NKL Homeobox Genes in Leukemia

Home , BARHL2, BCL11B, DBX1, DLX4, DLX6, EMX1

I Homminga, R Pieters and JPP Meijerink

Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands

NK-like (NKL) homeobox genes code for transcription factors, to homeotic transformations such as originally described by which can act as key regulators in fundamental cellular Bateson. The classification and nomenclature of homeobox processes. NKL genes have been implicated in divergent types genes have varied substantially between research groups and of cancer. In this review, we summarize the involvement of NKL 5 genes in cancer and leukemia in particular. NKL genes can act over time. In 2007, Holland et al. proposed a classification for as tumor-suppressor genes and as oncogenes, depending on all human homeobox genes based on their hypothetical tissue type. Aberrant expression of NKL genes is especially evolutionary shared ancestry. In all, 11 homeobox gene classes common in T-cell acute lymphoblastic leukemia (T-ALL). are recognized that comprise a total of 102 homeobox gene In T-ALL, 8 NKL genes have been reported to be highly families. The largest classes are the ANTP (analogous to the B expressed in specific T-ALL subgroups, and in 30% of cases, Drosophila antennapedia (antp) gene) and PRD (analogous to high expression is caused by chromosomal rearrangement of 1 of 5 NKL genes. Most of these NKL genes are normally the Drosophila paired (prd) gene) classes. The ANTP class can not expressed in T-cell development. We hypothesize that be divided into the HOX-like (HOXL, including the HOXA and the NKL genes might share a similar downstream effect that HOXB gene clusters) and the NKL (NK-like) subclasses. The promotes leukemogenesis, possibly due to mimicking a NKL involvement of HOXL genes in cancer and leukemia has been gene that has a physiological role in early hematopoietic extensively reviewed in literature,6–11 whereas the NKL genes development, such as HHEX. All eight NKL genes posses have received less attention. However, recent findings implicate a conserved Eh1 repressor motif, which has an important role in regulating downstream targets in hematopoiesis and an important role for various NKL genes in human cancer. possibly in leukemogenesis as well. Identification of a potential Therefore, this review will focus on the NKL homeobox genes, common leukemogenic NKL downstream pathway will provide their role in cancer and leukemia in particular. a promising subject for future studies. Leukemia (2012) 26, 572–581; doi:10.1038/leu.2011.330; published online 18 November 2011 NKL homeobox genes Keywords: NKL; homeobox genes; T-ALL NKL homeobox genes have important functions in cell-fate specification and embryologic organ development, and their expression is often cell type specific. They are key regulators in fundamental processes as differentiation, proliferation and apoptosis. The NKL subclass comprises 48 genes and 19 Introduction assumed pseudogenes (Table 1). All NKL genes have a similar homeodomain. The NKL genes are named after Kim and 12 Homeobox genes Nirenberg who identified NK1–NK4 genes in 1989 when In 1894, William Bateson described a distinct kind of searching for new genes containing homeobox sequences in transformation within species, which he terms ‘homeosis’, Drosophila melanogaster. The human NKL genes can be divided derived from the Greek ‘homoiosis’, which means ‘the same’. into gene families, which consist of genes that are all derived from a single gene of the latest common ancestor of Drosophila He defined it as ‘a change of something into the likeness of 5 something else’.1 Famous examples are the mutant fruit fly and humans (Table 1). In several cases, the gene names are Antennapedia, which grows a foot on the head where normally somewhat misleading, frequently suggesting higher similarity the antenna is located. Another example is a moth (Zygaena), than is actually the case. For instance, the human NKX2-3, which has an extra wing on the position of a hind leg. Later, the NKX2-5 and NKX2-6 genes belong to the NK4 family and are most similar to the D. melanogaster NK4 gene. NKX2-1 and genes held responsible for these variations were called homeotic genes.1 In 1983, two research groups independently identified a NKX 2-4 resemble the Drosophila scarecrow (scro) gene and comprise the Nk2.1 family; NKX2-2 and NKX2-8 are ortholo- 180-bp sequence that was conserved in all homeotic genes, 2,3 gous to the Drosophila ventral nervous system defective (vnd) denoted as the homeobox. The homeobox is strongly gene and part of the Nk2.2 family. In addition, not all NKL genes conserved among species and encodes for a DNA-binding 4 have names that start with NKX, other genes such as TLX1 and domain consisting of three a-helices called the homeodomain. TLX3 also belong to the NKL gene subclass (Table 1). The NKL By now, 4200 human homeodomain proteins are known. genes are not strongly clustered on the chromosomes in contrast Homeodomain proteins are transcription factors that are to the HOXA-D genes, although some NKL genes cluster in involved in many key processes such as body patterning, pairs. This linkage is conserved through species especially for embryonic organogenesis and cell-fate decisions. A mutation in LBX1-TLX1, LBX2-TLX2, NKX2-6-NKX3-1 and HMX2-HMX313 one of these genes can have far-fetching consequences and lead and to a lesser extend for NKX2-1-NKX2-8 and NKX2-2-NKX2-4.14 Initially, clustering is often the result of tandem duplications; Correspondence: Dr JPP Meijerink, Dr Molewaterplein 60 Sp2456, however, the conservation of some of these pairs is likely due to GJ Rotterdam 3015, The Netherlands. 13 E-mail: [email protected] shared regulatory regions. In humans, several NKL genes are Received 8 August 2011; revised 25 September 2011; accepted 13 loosely gathered over large areas on chromosome bands 2p13, October 2011; published online 18 November 2011 4p15–16, 5q35 and 10q23–26 (Table 1). NKL homeobox genes in leukemia I Homminga et al 573 Table 1 NKL homeobox genes

Family Drosophila homolog Gene Synonyms Location Eh-1 motif FxIxxIL

Nk2.1 Scarecrow NKX2-1 TITF1, TTF1, NKX2A, TEBP 14q13.3 FSVSDIL (scro) NKX2-4 NKX2D 20p11.2 FSVSDIL Nk2.2 Ventral nervous system NKX2-2 NKX2B 20p11.2 FSVKDIL Defective (vnd)/NK2 NKX2-8 NKX2H 14q13.3 F Nk3 Bagpipe NKX3-1 NKX3A, BAPX2 8p21.2 FLIQDIL (bap) NKX3-2 BAPX1 4p15.33 FSIQAIL Nk4 Tinman NKX2-3 NKX2C, CSX3 10q24.2 FSVKDIL (tin)/NK4 NKX2-5 NKX2E, CSX1, CSX 5q35.1 FSVKDIL NKX2-6 CSX2 8p21.2 FSVKDIL Nk5 H6-like-homeobox HMX1 NKX5-3 4p16.1 FLIENLL (hmx) HMX2 NKX5-2 10q26.1 FTIQSIL HMX3 NKX5-1 10q26.1 FSIKNLL Nk6 Hgtx NKX6-1 NKX6A 4q21.23 HGINDIL NKX6-2 NKX6B 10q26.1 HGISDIL NKX6-3 8p11.21 HGITDIL Tlx C15 TLX1 HOX11, TCL3 10q24.3 FGIDQIL TLX2 HOX11L1, NCX, ENX 2p13.1 FGIDQIL TLX3 HOX11L2, RNX 5q35.1 FGIDQIL

Msx Drop MSX1 HOX7 4p16.2 F (dr) MSX2 5q35.2 F Nanog F NANOG FLJ12581 12p13.3 F Dlx Distal-less DLX1 2q31.1 F (dll) DLX2 TES1 2q31.1 F DLX3 17q21.3 F DLX4 DLX7, BP1 17q21.3 F DLX5 7q21.3 F DLX6 7q21.3 F

Dbx CG12361 DBX1 11p15.1 FGVNAIL DBX2 12q12 FLIENLL Bsx Brain-speciﬁc homeobox gene (bsh) BSX BSX1 11q24.1 FFIEDIL Barx BARX1 9q22.32 FMIEEIL BARX2 11q24.3 FMIDEIL Barhl BarH1 BARHL1 9q34.13 FGIDSIL BarH2 BARHL2 1p22.2 FGIDTIL Lbx Ladybird early (lbe) LBX1 LBX1H 10q24.3 FSIEDIL Ladybird late(lbl) LBX2 2p13.1 LSIADIL Hlx Homeodomain protein 2.0 (H2.0) HLX HB24, HLX1 1q41 FCIADIL Hhex CG7056 HHEX PRH, PRHX 10q23.3 FYIEDIL

En Engrailed (en) EN1 2q14.2 FFIDNIL Invected (inv) EN2 7q36.3 FFIDNIL Vax F VAX1 10q26.1 F VAX2 2p13.3 F Ventx F VENTX 10q26.3 F Emx Empty Spiracles (ems) EMX1 2p13.2 F E5 EMX2 10q26.1 F Noto CG18599 NOTO 2p13.2 FSVEAIL

Nk1 Slouch NKX1-1 HSPX153, HPX153 4p16.3 FSVLDIL (slou) NKX1-2 C10orf121 10q26.1 F Each gray box corresponds to a separate branch in the phylogenetic tree as described by Holland et al.,5 and therefore contains genes that are more related with regard to the homeodomain. Pseudogenes are left out of the table.

