved. For example, human HOXA9, HOXA10 and HOXC6, and 2 Smith A, Howell D, Patmore R, Jack A, Roman E. Incidence of haematological their cofactor MEIS1 were upregulated in both MLL-ENL T-ALL and malignancy by sub-type: a report from the Haematological Malignancy Research MLL B-ALL.113 Human HOXA9 and MEIS1, and murine Hoxa5, Hoxa9 Network. Br J 2011; 105: 1684–1692. and Meis1 were likewise upregulated in MLL-AF4 B-ALL.114,115 A 3 Estey E, Do¨hner H. Acute myeloid leukaemia. The Lancet 2006; 368: 1894–1907. common signature of MLL rearrangements in AML and ALL is the 4Mro´ zek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD. Clinical relevance overexpression of HOXA genes including HOXA3, HOXA5, HOXA7, of mutations and gene-expression changes in adult acute myeloid leukemia 116 with normal cytogenetics: are we ready for a prognostically prioritized molecular HOXA9 and HOXA10. In addition, HOX genes can be involved in classification? Blood 2007; 109: 431–448. T-ALL through other translocations such as CALM-AF10 T-ALL, 5 Dohner K, Dohner H. Molecular characterization of acute myeloid leukemia. where human HOXA5, HOXA9, HOXA10 and MEIS1 are Haematologica 2008; 93: 976–982. 117 overexpressed. HOXA proteins can also form chimeric fusion 6 Renneville A, Roumier C, Biggio V, Nibourel O, Boissel N, Fenaux P et al. Coop- proteins with T-cell receptor (HOXA-TCR) in T-ALL, which results in erating gene mutations in acute myeloid leukemia: a review of the literature. the global overexpression of HOXA genes.118,119 Surprisingly, the Leukemia 2008; 22: 915–931. aberrant expression of CDX2 in ALL, an upstream regulator of HOX 7 Betz BL, Hess JL. Acute myeloid leukemia diagnosis in the 21st century. Arch genes, is not correlated with HOX gene dysregulation.120 Pathol Lab Med 2010; 134: 1427–1433. 8 Hox Garcia-FernandezJ. ParaHox, ProtoHox: facts and guesses. Heredity 2004; 94: 145–152. HOX genes as prognostic markers 9 Shah N, Sukumar S. The Hox genes and their roles in oncogenesis. Nat Rev HOX gene expression has become an important prognostic factor Cancer 2010; 10: 361–371. in AML. Overexpression of HOX genes is associated with an 10 Abramovich C, Pineault N, Ohta H, Humphries RK. Hox Genes: from leukemia to intermediate/unfavorable cytogenetic subset of AML. For exam- expansion. Ann N Y Acad Sci 2005; 1044: 109–116. 11 Amores A, Force A, Yan YL, Joly L, Amemiya C, Fritz A et al. Zebrafish hox clusters ple, among 6817 genes that have been investigated in AML and genome . Science 1998; 282: 1711–1714. patients, the single gene correlated with the worst outcome and 12 Rice KL, Licht JD. HOX deregulation in acute myeloid leukemia. J Clin Invest 2007; 121 relapse of disease as well as short survival was HOXA9. 117: 865–868. Correspondingly, low HOXA9 expression was found to correlate 13 He H, Hua X, Yan J. Epigenetic regulations in hematopoietic Hox code. Oncogene with the best outcome and response to therapy.122 It is also 2011; 30: 379–388. noteworthy that low expression of HOXA4 and MEIS1 are also 14 Knittel T, Kessel M, Kim MH, Gruss P. A conserved enhancer of the human and favorable predictors for AML patient outcome.123 The global levels murine Hoxa-7 gene specifies the anterior boundary of expression during of HOX expression also seem to reflect the outcome of disease, embryonal development. Development 1995; 121: 1077–1088. possibly reflecting the functional redundancy exhibited by many 15 Phelan M, Rambaldi I, Featherstone