NKL homeobox genes in cancer function as a tumor-suppressor gene and as an oncogene in lung cancer depending on the subtype but can also have a dual Aberrant expression of NKL homeobox genes may have an function within a lung cancer subtype.18 Moreover, in the important role in oncogenesis. Different abnormalities of NKL absence of genetic hits, certain NKL homeobox genes have been genes have been associated with cancer (Table 2). For example, associated with oncogenesis as crucial downstream targets of NKX2-1 is amplified in 3–12% of primary lung adenocarcino- established oncogenes, or by effects on proliferation, differ- mas15,16 and in 33% of lung cancer cell lines.15 However, entiation or apoptosis (Table 2). For example, NKX2-2 is a amplification is significantly less frequent in other types of lung critical target of the EWS/FLI fusion protein in Ewing’s cancer, and deletion of NKX2-1 has been described in sarcoma,19 and in clear cell sarcoma, a fusion of EWS/ATF-1 squamous lung carcinomas.17 A recent study demonstrated a is responsible for NKX6-1 upregulation.20 Hypermethylation of suppressive effect of NKX2-1 expression on the progression of promoter regions of specific NKL genes has also been reported lung adenocarcinoma.18 This suggests that NKX2-1 can not only in human cancer, but in most cases, their significance in

Leukemia NKL homeobox genes in leukemia I Homminga et al 574 Table 2 NKL aberrations associated with cancer oncogenesis remains poorly understood (Table 2). Promoter hypermethylation of NKL genes has also been interpreted as a Aberration type Gene and cancer type sign of mitotic cell age possibly predisposing for certain types of cancer.21,22 Chromosomal HHEX in AML (t(10;11)(q23;p15)77) rearrangement NKX2-1 in pediatric T-ALL (inv(14)(q11.2q13), inv(14)(q13q32.33), t(7;14)(q34;q13))24 NKX2-2 in pediatric T-ALL (t(14;20)(q11;p11))24 NKL homeobox genes in leukemia NKX2-5 in T-ALL64,65 and CLL66 (t(5;14)(q34;q32.2), t(5;14)(q35;q11)) Leukemia can be divided into myeloid, B- or T-cell leukemia TLX1 in T-ALL (t(10;14)(q24;q11),t(7;10)(q35;q24)) TLX3 in T-ALL (t(5;14)(q35;q32)31,33,55,59 depending on the precursor cell that gave rise to the leukemia. t(5;14)(q32;q11)(60), t(5;7)(q35;q21),59 del5(q35.1)61) In normal B- and T-cell development, immunoglobulin (heavy and or k-light chain) and T-cell receptors (TCR-a, TCR-b, TCR-g Deletions BARX2 as part of a del(11)(q24a25) in ovarian carcinoma120 – 122 or TCR-d) are rearranged to provide a wide diversity in antigen EMX2 as part of a del(10)(q25.3q26.1) in endometrial recognition. This process involves double-stranded DNA breaks, cancer123 deletion, random addition of nucleic acids and ligation of DNA 17 NKX2-1 and NKX2-8 in lung carcinoma ends. Dysregulation of this process can result in chromosomal NKX2-3 as part of a del(10)(q23.31q24.33) in sporadic colorectal cancer124 translocations or inversions that may lead to ectopic expression of oncogenes. Such oncogenic translocations are common in 125 Amplifications DLX4 in breast cancer hematopoietic malignancies; in fact, 450% of lymphatic NKX2-1 and NKX2-8 in lung adenocarcinoma15,16 leukemias and lymphomas carry chromosomal translocations Mutations EMX2 in endometrial cancer123 that involve immunoglobulin or TCR genes.23 To date, many SNP HLX increased risk of therapy-related AML94 different oncogenes have been identified that are ectopically MSX1 increased risk of breast cancer 126 expressed as a consequence of such translocations. Among NKX3-1 increased risk of prostate cancer127 these are homeobox genes such as HOXA and HOXB (for review of HOX genes in leukemia, see 7–10). Thus far, six NKL Overexpression BARX2 in estrogen-dependent breast cancer cell lines128 homeobox genes have been described to be involved in DLX4 in prostate cancer129 lung cancer,130 chromosomal translocations or inversions in leukemia and an promyelocytic leukemia,131 AML and T-ALL132 additional four have been implicated in leukemogenesis by 133 and breast cancer aberrant expression (Table 2). DLX5 in ovarian cancer134 and lung cancer135 LBX1 in breast cancer136 MSX2 in pancreatic cancer137 – 139 NKX2-2 in Ewing’s sarcoma.19 NKX2-1 and NKX2-2 140 NKX2-5 in ovarian yolk sac tumors Our group recently reported on the NKX2-1 and NKX2-2 genes NKX3-1 in lobular breast carcinomas141,142 62,83 that are amplified (o1%) or involved in TCR or immunoglobulin- NKX3-1 in TAL1 rearranged T-ALL B NKX6-1 in clear cell sarcoma20 driven chromosomal translocations or inversions in 5% TLX2 in T-ALL54 of pediatric T-cell acute lymphoblastic leukemia (T-ALL) VENTX in AML95 patients.24 Repositioning of enhancer regions from TCR or Downregulation DLX4 in colorectal cancer143 immunoglobulin loci adjacent to NKX2-1 or NKX2-2 results in HLX in colorectal cancer143 aberrant activation of these genes. NKX2-1 and NKX2-2 are MSX1 in cervical cancer144 very-related homeobox genes forming gene families Nk2.1 and NKX2-3 in liver metastases of gastrointestinal 145 Nk2.2 together with NKX2-4 and NKX2-8. Normally, these carcinomas genes are not expressed in T cells or their precursors,25 and the Promoter BARHL1 and TLX2 in melanoma cell lines146 mechanisms by which they can contribute to leukemia is not hypermethylation DLX1 and LBX1 in lung cancer147 yet clear. T-ALL cases harboring these NKX2-1 or NKX2-2 DLX1 and DLX4 in chronic lymphocytic 148 rearrangements shared a gene-expression signature with T-ALL leukemia patients harboring a translocation involving TLX1, another NKL DLX2-4 in AML DLX4 in breast cancer149 gene. It is interesting to note that various TLX1-rearranged T-ALL EMX2 in lung carcinoma150 cases express NKX2-1, although to a lower level compared with EN1, LBX2, NKX2-4 and NKX2-5 in salivary gland NKX2-1-rearranged T-ALL cases. This signature was character- 151 carcinoma ized by a high expression of genes involved in proliferation.24 In HMX2 in colorectal cancer cell lines152 MSX1 in T-ALL153 lung, breast, colon, prostate most cases, lymphoblasts of these patients are arrested in the 26 – 29 and gastric cancer154,155 early cortical stage of thymocyte development. MSX2 in ALL156 NKX2-2 and DLX2 in luminal breast cancer157 NKX2-3 in melanoma cell lines158 NKX2-5 in MDS and acute leukemia21 TLX1 (HOX11) NKX2-5 in prostate cancer22 The TLX1 gene is upregulated by translocations in B7% of NKX3-1 in prostate cancer159 pediatric and B30% of adult T-ALL patients.30 – 33 TLX1 NKX6-1 in cervical cancer,160,161 ALL162 and small 163 aberrations have a been associated with a relative good B-cell lymphoma prognosis.30,32,34,35 Normally, TLX1 is not expressed in adult NKX6-1, BARHL2, NKX2-6, DLX2, EN1 and NKX2-8 36,37,25 in astrocytomas164 T cells, thymocytes or hematopoietic stem cells but has a TLX3 in prostate cancer165 role in spleen and neural development.38 – 40 TLX1 immortalizes 41 – 44 Abbreviations: AML, acute myeloid leukemia; CLL, chronic lymphocy- different hematopoietic cell lineages including T cells, and tic leukemia; SNP, single-nucleotide polymorphism; T-ALL, T-cell acute overexpression of TLX1 in transgenic mice results in lymphoid 42,45 lymphoblastic leukemia. tumors with long latency. TLX1 promotes proliferation and In bold: NKL genes associated with leukemia. blocks differentiation of hematopoietic precursors, thereby

Leukemia NKL homeobox genes in leukemia I Homminga et al 575 contributing to leukemogenesis,41 – 43,46 – 51 and TLX1-deregu- T-cell development, MEF2C is downregulated and ectopic lated T-ALL patients highly express genes that are involved in MEF2C expression has been shown to provide a differentiation proliferation.24,31 TLX1 enhances MYC protein levels by block.24 MEF2C can also inhibit apoptosis by repressing NR4A1/ posttranscriptional regulation.52 It also has abrogating effects NUR77, which subsequently prevents transformation of BCL2 on cell-cycle check points, for instance, through downregula- into a pro-apoptotic factor.71 tion of CHEK145 or inhibition of protein serine-threonine phosphatases PP1 and PP2A.51,53 In addition, TLX1 may increase genomic instability.45 HHEX ETP-ALL is characterized by early T-cell developmental arrest24,31,72 and ectopic expression of LMO2, LYL1 and the TLX2 (HOX11L1) NKL homeobox gene HHEX.24,31 We have previously demon- TLX2 is normally not expressed in thymocytes,25 but was highly strated that LMO, LYL1 and HHEX are transcriptional targets of upregulated in a single T-ALL patient in a cohort of 92 MEF2C.24 HHEX may represent an important transcriptional patients.54 This patient co-clustered with TLX3-rearranged cases target gene for this T-ALL subtype, as HHEX itself is sufficient in hierarchical cluster analysis based on microarray gene- to initiate self-renewal in thymocytes73 and Hex can induce T expression data,54 suggesting the existence of sporadic T-ALL cell-derived lymphomas when overexpressed in hematopoietic cases in which the TLX2 gene has an oncogenic role, which is precursor cells in mice.74 HHEX is highly expressed in normal homologous to TLX3-rearranged cases. So far however, no hematopoietic stem cells and downregulation is necessary for translocation of this gene has been identified in this patient;54 normal T-cell development,74,75 whereas most other hemato- therefore, the role of TLX2 in T-ALL is not yet clear. poietic lineages maintain HHEX expression.76,25 So far, no genetic abnormalities of the HHEX gene itself have been found in human T-ALL, although fluorescence in situ hybridization analysis for TLX3 (HOX11L2) possible translocations involving the HHEX locus has been Approximately 20% of pediatric and 5% of adult T-ALL patients extensively performed in patients24 and T-ALL cell lines.67 are characterized by TLX3 rearrangements and ectopic expression In acute myeloid leukemia (AML), a NUP98/HHEX fusion has mostly due to a cryptic translocation t(5;14)(q35;q32),33,55 – 58 been described due to a t(10;11)(q23;p15).77 NUP98 is often placing TLX3 under the influence of the distal enhancer region involved in translocations that result in fusion genes and 420 downstream of BCL11B.31,33,55,59 In sporadic cases, variant different partner genes have been described. Among these are TLX3 aberrations have been reported such as translocations to several other Antp homeobox genes such as HOXA9,78,79 the TRA/D@60 or CDK659 gene, a del5(q35.1)61 or more complex HOXD1180 and HOXC13.81 For most NUP98–homeodomain rearrangements involving the 5q35region.59 Alike TLX1 and fusion proteins, transforming capacities have been demonstrated. TLX2, TLX3 is not expressed in normal T-cell development. The transforming activity seems to depend primarily on the NUP98 Leukemogenic modes of actions of TLX3 have not been N terminus and at least in part on an intact homeodomain.77,82 extensively studied. As TLX1 and TLX3 are closely related genes, a common oncogenic pathway is expected. This is supported by the fact that they can cluster together in NKX3-1 gene expression-based hierarchical cluster analysis.24,31,62 On NKX3-1 has been found to be overexpressed in TAL1/LMO- the other hand, supervised gene-expressing profiling shows rearranged T-ALL.62,83 Normally, TAL1 is only expressed in the distinct profiles for TLX1- and TLX3-rearranged cases,54 and early thymocyte differentiation stages (CD34 þ , CD1a, CD4 and both groups have a different clinical outcome; TLX1-rearranged CD8)84 but it is aberrantly upregulated by interstitial deletions, patients have a better prognosis than do TLX3-rearranged translocations or unknown mechanisms in 440% T-ALL patients.30 – 32,34,35,56 – 58,63 In addition, TLX3 cases are asso- cases.30,31,57,85,86 TAL1 can bind to many target genes. One of ciated with the gd-lineage and immature T-cell development, its target genes is the NKL homeobox gene NKX3-1. TAL1 binds whereas TLX1 cases are associated with ab-lineage commit- to the NKX3-1 promoter in a complex with LMO, Ldb1 and ment.26,27,57 TLX1-3 chromosomal aberrations are not reported GATA3 and activates its transcription.83 NKX3-1 in turn seems in other types of cancer. to be required for T-ALL proliferation and accordingly, genes associated with NKX3-1 expression found by gene-expression analysis were involved in proliferation.83 NKX3-1 was shown to NKX2-5 potentially downregulate microRNAs of the miR-17-92 cluster NKX2-5 translocations to either TRD@ or BCL11B sites are seen and NKX3-1 and miR-17-92 expressions were inversely in sporadic T-ALL cases.64,65 A single case of atypical B-cell correlated.83 However, T-ALL oncogenes NKX2-5 and TLX1 chronic proliferative disorder has also been described to carry a were reported to upregulate these miRNAs in T-ALL70 in line NKX2-5 translocation.66 NKX2-5 is normally not expressed in with reported leukemogenic activity of this miRNA cluster in thymocytes25,65 but is involved in spleen and muscle formation. T-ALL models.87 Therefore, the role of miR-17-92 suppression Different pathways that are targeted by NKX2-5 have been by NKX3-1 in T-ALL is not clear. Normally, NKX3-1 is not proposed to have a role in leukemogenesis. NKX2-5 activates expressed in adult tissues except in prostate and testis.88 NOTCH3,67 which can enhance survival68,69 and NKX2-5 causes upregulation of miR-17-92, which may lead to increased proliferation.70 In addition, NKX2-5 binds the promoter of MEF2C HLX and activates MEF2C transcription in T-ALL cell lines.24,71 The NKL gene HLX has been suggested as a potential oncogene Recently, we identified MEF2C as a central oncogene in an in AML and T-ALL. HLX is normally expressed in activated immature T-ALL subgroup that shares characteristics with early T cells and early hematopoietic progenitors.89,90 Knock down of T-cell precursors (ETP-ALL). In these patients, genetic aberra- HLX in CD34 þ bone marrow cells inhibits proliferation, tions were identified that target MEF2C or MEF2C-regulating whereas overexpression of HLX impairs differentiation into transcription factors, including NKX2-5.24 During early normal mature hematopoietic lineages.90 HLX promotes proliferation in

Leukemia NKL homeobox genes in leukemia I Homminga et al 576 the T-ALL cell line Jurkat91 and HLX stably transfected Jurkat types of cancer (Table 2). Approximately 30% of pediatric T-ALL cells produce tumors in mice.92 High levels of HLX have been cases harbor a genetic aberration involving a NKL gene (5% demonstrated in AML patient samples,93 and a single-nucleotide NKX2-1,1%NKX2-2,6%TLX1, 19% TLX3,1%NKX2-5). The polymorphism in the 30-untranslated region of HLX is associated percentage of cases that overexpress NKL genes is even higher, with increased risk of therapy-related AML.94 No genetic as TAL1-rearranged cases overexpress NKX3-1 and immature aberrations of HLX have been found in T-ALL or AML. HLX is T-ALL cases overexpress HHEX. In addition, high expression of not associated with other types of cancer as oncogene or tumor- some NKL genes, such as NKX2-1, is observed in some cases in suppressor gene. which no genetic aberration was identified. Seven out of eight NKL homeobox genes that are association with T-ALL (NKX2-1, NKX2-2, NKX2-5, NKX3-1, TLX1, TLX2 and TLX3) are part of a VENTX separate branch in the phylogenetic tree of the NKL homeobox VENTX is highly expressed in a small portion of acute myeloid genes and therefore have a similar homeodomain5 (boxed in leukemia patients, especially those with translocation t(8,21) or Table 1). All these seven genes are not expressed in normal 95 a normal karyotype, but whether it actually has a role in T-cell development. The other gene, HHEX, is part of a separate leukemogenesis is not clear. There are inconsistencies in the phylogenetic tree branch and is expressed in early hematopoie- reports on the expression levels of VENTX in normal hemato- tic stages and downregulated upon further T-cell maturation. poietic lineages. Healthy CD34 þ early hematopoietic cells Two different but compatible lineage-dependency models have express VENTX at low-to-undetectable levels, whereas mature been proposed for oncogenes, that is, the ‘oncogene-addiction’ 25,95 myeloid cells highly express VENTX. Some reports describe model and the ‘lineage-survival model’. As the oncogene- 96 VENTX expression in mature B and T cells, whereas others addiction model states that an oncogene will provide a tumor- have reported a low-to-absent expression in these cell specific gain of function, the lineage-survival model poses that 95,25 types. Enforced expression of VENTX in CD34 þ progenitor tumors may become dependent on survival pathways that are cells impairs B- and T-cell development, but promotes the already present in precursor cells of the specific cell lineage. The 95 development of myeloid cells. VENTX knockdown in AML seven NKX and TLX genes mentioned above might be involved cell lines impairs proliferation, suggesting a possible oncogenic in downstream pathways that are normally not involved in T-cell role for VENTX in AML or a role in promoting myeloid development, but are nonetheless beneficial for survival of phenotype over lymphoid phenotype in preleukemic or thymocytes, in line with the ‘oncogene-addiction model’. On 95 leukemic clones. In contrast, in chronic lymphoid leukemia, the other hand, these genes might mimic NKL genes that have a VENTX has been suggested as a potential tumor-suppressor role in normal T-cell development, thereby making use of 96 gene that functions by inducing cell senescence through the existing pathways, in line with a ‘lineage-survival model’. ink4a 97 activation of p53 and p16 . HHEX49 and MSX267 have both been proposed as candidate genes that might be mimicked by ectopically expressed NKL DLX2, DLX3 and DLX4 genes. HHEX is highly expressed in early hematopoietic progenitors and downregulated upon T-cell differentiation. In pediatric precursor B-ALL patients carrying a mixed lineage HHEX has also been implicated in leukemogenesis (see above), leukemia – ALL1-fused gene from chromosome 4 translocation, which makes HHEX a more likely candidate to be mimicked by decreased levels of DLX2, 3 and 498 have been reported, NKL genes in T-ALL. possibly due to promoter hypermethylation of these genes.99 This points to a tumor-suppressor-like role of these genes in this leukemia type. However, the role of these specific NKL genes in NKLs modes of action oncogenesis is not clear, as extensive promoter hypermethylation of many genes was recently demonstrated in mixed lineage NKLs have been implied in different processes essential for leukemia – ALL1-fused gene from chromosome 4 translocated oncogenesis especially differentiation, proliferation and apop- infant B-ALL.100 DLX2 and DLX4 are normally highly expressed in tosis (see above). The exact mechanisms by which these healthy B-cell progenitors and downregulated during maturation. processes are regulated remain poorly understood. In general, NKL genes function predominantly as transcriptional repressors, NKL as an oncogene or a tumor-suppressor gene? although activating properties have also been described.49,102,103 More than half (32/48) of the NKL home- As homeobox genes have been initially found to be overexpressed, odomain proteins contain a conserved Engrailed homology 1 104,105 it has been concluded that homeobox genes act as oncogenes. (Eh1) motif (FxIxxIL, whereby x can be any amino acid) at Nowadays, studies have also reported deletions and down- their N terminal (defined here as having at least 3 out of 4 regulation of homeobox genes as important oncogenic events. conserved amino acids present at the correct position at the Hence, NKL genes cannot be considered as ‘classic oncogenes’. N-terminal side, Table 1). The Eh1 domain functions as a strong Apparently, the same NKL gene can act as an oncogene or as a transcriptional repressor. It can interact with Transducin-like 106 tumor-suppressor gene depending on the cellular context. In Enhancer-of-split (TLE) co-repressor proteins, and these general, it can be stated that homeobox genes that are normally proteins can induce transcriptional repression by recruiting 107,108 expressed in undifferentiated cells are upregulated in cancer, histone deacetylases. For HHEX and TLX1, it has been whereas homeobox genes that are normally expressed in shown that the interaction with TLE proteins is important in differentiated tissues are downregulated in cancer.101 regulating downstream targets in hematopoietic lineages and in T-ALL.52,109 Binding of TLE can result in repression of transcription of NKL targets, but it can also act as a competitive NKL overexpression as a common theme in T-ALL substrate for TLE proteins relieving other factors from TLE-mediated repression and activation of transcription.110 For It is remarkable that in T-ALL, many different NKL genes are example, TLX1 expression enhances NOTCH1 signaling in a implied in leukemogenesis, especially compared with other T-ALL cell line, partly by sequestering TLE from the