M. Cooperative interactions between HOX and PBX proteins mediated by a conserved motif. Mol Cell Biol 1995; 15: of the HOX genes. Thus, the highest levels of HOX genes are seen 3989–3997. in FLT3 mutation cases, which have unfavorable outcomes, and 16 Moens CB, Selleri L. Hox cofactors in vertebrate development. Dev Biol 2006; 291: generally the HOX genes are expressed only at a very low level in 193–206. favorable subsets of AML. 17 Mann RS, Lelli KM, Joshi R. Hox Specificity: Unique Roles for Cofactors and Collaborators. Chapter 3 Current Topics in , 2009. Academic Press. FUTURE DIRECTIONS 18 Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G. Defining roles for Although recent work has established the importance of HOX HOX and MEIS1 genes in induction of acute myeloid leukemia. Mol Cell Biol 2001; 21: 224–234. genes in the development of AML, it is still not clear exactly what 19 Dimartino JF, Selleri L, Traver D, Firpo MT, Rhee J, Warnke R et al. The Hox the functions of these genes are beyond a general inhibition of cofactor and proto-oncogene Pbx1 is required for maintenance of definitive differentiation and an increase in cell proliferation. This lack of hematopoiesis in the fetal liver. Blood 2001; 98: 618–626. precise mechanistic knowledge for individual HOX genes may be 20 Hisa T, Spence SE, Rachel RA, Fujita M, Nakamura T, Ward JM et al. Hemato- owing to the functional redundancy showed by many members of poietic, angiogenic and eye defects in Meis1 mutant . EMBO J 2004; 23: this family, making the knockdown of single HOX genes generally 450–459. difficult to interpret, and it may also help to explain the contrast in 21 Pillay LM, Forrester AM, Erickson T, Berman JN, Waskiewicz AJ. The Hox cofactors gene knockin and knockout results. In myeloma and some solid Meis1 and Pbx act upstream of to regulate primitive hematopoiesis. Dev malignancies this has been addressed by targeting the HOX genes Biol 2010; 340: 306–317. 22 Shen WF, Chang CP, Rozenfeld S, Sauvageau G, Humphries RK, Lu M et al. Hox as a group by antagonizing their interactions with the PBX cofactor 124–128 homeodomain proteins exhibit selective complex stabilities with Pbx and DNA. using the peptide inhibitor HXR9. A similar approach may Nucleic Acids Res 1996; 24: 898–906. be useful in AML for addressing redundant functions of HOX 23 Shen W, Montgomery J, Rozenfeld S, Moskow J, Lawrence H, Buchberg AC et al. genes. HXR9 is cytotoxic to cells, predominantly through the AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain induction of , which in turn depends upon a rapid proteins. Mol Cell Biol 1997; 17: 6448–6458. increase in expression of c-Fos.124–129 It seems likely that AML 24 Giampaolo A, Sterpetti P, Bulgarini D, Samoggia P, Pelosi E, Valtieri M et al. would also be sensitive to killing by HXR9, and indeed it may be Key functional role and lineage-specific expression of selected HOXB genes in that the HOX genes could represent a useful therapeutic target, purified hematopoietic progenitor differentiation. Blood 1994; 84: 3637–3647. especially in AMLs that show high levels of HOX expression and a 25 Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined correspondingly poor prognosis. subpopulations of leukemic and normal human hematopoietic cells. Leukemia 1999; 13: 687–698. 26 Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of CONFLICT OF INTEREST Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic The authors declare no conflict of interest. . Exp Hematol 2002; 30: 49–57.