Leukemia NKL homeobox genes in leukemia I Homminga et al 577 Hairy/Enhancer of split 1 -TLE-mediated transcriptional repres- by NMR spectroscopy in solution: comparison with prokaryotic sion complex.52,111 In line with this hypothesis are studies that repressors. Cell 1989; 59: 573–580. have identified direct downregulation of TLE proteins by 5 Holland PW, Booth HA, Bruford EA. Classification and nomen- promoter hypermethylation and deletions in acute myeloid clature of all human homeobox genes. BMC Biol 2007; 5: 47. 112,113 6 Shah N, Sukumar S. The Hox genes and their roles in leukemia. All NKL genes associated with T-ALL have an oncogenesis. Nat Rev Cancer 2010; 10: 361–371. Eh1-like motif at their N terminal. Therefore, it is tempting to 7 McGonigle GJ, Lappin TR, Thompson A. Grappling with the HOX speculate that transcriptional repression through the Eh1 motif network in hematopoiesis and leukemia. Front Biosci 2008; 13: and TLE scavenging may be general mechanisms by which NKLs 4297–4308. promote leukemogenesis in T-ALL. Besides TLE, GATA proteins 8 Argiropoulos B, Humphries RK. Hox genes in hematopoiesis and also interact with NKL proteins. Together they can activate leukemogenesis. Oncogene 2007; 26: 6766–6776. 114 – 118 9 Abramovich C, Humphries RK. Hox regulation of normal and target genes in muscle and lung tissues. What could be leukemic hematopoietic stem cells. Curr Opin Hematol 2005; 12: important downstream factors of NKL genes, or proteins affected 210–216. by NKL genes in leukemogenesis? In general, gene-expression 10 Abramovich C, Pineault N, Ohta H, Humphries RK. Hox genes: analyses have shown enrichment of genes involved in prolifera- from leukemia to hematopoietic stem cell expansion. Ann NY tion in TLX1-, TLX3-, NKX2-1/2-2- and NKX3-1-rearranged T- Acad Sci 2005; 1044: 109–116. ALL patients.24,31,62,83 Besides a general profile, specific genes 11 Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halli- day HL, Lappin TR. The pathophysiology of HOX genes and their or miRNAs have been identified for each of these NKL genes. role in cancer. J Pathol 2005; 205: 154–171. NOTCH3 has been identified as a recurrent target of NKL 12 Kim Y, Nirenberg M. Drosophila NK-homeobox genes. Proc Natl 67 proteins TLX1, MSX2 and NKX2-5. NOTCH3 overexpression Acad Sci USA 1989; 86: 7716–7720. induces T-cell lymphomas in mice68 and is highly expressed in 13 Wotton KR, Weierud FK, Juarez-Morales JL, Alvares LE, Dietrich all 30 T-ALL cases examined in a study by Bellavia et al.119 S, Lewis KE. Conservation of gene linkage in dispersed vertebrate posing a possible important NKL downstream target. NK homeobox clusters. Dev Genes Evol 2009; 219: 481–496. 14 Wang CC, Brodnicki T, Copeland NG, Jenkins NA, Harvey RP. Conserved linkage of NK-2 homeobox gene pairs Nkx2-2/2-4 and Summary and future implications Nkx2-1/2-9 in mammals. Mamm Genome 2000; 11: 466–468. 15 Kwei KA, Kim YH, Girard L, Kao J, Pacyna-Gengelbach M, Salari K et al. Genomic profiling identifies TITF1 as a lineage- NKLs are implicated in divergent types of cancer and can specific oncogene amplified in lung cancer. Oncogene 2008; 27: function as oncogenes or tumor-suppressor genes, depending on 3635–3640. the tissue type. Oncogenes are downregulated during tissue 16 Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R et al. development in normal tissues, whereas tumor-suppressor genes Characterizing the cancer genome in lung adenocarcinoma. are normally upregulated during differentiation. Many different Nature 2007; 450: 893–898. 17 Harris T, Pan Q, Sironi J, Lutz D, Tian J, Sapkar J et al. Both gene NKLs are involved in T-ALL compared with other types of amplification and allelic loss occur at 14q13.3 in lung cancer. cancer. The overexpression of eight different NKL genes has Clin Cancer Res 2011; 17: 690–699. been associated with T-ALL covering the majority of pediatric 18 Winslow MM, Dayton TL, Verhaak RG, Kim-Kiselak C, Snyder T-ALL cases. Most of these NKLs are normally not expressed in EL, Feldser DM et al. Suppression of lung adenocarcinoma T-cell development. This suggests a potential similar down- progression by Nkx2-1. Nature 2011; 473: 101–104. stream effect that promotes leukemogenesis in T-cell progeni- 19 Smith R, Owen LA, Trem DJ, Wong JS, Whangbo JS, Golub TR et al. Expression profiling of EWS/FLI identifies NKX2.2 as a tors, possibly due to mimicking HHEX. As all eight NKL genes critical target gene in Ewing’s sarcoma. Cancer Cell 2006; 9: posses a conserved Eh1 repressor motif, this might also have an 405–416. important role. A common downstream pathway of NKLs might 20 Jishage M, Fujino T, Yamazaki Y, Kuroda H, Nakamura T. prove difficult to be discovered, especially when most gene- Identification of target genes for EWS/ATF-1 chimeric transcrip- expression analyses are focused on comparisons between tion factor. Oncogene 2003; 22: 41–49. subgroups in T-ALL. To elucidate a potential common role of 21 Mossner M, Hopfer O, Nowak D, Baldus CD, Neumann U, NKL genes in T-ALL, comparisons with normal thymocytes Kmetsch A et al. Detection of differential mitotic cell age in bone marrow CD34(+) cells from patients with myelodysplastic subsets in combination with ChIP (chromatin immunoprecipita- syndrome and acute leukemia by analysis of an epigenetic tion)-on-ChIP or ChIP-seq analysis and functional knockdown and molecular clock DNA signature. Exp Hematol 2010; 38: knock-in experiments will be essential. 661–665. 22 Kwabi-Addo B, Chung W, Shen L, Ittmann M, Wheeler T, Jelinek J et al. Age-related DNA methylation changes in normal human Conflict of interest prostate tissues. Clin Cancer Res 2007; 13: 3796–3802. 23 Zhang Y, Rowley JD. Chromatin structural elements and The authors declare no conflict of interest. chromosomal translocations in leukemia. DNA Repair (Amst) 2006; 5: 1282–1297. 24 Homminga I, Pieters R, Langerak AW, de Rooi JJ, Stubbs A, Verstegen M et al. Integrated transcript and genome analyses References reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia. Cancer Cell 2011; 19: 484–497. 1 Gehring WJ. Master Control Genes in Development and 25 Novershtern N, Subramanian A, Lawton LN, Mak RH, Haining Evolution: The Homeobox Story. 1st edn. Yale University Press: WN, McConkey ME et al. Densely interconnected transcriptional New Haven, 1998. circuits control cell states in human hematopoiesis. Cell 2011; 2 Garber RL, Kuroiwa A, Gehring WJ. Genomic and cDNA clones 144: 296–309. of the homeotic locus Antennapedia in Drosophila. EMBO J 26 Asnafi V, Beldjord K, Libura M, Villarese P, Millien C, Ballerini P 1983; 2: 2027–2036. et al. Age-related phenotypic and oncogenic differences in T-cell 3 Scott MP, Weiner AJ, Hazelrigg TI, Polisky BA, Pirrotta V, acute lymphoblastic leukemias may reflect thymic atrophy. Blood Scalenghe F et al. The molecular organization of the Antenna- 2004; 104: 4173–4180. pedia locus of Drosophila. Cell 1983; 35 (3 Part 2): 763–776. 27 van Grotel M, Meijerink JP, van Wering ER, Langerak AW, 4 Qian YQ, Billeter M, Otting G, Muller M, Gehring WJ, Wuthrich Beverloo HB, Buijs-Gladdines JG et al. Prognostic significance of K. The structure of the Antennapedia homeodomain determined molecular-cytogenetic abnormalities in pediatric T-ALL is not