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1000 – 1008 The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1006 27 Grier DG, Thompson A, Kwasniewska A, Mcgonigle GJ, Halliday HL, Lappin TR. 50 Cellot S, Krosl J, Chagraoui J, Meloche S, Humphries RK, Sauvageau G. Sustained The pathophysiology of HOX genes and their role in cancer. J Pathol 2005; 205: in vitro trigger of self-renewal divisions in Hoxb4(hi)Pbx1(lo) hematopoietic stem 154–171. cells. Exp Hematol 2007; 35: 802–816. 28 Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, Reid DS et al. 51 Beuchle D, Struhl G, Muller J. Polycomb group proteins and heritable silencing of Differential expression of homeobox genes in functionally distinct CD34 þ Drosophila Hox genes. Development 2001; 128: 993–1004. subpopulations of human bone marrow cells. Proc Natl Acad Sci USA 1994; 91: 52 Ernst P, Mabon M, Davidson AJ, Zon LI, Korsmeyer SJ. An Mll-dependent 12223–12227. Hox program drives hematopoietic progenitor expansion. Curr Biol 2004; 14: 29 Taghon T, Thys K, De Smedt M, Weerkamp F, Staal FJT, Plum J et al. Homeobox 2063–2069. gene expression profile in human hematopoietic multipotent stem cells and 53 Jude CD, Climer L, Xu D, Artinger E, Fisher JK, Ernst P. Unique and independent T-cell progenitors: implications for human T-cell development. Leukemia 2003; roles for MLL in adult hematopoietic stem cells and progenitors. Cell stem cell 17: 1157–1163. 2007; 1: 324–337. 30 Crooks GM, Fuller J, Petersen D, Izadi P, Malik P, Pattengale PK et al. Constitutive 54 Davidson AJ, Zon LI. The caudal-related homeobox genes cdx1a and act HOXA5 expression inhibits erythropoiesis and increases myelopoiesis from redundantly to regulate hox gene expression and the formation of putative human hematopoietic progenitors. Blood 1999; 94: 519–528. hematopoietic stem cells during zebrafish embryogenesis. Dev Biol 2006; 292: 31 Fuller JF, Mcadara J, Yaron Y, Sakaguchi M, Fraser JK, Gasson JC. Characterization 506–518. of HOX gene expression during myelopoiesis: role of HOX A5 in lineage com- 55 Wang Y, Yabuuchi A, Mckinney-Freeman S, Ducharme DMK, Ray MK, mitment and maturation. Blood 1999; 93: 3391–3400. Chawengsaksophak K et al. Cdx gene deficiency compromises embryonic 32 So CW, Karsunky H, Wong P, Weissman IL, Cleary ML. Leukemic transformation of hematopoiesis in the mouse. Proc Natl Acad Sci USA 2008; 105: 7756–7761. hematopoietic progenitors by MLL-GAS7 in the absence of Hoxa7 or Hoxa9. 56 Koo S, Huntly BJ, Wang Y, Chen J, Brumme K, Ball B et al. Cdx4 is dispensable for Blood 2004; 103: 3192–3199. murine adult hematopoietic stem cells but promotes MLL-AF9-mediated leu- 33 Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Hoxa9 kemogenesis. Haematologica 2010; 95: 1642–1650. transforms primary bone marrow cells through specific collaboration with 57 Scholl C, Bansal D, Dohner K, Eiwen K, Huntly BJP, Lee BH et al. The homeobox Meis1a but not Pbx1b. EMBO J 1998; 17: 3714–3725. gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and 34 Thorsteinsdottir U, Mamo A, Kroon E, Jerome L, Bijl J, Lawrence HJ et al. Over- promotes leukemogenesis. J Clin Invest 2007; 117: 1037–1048. expression of the myeloid leukemia–associatedHoxa9 gene in bone marrow cells 58 Rawat VPS, Thoene S, Naidu VM, Arseni N, Heilmeier B, Metzeler K et al. Over- induces stem cell expansion. Blood 2002; 99: 121–129. expression of CDX2 perturbs HOX gene expression in murine progenitors 35 Magnusson M, Brun ACM, Lawrence HJ, Karlsson S. Hoxa9// com- depending on its N-terminal domain and is closely correlated with deregulated pound null mice display severe hematopoietic defects. Exp Hematol 2007a; 35: HOX gene expression in human acute myeloid leukemia. Blood 2008; 111: 1421–1428. 