Leukemia NKL homeobox genes in leukemia I Homminga et al 578 explained by immunophenotypic differences. Leukemia 2008; 46 Greene WK, Ford J, Dixon D, Tilbrook PA, Watt PM, Klinken SP 22: 124–131. et al. Enforced expression of HOX11 is associated with an 28 Ludwig WD, Harbott J, Bartram CR, Komischke B, Sperling C, immature phenotype in J2E erythroid cells. Br J Haematol 2002; Teichmann JV et al. Incidence and prognostic significance of 118: 909–917. immunophenotypic subgroups in childhood acute lymphoblastic 47 Riz I, Hawley TS, Johnston H, Hawley RG. Role of TLX1 in T-cell leukemia: experience of the BFM study 86. Recent Results Cancer acute lymphoblastic leukaemia pathogenesis. Br J Haematol Res 1993; 131: 269–282. 2009; 145: 140–143. 29 Pullen J, Shuster JJ, Link M, Borowitz M, Amylon M, Carroll AJ 48 Riz I, Akimov SS, Eaker SS, Baxter KK, Lee HJ, Marino-Ramirez L et al. Significance of commonly used prognostic factors differs for et al. TLX1/HOX11-induced hematopoietic differentiation block- children with T cell acute lymphocytic leukemia (ALL), as ade. Oncogene 2007; 26: 4115–4123. compared to those with B-precursor ALL. A Pediatric Oncology 49 Dixon DN, Izon DJ, Dagger S, Callow MJ, Taplin RH, Kees UR Group (POG) study. Leukemia 1999; 13: 1696–1707. et al. TLX1/HOX11 transcription factor inhibits differentiation and 30 Ferrando AA, Neuberg DS, Dodge RK, Paietta E, Larson RA, promotes a non-haemopoietic phenotype in murine bone marrow Wiernik PH et al. Prognostic importance of TLX1 (HOX11) cells. Br J Haematol 2007; 138: 54–67. oncogene expression in adults with T-cell acute lymphoblastic 50 Owens BM, Hawley TS, Spain LM, Kerkel KA, Hawley RG. TLX1/ leukaemia. Lancet 2004; 363: 535–536. HOX11-mediated disruption of primary thymocyte differentiation 31 Ferrando AA, Neuberg DS, Staunton J, Loh ML, Huard C, prior to the CD4+CD8+ double-positive stage. Br J Haematol Raimondi SC et al. Gene expression signatures define novel 2006; 132: 216–229. oncogenic pathways in T cell acute lymphoblastic leukemia. 51 Kawabe T, Muslin AJ, Korsmeyer SJ. HOX11 interacts with Cancer Cell 2002; 1: 75–87. protein phosphatases PP2A and PP1 and disrupts a G2/M cell- 32 Kees UR, Heerema NA, Kumar R, Watt PM, Baker DL, La MK cycle checkpoint. Nature 1997; 385: 454–458. et al. Expression of HOX11 in childhood T-lineage acute 52 Riz I, Hawley TS, Luu TV, Lee NH, Hawley RG. TLX1 and lymphoblastic leukaemia can occur in the absence of cytogenetic NOTCH coregulate transcription in T cell acute lymphoblastic aberration at 10q24: a study from the Children’s Cancer Group leukemia cells. Mol Cancer 2010; 9: 181. (CCG). Leukemia 2003; 17: 887–893. 53 Riz I, Hawley RG. G1/S transcriptional networks modulated by 33 Berger R, Dastugue N, Busson M, Van Den Akker J, Perot C, the HOX11/TLX1 oncogene of T-cell acute lymphoblastic Ballerini P et al. t(5;14)/HOX11L2-positive T-cell acute lympho- leukemia. Oncogene 2005; 24: 5561–5575. blastic leukemia. A collaborative study of the Groupe Francais 54 Van Vlierberghe P, van Grotel M, Tchinda J, Lee C, Beverloo HB, de Cytogenetique Hematologique (GFCH). Leukemia 2003; 17: van der Spek PJ et al. The recurrent SET-NUP214 fusion as a new 1851–1857. HOXA activation mechanism in pediatric T-cell acute lympho- 34 Cave H, Suciu S, Preudhomme C, Poppe B, Robert A, blastic leukemia. Blood 2008; 111: 4668–4680. Uyttebroeck A et al. Clinical significance of HOX11L2 expression 55 Bernard OA, Busson-LeConiat M, Ballerini P, Mauchauffe M, linked to t(5;14)(q35;q32), of HOX11 expression, and of SIL-TAL Della Valle V, Monni R et al. A new recurrent and specific cryptic fusion in childhood T-cell malignancies: results of EORTC studies translocation, t(5;14)(q35;q32), is associated with expression of 58881 and 58951. Blood 2004; 103: 442–450. the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia 35 Schneider NR, Carroll AJ, Shuster JJ, Pullen DJ, Link MP, 2001; 15: 1495–1504. Borowitz MJ et al. New recurring cytogenetic abnormalities 56 Mauvieux L, Leymarie V, Helias C, Perrusson N, Falkenrodt A, and association of blast cell karyotypes with prognosis in Lioure B et al. High incidence of Hox11L2 expression in children childhood T-cell acute lymphoblastic leukemia: a pediatric with T-ALL. Leukemia 2002; 16: 2417–2422. oncology group report of 343 cases. Blood 2000; 96: 57 van Grotel M, Meijerink JP, Beverloo HB, Langerak AW, Buys- 2543–2549. Gladdines JG, Schneider P et al. The outcome of molecular- 36 Salvati PD, Ranford PR, Ford J, Kees UR. HOX11 expression in cytogenetic subgroups in pediatric T-cell acute lymphoblastic pediatric acute lymphoblastic leukemia is associated with T-cell leukemia: a retrospective study of patients treated according phenotype. Oncogene 1995; 11: 1333–1338. to DCOG or COALL protocols. Haematologica 2006; 91: 37 Yamamoto H, Hatano M, Iitsuka Y, Mahyar NS, Yamamoto M, 1212–1221. Tokuhisa T. Two forms of Hox11 a T cell leukemia oncogene, are 58 Ballerini P, Blaise A, Busson-Le Coniat M, Su XY, Zucman-Rossi J, expressed in fetal spleen but not in primary lymphocytes. Mol Adam M et al. HOX11L2 expression defines a clinical subtype of Immunol 1995; 32: 1177–1182. pediatric T-ALL associated with poor prognosis. Blood 2002; 100: 38 Kanzler B, Dear TN. Hox11 acts cell autonomously in spleen 991–997. development and its absence results in altered cell fate of 59 Su XY, Busson M, Della Valle V, Ballerini P, Dastugue N, Talmant mesenchymal spleen precursors. Dev Biol 2001; 234: 231–243. P et al. Various types of rearrangements target TLX3 locus in 39 Cheng L, Arata A, Mizuguchi R, Qian Y, Karunaratne A, Gray PA T-cell acute lymphoblastic leukemia. Genes Chromosomes et al. Tlx3 and Tlx1 are post-mitotic selector genes determining Cancer 2004; 41: 243–249. glutamatergic over GABAergic cell fates. Nat Neurosci 2004; 7: 60 Hansen-Hagge TE, Schafer M, Kiyoi H, Morris SW, Whitlock JA, 510–517. Koch P et al. Disruption of the RanBP17/Hox11L2 region 40 Holland PW, Takahashi T. The evolution of homeobox genes: by recombination with the TCRdelta locus in acute lympho- Implications for the study of brain development. Brain Res Bull blastic leukemias with t(5;14)(q34;q11). Leukemia 2002; 16: 2005; 66: 484–490. 2205–2212. 41 Hawley RG, Fong AZ, Reis MD, Zhang N, Lu M, Hawley TS. 61 Van Vlierberghe P, Homminga I, Zuurbier L, Gladdines-Buijs J, Transforming function of the HOX11/TCL3 homeobox gene. van Wering ER, Horstmann M et al. Cooperative genetic Cancer Res 1997; 57: 337–345. defects in TLX3 rearranged pediatric T-ALL. Leukemia 2008; 42 Hough MR, Reis MD, Singaraja R, Bryce DM, Kamel-Reid S, 22: 762–770. Dardick I et al. A model for spontaneous B-lineage lymphomas in 62 Soulier J, Clappier E, Cayuela JM, Regnault A, Garcia-Peydro M, IgHmu-HOX11 transgenic mice. Proc Natl Acad Sci USA 1998; Dombret H et al. HOXA genes are included in genetic and 95: 13853–13858. biologic networks defining human acute T-cell leukemia (T-ALL). 43 Keller G, Wall C, Fong AZ, Hawley TS, Hawley RG. Over- Blood 2005; 106: 274–286. expression of HOX11 leads to the immortalization of embryonic 63 Baak U, Gokbuget N, Orawa H, Schwartz S, Hoelzer D, Thiel E precursors with both primitive and definitive hematopoietic et al. Thymic adult T-cell acute lymphoblastic leukemia stratified potential. Blood 1998; 92: 877–887. in standard- and high-risk group by aberrant HOX11L2 expres- 44 Hawley RG, Fong AZ, Lu M, Hawley TS. The HOX11 homeobox- sion: experience of the German multicenter ALL study group. containing gene of human leukemia immortalizes murine Leukemia 2008; 22: 1154–1160. hematopoietic precursors. Oncogene 1994; 9: 1–12. 64 Nagel S, Kaufmann M, Drexler HG, MacLeod RA. The cardiac 45 De Keersmaecker K, Real PJ, Gatta GD, Palomero T, Sulis ML, homeobox gene NKX2-5 is deregulated by juxtaposition with Tosello V et al. The TLX1 oncogene drives aneuploidy in T cell BCL11B in pediatric T-ALL cell lines via a novel transformation. Nat Med 2010; 16: 1321–1327. t(5;14)(q35.1;q32.2). Cancer Res 2003; 63: 5329–5334.