309–319. 36 Buske C, Feuring-Buske M, Antonchuk J, Rosten P, Hogge DE, Eaves CJ et al. 59 Riedt T, Ebinger M, Salih HR, Tomiuk J, Handgretinger R, Kanz L et al. Aberrant Overexpression of HOXA10 perturbs human lymphomyelopoiesis in vitro and expression of the homeobox gene CDX2 in pediatric acute lymphoblastic leu- in vivo. Blood 2001; 97: 2286–2292. kemia. Blood 2009; 113: 4049–4051. 37 Sauvageau G, Thorsteinsdottir U, Hough MR, Hugo P, Lawrence HJ, Largman C et al. 60 Faber J, Krivtsov AV, Stubbs MC, Wright R, Davis TN, Van Den Heuvel-Eibrink M Overexpression of HOXB3 in hematopoietic cells causes defective lymphoid et al. HOXA9 is required for survival in human MLL-rearranged acute leukemias. development and progressive myeloproliferation. Immunity 1997; 6: 13–22. Blood 2009; 113: 2375–2385. 38 Ko KH, Kwan Lam QL, Zhang M, Yen Wong CK, Chun LoCK, Kahmeyer-Gabbe M 61 Hu YL, Passegue´ E, Fong S, Largman C, Lawrence HJ. Evidence that the Pim1 et al. Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow. kinase gene is a direct target of HOXA9. Blood 2007; 109: 4732–4738. Exp Hematol 2007; 35: 465–475. 62 Nagel S, Venturini L, Marquez V, Meyer C, Kaufmann M, Scherr M et al. Polycomb 39 Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp repressor complex 2 regulates HOXA9 and HOXA10, activating ID2 in NK/T-cell PM et al. Overexpression of HOXB4 in hematopoietic cells causes the selective lines. Mol Cancer 2010; 9: 151. expansion of more primitive populations in vitro and in vivo. Genes Dev 1995; 9: 63 Bei L, Lu Y, Eklund EA. HOXA9 activates transcription of the gene encoding 1753–1765. gp91Phox during myeloid differentiation. J Biol Chem 2005; 280: 12359–12370. 40 Amsellem S, Pflumio F, Bardinet D, Izac B, Charneau P, Romeo PH et al. Ex vivo 64 Gwin K, Frank E, Bossou A, Medina KL. Hoxa9 Regulates Flt3 in Lymphohema- expansion of human hematopoietic stem cells by direct delivery of the HOXB4 topoietic Progenitors. J Immunol 2010; 185: 6572–6583. homeoprotein. Nat Med 2003; 9: 1423–1427. 65 Hu YL, Fong S, Ferrell C, Largman C, Shen WF. HOXA9 modulates its oncogenic 41 Brun ACM, Bjo¨rnsson JM, Magnusson M, Larsson N, Levee´n P, Ehinger M partner Meis1 to influence normal hematopoiesis. Mol Cell Biol 2009; 29: et al. Hoxb4-deficient mice undergo normal hematopoietic development but 5181–5192. exhibit a mild proliferation defect in hematopoietic stem cells. Blood 2004; 103: 66 Huang Y, Sitwala K, Bronstein J, Sanders D, Dandekar M, Collins C et al. Identi- 4126–4133. fication and characterization of Hoxa9 binding sites in hematopoietic cells. Blood 42 Bijl J, Thompson A, Ramirez-Solis R, Krosl J, Grier DG, Lawrence HJ et al. Analysis 2012; 119: 388–398. of HSC activity and compensatory Hox gene expression profile in Hoxb cluster 67 Hess JL, Bittner CB, Zeisig DT, Bach C, Fuchs U, Borkhardt A et al. c-Myb is an mutant fetal liver cells. Blood 2006; 108: 116–122. essential downstream target for homeobox-mediated transformation of hema- 43 Bjornsson JM, Larsson N, Brun ACM, Magnusson M, Andersson E, Lundstrom P et al. topoietic cells. Blood 2006; 108: 297–304. Reduced proliferative