Leukemia NKL homeobox genes in leukemia I Homminga et al 579 65 Przybylski GK, Dik WA, Grabarczyk P, Wanzeck J, Chudobska P, of TAL1-expressing human T cell acute lymphoblastic leukemia. Jankowski K et al. The effect of a novel recombination between J Exp Med 2010; 207: 2141–2156. the homeobox gene NKX2-5 and the TRD locus in T-cell acute 84 Herblot S, Steff AM, Hugo P, Aplan PD, Hoang T. SCL and lymphoblastic leukemia on activation of the NKX2-5 gene. LMO1 alter thymocyte differentiation: inhibition of E2A-HEB Haematologica 2006; 91: 317–321. function and pre-T alpha chain expression. Nat Immunol 2000; 1: 66 Su X, Della-Valle V, Delabesse E, Azgui Z, Berger R, Merle-Beral 138–144. H et al. Transcriptional activation of the cardiac homeobox gene 85 Bash RO, Hall S, Timmons CF, Crist WM, Amylon M, Smith RG CSX1/NKX2-5 in a B-cell chronic lymphoproliferative disorder. et al. Does activation of the TAL1 gene occur in a majority of Haematologica 2008; 93: 1081–1085. patients with T-cell acute lymphoblastic leukemia? A pediatric 67 Nagel S, Venturini L, Przybylski GK, Grabarczyk P, Meyer C, oncology group study. Blood 1995; 86: 666–676. Kaufmann M et al. NK-like homeodomain proteins activate 86 Armstrong SA, Look AT. Molecular genetics of acute lympho- NOTCH3-signaling in leukemic T-cells. BMC Cancer 2009; 9: blastic leukemia. J Clin Oncol 2005; 23: 6306–6315. 371. 87 Mavrakis KJ, Wolfe AL, Oricchio E, Palomero T, de Keersmaecker 68 Bellavia D, Campese AF, Alesse E, Vacca A, Felli MP, K, McJunkin K et al. Genome-wide RNA-mediated interference Balestri A et al. Constitutive activation of NF-kappaB and screen identifies miR-19 targets in Notch-induced T-cell acute T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J lymphoblastic leukaemia. Nat Cell Biol 2010; 12: 372–379. 2000; 19: 3337–3348. 88 Korkmaz KS, Korkmaz CG, Ragnhildstveit E, Kizildag S, Pretlow 69 Doucas H, Mann CD, Sutton CD, Garcea G, Neal CP, Berry DP TG, Saatcioglu F. Full-length cDNA sequence and genomic et al. Expression of nuclear Notch3 in pancreatic adenocarcino- organization of human NKX3A–alternative forms and regulation mas is associated with adverse clinical features, and correlates by both androgens and estrogens. Gene 2000; 260: 25–36. with the expression of STAT3 and phosphorylated Akt. J Surg 89 Deguchi Y, Kehrl JH. Selective expression of two homeobox Oncol 2008; 97: 63–68. genes in CD34-positive cells from human bone marrow. Blood 70 Nagel S, Venturini L, Przybylski GK, Grabarczyk P, Schmidt CA, 1991; 78: 323–328. Meyer C et al. Activation of miR-17-92 by NK-like homeo- 90 Deguchi Y, Thevenin C, Kehrl JH. Stable expression of HB24, a domain proteins suppresses apoptosis via reduction of E2F1 in diverged human homeobox gene, in T lymphocytes induces T-cell acute lymphoblastic leukemia. Leuk Lymphoma 2009; 50: genes involved in T cell activation and growth. J Biol Chem 1992; 101–108. 267: 8222–8229. 71 Nagel S, Meyer C, Quentmeier H, Kaufmann M, Drexler HG, 91 Deguchi Y, Moroney JF, Wilson GL, Fox CH, Winter HS, Kehrl MacLeod RA. MEF2C is activated by multiple mechanisms in a JH. Cloning of a human homeobox gene that resembles a subset of T-acute lymphoblastic leukemia cell lines. Leukemia diverged Drosophila homeobox gene and is expressed in 2008; 22: 600–607. activated lymphocytes. New Biol 1991; 3: 353–363. 72 Coustan-Smith E, Mullighan CG, Onciu M, Behm FG, Raimondi 92 Deguchi Y, Kehrl JH. High level expression of the homeobox SC, Pei D et al. Early T-cell precursor leukaemia: a subtype of gene HB24 in a human T-cell line confers the ability to form very high-risk acute lymphoblastic leukaemia. Lancet Oncol tumors in nude mice. Cancer Res 1993; 53: 373–377. 2009; 10: 147–156. 93 Deguchi Y, Kirschenbaum A, Kehrl JH. A diverged homeobox 73 McCormack MP, Young LF, Vasudevan S, de Graaf CA, gene is involved in the proliferation and lineage commitment of Codrington R, Rabbitts TH et al. The Lmo2 oncogene initiates human hematopoietic progenitors and highly expressed in acute leukemia in mice by inducing thymocyte self-renewal. Science myelogenous leukemia. Blood 1992; 79: 2841–2848. 2010; 327: 879–883. 94 Jawad M, Seedhouse CH, Russell N, Plumb M. Polymorphisms in 74 George A, Morse 3rd HC, Justice MJ. The homeobox gene Hex human homeobox HLX1 and DNA repair RAD51 genes increase induces T-cell-derived lymphomas when overexpressed in the risk of therapy-related acute myeloid leukemia. Blood 2006; hematopoietic precursor cells. Oncogene 2003; 22: 6764–6773. 108: 3916–3918. 75 Mack DL, Leibowitz DS, Cooper S, Ramsey H, Broxmeyer HE, 95 Rawat VP, Arseni N, Ahmed F, Mulaw MA, Thoene S, Heilmeier Hromas R. Down-regulation of the myeloid homeobox protein B et al. The vent-like homeobox gene VENTX promotes human Hex is essential for normal T-cell development. Immunology myeloid differentiation and is highly expressed in acute myeloid 2002; 107: 444–451. leukemia. Proc Natl Acad Sci USA 2010; 107: 16946–16951. 76 Manfioletti G, Gattei V, Buratti E, Rustighi A, De Iuliis A, 96 Gao H, Le Y, Wu X, Silberstein LE, Giese RW, Zhu Z. VentX, a Aldinucci D et al. Differential expression of a novel proline-rich novel lymphoid-enhancing factor/T-cell factor-associated tran- homeobox gene (Prh) in human hematolymphopoietic cells. scription repressor, is a putative tumor suppressor. Cancer Res Blood 1995; 85: 1237–1245. 2010; 70: 202–211. 77 Jankovic D, Gorello P, Liu T, Ehret S, La Starza R, Desjobert C 97 Wu X, Gao H, Ke W, Hager M, Xiao S, Freeman MR et al. VentX et al. Leukemogenic mechanisms and targets of a NUP98/HHEX trans-activates p53 and p16ink4a to regulate cellular senescence. fusion in acute myeloid leukemia. Blood 2008; 111: 5672–5682. J Biol Chem 2011; 286: 12693–12701. 78 Borrow J, Shearman AM, Stanton Jr VP, Becher R, Collins T, 98 Ferrari N, Palmisano GL, Paleari L, Basso G, Mangioni M, Williams AJ et al. The t(7;11)(p15;p15) translocation in acute Fidanza V et al. DLX genes as targets of ALL-1: DLX 2,3,4 down- myeloid leukaemia fuses the genes for nucleoporin NUP98 and regulation in t(4;11) acute lymphoblastic leukemias. J Leukoc Biol class I homeoprotein HOXA9. Nat Genet 1996; 12: 159–167. 2003; 74: 302–305. 79 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, 99 Campo Dell’Orto M, Banelli B, Giarin E, Accordi B, Trentin L, Toyama K et al. Fusion of the nucleoporin gene NUP98 to Romani M et al. Down-regulation of DLX3 expression in HOXA9 by the chromosome translocation t(7;11)(p15;p15) in MLL-AF4 childhood lymphoblastic leukemias is mediated by human myeloid leukaemia. Nat Genet 1996; 12: 154–158. promoter region hypermethylation. Oncology Rep 2007; 18: 80 Taketani T, Taki T, Shibuya N, Ito E, Kitazawa J, Terui K et al. 417–423. The HOXD11 gene is fused to the NUP98 gene in acute 100 Stumpel DJ, Schneider P, van Roon EH, Boer JM, de Lorenzo P, myeloid leukemia with t(2;11)(q31;p15). Cancer Res 2002; 62: Valsecchi MG et al. Specific promoter methylation identifies 33–37. different subgroups of MLL-rearranged infant acute lymphoblastic 81 Panagopoulos I, Isaksson M, Billstrom R, Strombeck B, Mitelman leukemia, influences clinical outcome, and provides therapeutic F, Johansson B. Fusion of the NUP98 gene and the homeobox options. Blood 2009; 114: 5490–5498. gene HOXC13 in acute myeloid leukemia with t(11;12)(p15;q13). 101 Abate-Shen C. Deregulated homeobox gene expression in cancer: Genes Chromosomes Cancer 2003; 36: 107–112. cause or consequence? Nat Rev Cancer 2002; 2: 777 – 785. 82 Yassin ER, Sarma NJ, Abdul-Nabi AM, Dombrowski J, Han Y, 102 Greene WK, Bahn S, Masson N, Rabbitts TH. The T-cell Takeda A et al. Dissection of the transformation of primary oncogenic protein HOX11 activates Aldh1 expression in NIH human hematopoietic cells by the oncogene NUP98-HOXA9. 3T3 cells but represses its expression in mouse spleen develop- PloS One 2009; 4: e6719. ment. Mol Cell Biol 1998; 18: 7030–7037. 83 Kusy S, Gerby B, Goardon N, Gault N, Ferri F, Gerard D et al. 103 Muhr J, Andersson E, Persson M, Jessell TM, Ericson J. Groucho- NKX3.1 is a direct TAL1 target gene that mediates proliferation mediated transcriptional repression establishes progenitor cell

Leukemia NKL homeobox genes in leukemia I Homminga et al 580 pattern and neuronal fate in the ventral neural tube. Cell 2001; 123 Noonan FC, Mutch DG, Ann Mallon M, Goodfellow PJ. 104: 861–873. Characterization of the homeodomain gene EMX2: sequence 104 Smith ST, Jaynes JB. A conserved region of engrailed, shared conservation, expression analysis, and a search for mutations in among all en-, gsc-, Nk1-, Nk2- and msh-class homeoproteins, endometrial cancers. Genomics 2001; 76: 37–44. mediates active transcriptional repression in vivo. Dev (Cam- 124 Wang X, Zbou C, Qiu G, Fan J, Tang H, Peng Z. Screening of new bridge, England) 1996; 122: 3141–3150. tumor suppressor genes in sporadic colorectal cancer patients. 105 Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP. Nkx-2.5: a Hepato-Gastroenterology 2008; 55: 2039–2044. novel murine homeobox gene expressed in early heart progenitor 125 Cavalli LR, Man YG, Schwartz AM, Rone JD, Zhang Y, Urban CA cells and their myogenic descendants. Development (Cambridge, et al. Amplification of the BP1 homeobox gene in breast cancer. England) 1993; 119: 969. Cancer Genet Cytogenet 2008; 187: 19–24. 106 Desjobert C, Noy P, Swingler T, Williams H, Gaston K, 126 Sliwinski T, Synowiec E, Czarny P, Gomulak P, Forma E, Jayaraman PS. The PRH/Hex repressor protein causes nuclear Morawiec Z et al. The c.469+46_56del mutation in the homeo- retention of Groucho/TLE co-repressors. Biochem J 2009; 417: box MSX1 gene–a novel risk factor in breast cancer? Cancer 121–132. Epidemiol 2010; 34: 652–655. 107 Chen G, Fernandez J, Mische S, Courey AJ. A functional 127 Zheng SL, Ju JH, Chang BL, Ortner E, Sun J, Isaacs SD et al. Germ- interaction between the histone deacetylase Rpd3 and the line mutation of NKX3.1 cosegregates with hereditary prostate corepressor groucho in Drosophila development. Genes Dev cancer and alters the homeodomain structure and function. 1999; 13: 2218–2230. Cancer Res 2006; 66: 69–77. 108 Choi CY, Kim YH, Kwon HJ, Kim Y. The homeodomain protein 128 Stevens TA, Meech R. BARX2 and estrogen receptor-alpha (ESR1) NK-3 recruits Groucho and a histone deacetylase complex to coordinately regulate the production of alternatively spliced ESR1 repress transcription. J Biol Chem 1999; 274: 33194–33197. isoforms and control breast cancer cell growth and invasion. 109 Swingler TE, Bess KL, Yao J, Stifani S, Jayaraman PS. The proline- Oncogene 2006; 25: 5426–5435. rich homeodomain protein recruits members of the Groucho/ 129 Schwartz AM, Man YG, Rezaei MK, Simmens SJ, Berg PE. BP1, a Transducin-like enhancer of split protein family to co-repress homeoprotein, is significantly expressed in prostate adenocarci- transcription in hematopoietic cells. J Biol Chem 2004; 279: noma and is concordant with prostatic intraepithelial neoplasia. 34938–34947. Mod Pathol 2009; 22:1–6. 110 Rice KL, Kees UR, Greene WK. Transcriptional regulation 130 Yu M, Wan Y, Zou Q. Prognostic significance of BP1 mRNA of FHL1 by TLX1/HOX11 is dosage, cell-type and promoter expression level in patients with non-small cell lung cancer. Clin context-dependent. Biochem Biophys Res Commun 2008; 367: Biochem 2008; 41: 824–830. 707–713. 131 Awwad RT, Do K, Stevenson H, Fu SW, Lo-Coco F, Costello M 111 Riz I, Lee HJ, Baxter KK, Behnam R, Hawley TS, Hawley RG. et al. Overexpression of BP1, a homeobox gene, is associated Transcriptional activation by TLX1/HOX11 involves Gro/TLE with resistance to all-trans retinoic acid in acute promyelocytic corepressors. Biochem Biophys Res Commun 2009; 380: leukemia cells. Ann Hematol 2008; 87: 195–203. 361–365. 132 Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD 112 Dayyani F, Wang J, Yeh JR, Ahn EY, Tobey E, Zhang DE et al. Loss et al. BP1, a new homeobox gene, is frequently expressed in of TLE1 and TLE4 from the del(9q) commonly deleted region in acute leukemias. Leukemia 2000; 14: 1867–1875. AML cooperates with AML1-ETO to affect myeloid cell prolifera- 133 Fu SW, Schwartz A, Stevenson H, Pinzone JJ, Davenport GJ, tion and survival. Blood 2008; 111: 4338–4347. Orenstein JM et al. Correlation of expression of BP1, a homeobox 113 Fraga MF, Berdasco M, Ballestar E, Ropero S, Lopez-Nieva P, gene, with estrogen receptor status in breast cancer. Breast Lopez-Serra L et al. Epigenetic inactivation of the Groucho Cancer Res 2003; 5: R82–R87. homologue gene TLE1 in hematologic malignancies. Cancer Res 134 Tan Y, Cheung M, Pei J, Menges CW, Godwin AK, Testa JR. 2008; 68: 4116–4122. Upregulation of DLX5 promotes ovarian cancer cell prolifera- 114 Liu C, Glasser SW, Wan H, Whitsett JA. GATA-6 and thyroid tion by enhancing IRS-2-AKT signaling. Cancer Res 2010; 70: transcription factor-1 directly interact and regulate surfactant 9197–9206. protein-C gene expression. J Biol Chem 2002; 277: 4519–4525. 135 Kato T, Sato N, Takano A, Miyamoto M, Nishimura H, Tsuchiya E 115 Nishida W, Nakamura M, Mori S, Takahashi M, Ohkawa Y, et al. Activation of placenta-specific transcription factor distal- Tadokoro S et al. A triad of serum response factor and the GATA less homeobox 5 predicts clinical outcome in primary lung and NK families governs the transcription of smooth and cardiac cancer patients. Clin Cancer Res 2008; 14: 2363–2370. muscle genes. J Biol Chem 2002; 277: 7308–7317. 136 Yu M, Smolen GA, Zhang J, Wittner B, Schott BJ, Brachtel E 116 Sepulveda JL, Belaguli N, Nigam V, Chen CY, Nemer M, et al. A developmentally regulated inducer of EMT, LBX1, Schwartz RJ. GATA-4 and Nkx-2.5 coactivate Nkx-2 DNA contributes to breast cancer progression. Genes Dev 2009; 23: binding targets: role for regulating early cardiac gene expression. 1737–1742. Mol Cell Biol 1998; 18: 3405–3415. 137 Satoh K, Hamada S, Kanno A, Hirota M, Umino J, Ito H et al. 117 Sepulveda JL, Vlahopoulos S, Iyer D, Belaguli N, Schwartz RJ. Expression of MSX2 predicts malignancy of branch duct Combinatorial expression of GATA4, Nkx2-5, and serum intraductal papillary mucinous neoplasm of the pancreas. response factor directs early cardiac gene activity. J Biol Chem J Gastroenterol 2010; 45: 763–770. 2002; 277: 25775–25782. 138 Satoh K, Hamada S, Kanno A, Ishida K, Ito H, Hirota M et al. 118 Weidenfeld J, Shu W, Zhang L, Millar SE, Morrisey EE. The Evaluation of MSX2 mRNA in brush cytology specimens WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in distinguished pancreatic carcinoma from chronic pancreatitis. lung epithelium. J Biol Chem 2002; 277: 21061–21070. Cancer Sci 2011; 102: 157–161. 119 Bellavia D, Campese AF, Checquolo S, Balestri A, Biondi A, 139 Satoh K, Hamada S, Kimura K, Kanno A, Hirota M, Umino J et al. Cazzaniga G et al. Combined expression of pTalpha and Notch3 Up-regulation of MSX2 enhances the malignant phenotype and is in T cell leukemia identifies the requirement of preTCR for associated with twist 1 expression in human pancreatic cancer leukemogenesis. Proc Natl Acad Sci USA 2002; 99: 3788–3793. cells. Am J Pathol 2008; 172: 926–939. 120 Sellar GC, Li L, Watt KP, Nelkin BD, Rabiasz GJ, Stronach EA 140 Shibata K, Kajiyama H, Yamamoto E, Terauchi M, Ino K, Nawa A et al. BARX2 induces cadherin 6 expression and is a functional et al. Establishment and characterization of an ovarian yolk sac suppressor of ovarian cancer progression. Cancer Res 2001; 61: tumor cell line reveals possible involvement of Nkx2.5 in tumor 6977–6981. development. Oncology 2008; 74: 104–111. 121 Davis M, Hitchcock A, Foulkes WD, Campbell IG. Refinement of 141 Turashvili G, Bouchal J, Burkadze G, Kolar Z. Differentiation of two chromosome 11q regions of loss of heterozygosity in ovarian tumours of ductal and lobular origin: I. Proteomics of invasive cancer. Cancer Res 1996; 56: 741–744. ductal and lobular breast carcinomas. Biomedical Papers of the 122 Gabra H, Watson JE, Taylor KJ, Mackay J, Leonard RC, Steel CM Medical Faculty of the University Palacky, Olomouc, Czecho- et al. Definition and refinement of a region of loss of slovakia 2005; 149: 57–62. heterozygosity at 11q23.3-q24.3 in epithelial ovarian cancer 142 Gelmann EP, Bowen C, Bubendorf L. Expression of NKX3.1 in associated with poor prognosis. Cancer Res 1996; 56: 950–954. normal and malignant tissues. Prostate 2003; 55: 111–117.