capacity of hematopoietic stem cells deficient in Hoxb3 and 68 Takeda A, Goolsby C, Yaseen NR. NUP98-HOXA9 induces long-term proliferation Hoxb4. Mol Cell Biol 2003; 23: 3872–3883. and blocks differentiation of primary human CD34 þ hematopoietic cells. Cancer 44 Fischbach NA, Rozenfeld S, Shen W, Fong S, Chrobak D, Ginzinger D et al. HOXB6 Res 2006; 66: 6628–6637. overexpression in murine bone marrow immortalizes a myelomonocytic pre- 69 Magnusson M, Brun ACM, Miyake N, Larsson J, Ehinger M, Bjornsson JM et al. cursor in vitro and causes hematopoietic stem cell expansion and acute myeloid HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/ leukemia in vivo. Blood 2005; 105: 1456–1466. development. Blood 2007; 109: 3687–3696. 45 Kappen C. Disruption of the homeobox gene Hoxb-6 in mice results in 70 Bei L, Lu Y, Bellis SL, Zhou W, Horvath E, Eklund EA. Identification of increased numbers of early erythrocyte progenitors. Am J Hematol 2000; 65: a HoxA10 activation domain necessary for transcription of the gene 111–118. encoding b3 integrin during myeloid differentiation. J Biol Chem 2007; 282: 46 Takeshita K, Bollekens JA, Hijiya N, Ratajczak M, Ruddle FH, Gewirtz AM. A 16846–16859. homeobox gene of the antennapedia class is required for human adult ery- 71 Wang H, Lu Y, Huang W, Papoutsakis ET, Fuhrken P, Eklund EA. HoxA10 activates thropoiesis. Proc Natl Acad Sci USA 1993; 90: 3535–3538. transcription of the gene encoding mitogen-activated protein kinase phospha- 47 Daga A, Podesta M, Capra MC, Piaggio G, Frassoni F, Corte G. The retroviral tase 2 (Mkp2) in myeloid cells. J Biol Chem 2007; 282: 16164–16176. transduction of HOXC4 into human CD34 þ cells induces an in vitro expansion 72 Bei L, Huang W, Wang H, Shah C, Horvath E, Eklund E. HoxA10 activates CDX4 of clonogenic and early progenitors. Exp Hematol 2000; 28: 569–574. transcription and Cdx4 activates HOXA10 transcription in myeloid cells. J Biol 48 Shimamoto T, Tang Y, Naot Y, Nardi M, Brulet P, Bieberich CJ et al. Hematopoietic Chem 2011; 286: 19047–19064. progenitor cell abnormalities in Hoxc-8 null mutant mice. J Exp Zool 1999; 283: 73 Bromleigh VC, Freedman LP. p21 is a transcriptional target of HOXA10 in dif- 186–193. ferentiating myelomonocytic cells. Genes Dev 2000; 14: 2581–2586. 49 Krosl J, Beslu N, Mayotte N, Humphries RK, Sauvageau G. The competitive nature 74 Eklund EA, Jalava A, Kakar R. Tyrosine phosphorylation of HoxA10 decreases of HOXB4-transduced HSC is limited by PBX1: the generation of ultra-competi- DNA binding and transcriptional repression during interferon g-induced tive stem cells retaining full differentiation potential. Immunity 2003; 18: differentiation of myeloid leukemia cell lines. J Biol Chem 2000; 275: 561–571. 20117–20126.

Leukemia (2013) 1000 – 1008 & 2013 Macmillan Publishers Limited The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1007 75 Palmqvist L, Pineault N, Wasslavik C, Humphries RK. Candidate genes for 100 Abdul-Nabi AM, Yassin ER, Varghese N, Deshmukh H, Yaseen NR. In vitro expansion and transformation of hematopoietic stem cells by NUP98-HOX transformation of primary human CD34 þ cells by AML fusion : early fusion genes. PLoS ONE 2007; 2: e768. gene expression profiling reveals possible drug target in AML. PLoS ONE 2010; 5: 76 Lee HM, Zhang H, Schulz V, Tuck DP, Forget BG. Downstream targets of HOXB4 e12464. in a cell line model of primitive hematopoietic progenitor cells. Blood 2010; 116: 101 Ayton PM, Cleary ML. Transformation of myeloid progenitors by MLL onco- 720–730. proteins is dependent on Hoxa7 and Hoxa9. Genes Dev 2003; 17: 2298–2307. 77 Pan Q, Simpson R. Antisense knockout of HOXB4 blocks 1,25-dihydroxyvitamin 102 Bansal D, Scholl C, Frohling S, Mcdowell E, Lee BH, Dohner K et al. Cdx4 dys- D3 inhibition of c-myc expression. J Endocrinol 2001; 169: 153–159. regulates Hox gene expression and generates acute myeloid leukemia alone and 78 Satoh Y, Matsumura I, Tanaka H, Ezoe S, Sugahara H, Mizuki M et al. Roles in cooperation with Meis1a in a murine model. Proc Natl Acad Sci USA 2006; 103: for c-Myc in self-renewal of hematopoietic stem cells. J Biol Chem 2004; 279: 16924–16929. 24986–24993. 103 Bansal D, Scholl C, Fro¨hling S, Mcdowell E, Lee BH, Do¨hner K et al. Cdx4 dys- 79 Krosl J, Sauvageau G. AP-1 complex is effector of Hox-induced cellular regulates Hox gene expression and generates acute myeloid leukemia alone and proliferation and transformation. Oncogene 2000; 19: 5134–5141. in cooperation with Meis1a in a murine model. Proc Natl Acad Sci USA 2006; 103: 80 Leverson JD, Koskinen PJ, Orrico FC, Rainio EM, Jalkanen KJ, Dash AB et al. Pim-1 16924–16929. Kinase and p100 cooperate to enhance c-Myb activity. Mol Cell 1998; 2: 417–425. 104 Rau R, Brown P. Nucleophosmin (NPM1) mutations in adult and childhood acute 81 Shah CA, Bei L, Wang H, Platanias LC, Eklund EA. HoxA10 protein regulates myeloid leukaemia: towards definition of a new leukaemia entity. Hematol Oncol transcription of gene encoding fibroblast growth factor 2 (FGF2) in myeloid cells. 2009; 27: 171–181. J Biol Chem 2012; 287: 18230–18248. 105 Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic 82 Shah CA, Wang H, Bei L, Platanias LC, Eklund EA. HoxA10 Regulates Transcription nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl of the Gene Encoding Transforming Growth Factor b2 (TGFb2) in Myeloid Cells. JMed2005; 352: 254–266. J Biol Chem 2011; 286: 3161–3176. 106 Thiede C, Koch S, Creutzig E, Steudel C, Lllmer T, Schiach M et al. Prevelance and 83 Oshima M, Endoh M, Endo TA, Toyoda T, Nakajima-Takagi Y, Sugiyama F et al. prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic leukemia (AML). Blood 2006; 107: 4011–4020. stem and progenitor cells developing from embryonic stem cells. Blood 2011; 107 Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Bullinger L et al. 117: e142–e150. Mutations and treatment outcome in cytogenetically normal acute myeloid 84 Fan R, Bonde S, Gao P, Sotomayor B, Chen C, Mouw T et al. Dynamic HoxB4 leukemia. N Engl J Med 2008; 358: 1909–1918. regulatory network during embryonic stem cell differentiation to hematopoietic 108 Brown P, Mcintyre E, Rau R, Meshinchi S, Lacayo N, Dahl G et al. The incidence cells. Blood 2012; 119: e139–e147. and clinical significance of nucleophosmin mutations in childhood AML. 85 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K et al. Blood 2007; 110: 979–985. Fusion of the nucleoporin gene NUP98 to HOXA9 by the 109 Hollink IHIM, Zwaan CM, Zimmermann M, Arentsen-Peters TCJM, Pieters R, translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 1996; 12: Cloos J et al. Favorable prognostic impact of NPM1 gene mutations in childhood 154–158. acute myeloid leukemia, with emphasis on cytogenetically normal AML. Leuke- 86 Borrow J, Shearman AM, Stanton VP, Becher R, Collins T, Williams AJ et al. mia 2009; 23: 262–270. The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes 110 Falini B, Bolli N, Liso A, Martelli MP, Mannucci R, Pileri S et al. Altered nucleo- for nucleoporin NUP96 and class I homeoprotein HOXA9. Nat Genet 1996; 12: phosmin transport in acute myeloid leukaemia with mutated NPM1: molecular 159–167. basis and clinical implications. Leukemia 2009; 23: 1731–1743. 87 Fujino T, Suzuki A, Ito Y, Ohyashiki K, Hatano Y, Miura I et al. Single-translocation 111 Mullighan CG, Kennedy A, Zhou X, Radtke I, Phillips LA, Shurtleff SA et al. and double-chimeric transcripts: detection of NUP98-HOXA9 in myeloid leuke- Pediatric acute myeloid leukemia with NPM1 mutations is characterized by a mias withHOXA11 or HOXA13 breaks of the chromosomal translocation gene expression profile with dysregulated HOX gene expression distinct from t(7;11)(p15;p15). Blood 2002; 99: 1428–1433. MLL-rearranged leukemias. Leukemia 2007; 21: 2000–2009. 88 Suzuki A, Ito Y, Sashida G, Honda S, Katagiri T, Fujino T et al. t(7;11)(p15;p15) 112 Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A et al. Mutant nucleophosmin chronic myeloid leukaemia developed into blastic transformation showing a and cooperating pathways drive leukemia initiation and progression in mice. novel NUP98/HOXA11 fusion. Br J Haematol 2002; 116: 170–172. Nat Genet 2011; 43: 470–475. 89 Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, Aplan PD. NUP98- 113 Ferrando AA, Armstrong SA, Neuberg DS, Sallan SE, Silverman LB, Korsmeyer SJ HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer et al. Gene expression signatures in MLL-rearranged T-lineage and B-precursor Res 1998; 58: 4269–4273. acute leukemias: dominance of HOX dysregulation. Blood 2003; 102: 262–268. 90 Taketani T, Taki T, Shibuya N, Ito E, Kitazawa J, Terui K et al. The HOXD11 gene is 114 Rozovskaia T, Feinstein E, Mor O, Foa R, Blechman J, Nakamura T et al. Upre- fused to the NUP98 gene in acute myeloid leukemia with t(2;11)(q31;p15). gulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4: 11) Cancer Res 2002; 62: 33–37. abnormality. Oncogene 2001; 20: 874–878. 91 Taketani T, Taki T, Shibuya N, Kikuchi A, Hanada R, Hayashi Y. Novel NUP98- 115 Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati S, Sinha AU et al. H3K79 HOXC11 resulted from a chromosomal break within exon 1 of profiles define murine and human MLL-AF4 Leukemias. Cancer Cell HOXC11 in acute myeloid leukemia with t(11;12)(p15;q13). Cancer Res 2002; 62: 2008; 14: 355–368. 4571–4574. 116 Zangrando A, Dell’orto MC, Kronnie GT, Basso G. MLL rearrangements in 92 Pineault N, Abramovich C, Ohta H, Humphries RK. Differential and common pediatric acute lymphoblastic and myeloblastic leukemias: MLL specific and leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with lineage specific signatures. BMC Med Genomics 2009; 2:36. Meis1. Mol Cell Biol 2004; 24: 1907–1917. 117 Dik WA, Brahim W, Braun C, Asnafi V, Dastugue N, Bernard OA et al. CALM- 93 Palmqvist L, Argiropoulos B, Pineault N, Abramovich C, Sly LM, Krystal G et al. The AF10 þ T-ALL expression profiles are characterized by overexpression of HOXA Flt3 collaborates with NUP98-HOX fusions in acute and BMI1 oncogenes. Leukemia 2005; 19: 1948–1957. myeloid leukemia. Blood 2006; 108: 1030–1036. 118 Soulier J, Clappier E, Cayuela JM, Regnault A, Garcı´a-Peydro´ M, Dombret H et al. 94 Kroon E, Thorsteinsdottir U, Mayotte N, Nakamura T, Sauvageau G. NUP98- HOXA genes are included in genetic and biologic networks defining human HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid acute T-cell leukemia (T-ALL). Blood 2005; 106: 274–286. leukemias in mice. EMBO J 2001; 20: 350–361. 119 Speleman F, Cauwelier B, Dastugue N, Cools J, Verhasselt B, Poppe B et al. Anew 95 Pineault N, Buske C, Feuring-Buske M, Abramovich C, Rosten P, Hogge DE et al. recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 Induction of acute myeloid leukemia in mice by the human leukemia-specific and HOXA11 in a subset of T-cell acute lymphoblastic leukemias. Leukemia 2005; fusion gene NUP98-HOXD13 in concert with Meis1. Blood 2003; 101: 4529–4538. 19: 358–366. 96 Tosic N, Stojiljkovic M, Colovic N, Colovic M, Pavlovic S. Acute myeloid leukemia 120 Thoene S, Rawat VPS, Heilmeier B, Hoster E, Metzeler KH, Herold T et al. The with NUP98-HOXC13 fusion and FLT3 internal tandem duplication mutation: homeobox gene CDX2 is aberrantly expressed and associated with an inferior case report and literature review. Cancer Genet Cytogenet 2009; 193: 98–103. prognosis in patients with acute lymphoblastic leukemia. Leukemia 2009; 23: 97 Slany RK. The molecular biology of mixed lineage leukemia. Haematol-Hematol J 649–655. 2009; 94: 984–993. 121 Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP et al. 98 Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J et al. New insights to Molecular classification of cancer: class discovery and class prediction by gene the MLL recombinome of acute leukemias. Leukemia 2009; 23: 1490–1499. expression monitoring. Science 1999; 286: 531–537. 99 Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD et al. MLL targets SET 122 Andreeff M, Ruvolo V, Gadgil S, Zeng C, Coombes K, Chen W et al. HOX domain methyltransferase activity to Hox gene promoters. Mol Cell 2002; 10: expression patterns identify a common signature for favorable AML. Leukemia 1107–1117. 2008; 22: 2041–2047.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1000 – 1008 The role of HOX genes in normal hematopoiesis and acute leukemia RA Alharbi et al 1008 123 Zangenberg M, Grubach L, Aggerholm A, Silkjaer T, Juhl-Christensen C, 126 Plowright L, Harrington KJ, Pandha HS, Morgan R. HOX transcription factors are Nyvold CG et al. The combined expression of HOXA4 and MEIS1 is an inde- potential therapeutic targets in non-small-cell lung cancer (targeting HOX genes pendent prognostic factor in patients with AML. Eur J Haematol 2009; 83: in lung cancer). Br J Cancer 2009; 100: 470–475. 439–448. 127 Shears L, Plowright L, Harrington K, Pandha H, Morgan R. Disrupting the inter- 124 Daniels TR, Neacato II, Rodriguez JA, Pandha HS, Morgan R, Penichet ML. action between HOX and PBX causes necrotic and apoptotic cell death in the Disruption of HOX activity leads to cell death that can be enhanced renal cancer lines CaKi-2 and 769-P. J Urol 2008; 180: N2196–N2201. by the interference of iron uptake in malignant B cells. Leukemia 2010; 24: 128 Morgan R, Plowright L, Harrington K, Michael A, Pandha H. Targeting HOX and 1555–1565. PBX transcription factors in ovarian cancer. BMC Cancer 2010; 10:89. 125 Morgan R, Pirard P, Shears L, Sohal S, Pettengell R, Pandha H. Antagonism of 129 Espinosa AV, Shinohara M, Porchia LM, Chung YJ, Mccarty S, Saji M et al. Reg- HOX/PBX dimer formation blocks the in vivo proliferation of melanoma. Cancer ulator of calcineurin 1 modulates cancer cell migration in vitro. Clin Exp Metas- Res 2007; 67: 5806–5813. tasis 2009; 26: 517–526.

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