Leukemia NKL homeobox genes in leukemia I Homminga et al 581 143 Hollington P, Neufing P, Kalionis B, Waring P, Bentel J, cancer identifies novel methylation markers for multiple malig- Wattchow D et al. Expression and localization of homeodomain nancies. PLoS Med 2006; 3: e486. proteins DLX4, HB9 and HB24 in malignant and benign human 155 Yamashita S, Tsujino Y, Moriguchi K, Tatematsu M, Ushijima T. colorectal tissues. Anticancer Res 2004; 24: 955– 962. Chemical genomic screening for methylation-silenced genes in 144 Shim C, Zhang W, Rhee CH, Lee JH. Profiling of differen- gastric cancer cell lines using 5-aza-20-deoxycytidine treatment tially expressed genes in human primary cervical cancer by and oligonucleotide microarray. Cancer Sci 2006; 97: 64–71. complementary DNA expression array. Clin Cancer Res 1998; 4: 156 Kuang SQ, Tong WG, Yang H, Lin W, Lee MK, Fang ZH et al. 3045–3050. Genome-wide identification of aberrantly methylated promoter 145 Leja J, Essaghir A, Essand M, Wester K, Oberg K, Totterman TH associated CpG islands in acute lymphocytic leukemia. Leukemia et al. Novel markers for enterochromaffin cells and gastro- 2008; 22: 1529–1538. intestinal neuroendocrine carcinomas. Mod Pathol 2009; 22: 157 Kamalakaran S, Varadan V, Giercksky Russnes HE, Levy D, 261–272. Kendall J, Janevski A et al. DNA methylation patterns in luminal 146 Furuta J, Nobeyama Y, Umebayashi Y, Otsuka F, Kikuchi K, breast cancers differ from non-luminal subtypes and can identify Ushijima T. Silencing of Peroxiredoxin 2 and aberrant methylation relapse risk independent of other clinical variables. Mol Oncol of 33 CpG islands in putative promoter regions in human malignant 2011; 5: 77–92. melanomas. Cancer Res 2006; 66: 6080–6086. 158 Tellez CS, Shen L, Estecio MR, Jelinek J, Gershenwald JE, Issa JP. 147 Rauch T, Li H, Wu X, Pfeifer GP. MIRA-assisted microarray CpG island methylation profiling in human melanoma cell lines. analysis, a new technology for the determination of DNA methy- Melanoma Res 2009; 19: 146–155. lation patterns, identifies frequent methylation of homeodomain- 159 Asatiani E, Huang WX, Wang A, Rodriguez Ortner E, Cavalli LR, containing genes in lung cancer cells. Cancer Res 2006; 66: Haddad BR et al. Deletion, methylation, and expression of the 7939–7947. NKX3.1 suppressor gene in primary human prostate cancer. 148 Tong WG, Wierda WG, Lin E, Kuang SQ, Bekele BN, Estrov Z Cancer Res 2005; 65: 1164–1173. et al. Genome-wide DNA methylation profiling of chronic 160 Lai HC, Lin YW, Huang TH, Yan P, Huang RL, Wang HC et al. lymphocytic leukemia allows identification of epigenetically Identification of novel DNA methylation markers in cervical repressed molecular pathways with clinical impact. Epigenetics cancer. Int J Cancer 2008; 123: 161–167. 2010; 5: 499–508. 161 Lai HC, Lin YW, Huang RL, Chung MT, Wang HC, Liao YP 149 Miyamoto K, Fukutomi T, Akashi-Tanaka S, Hasegawa T, Asahara et al. Quantitative DNA methylation analysis detects cervical T, Sugimura T et al. Identification of 20 genes aberrantly intraepithelial neoplasms type 3 and worse. Cancer 2010; 116: methylated in human breast cancers. Int J Cancer 2005; 116: 4266–4274. 407–414. 162 Taylor KH, Pena-Hernandez KE, Davis JW, Arthur GL, Duff DJ, 150 Okamoto J, Hirata T, Chen Z, Zhou HM, Mikami I, Li H et al. Shi H et al. Large-scale CpG methylation analysis identifies novel EMX2 is epigenetically silenced and suppresses growth in human candidate genes and reveals methylation hotspots in acute lung cancer. Oncogene 2010; 29: 5969–5975. lymphoblastic leukemia. Cancer Res 2007; 67: 2617–2625. 151 Bell A, Bell D, Weber RS, El-Naggar AK. CpG island methylation 163 Rahmatpanah FB, Carstens S, Guo J, Sjahputera O, Taylor KH, profiling in human salivary gland adenoid cystic carcinoma. Duff D et al. Differential DNA methylation patterns of small B- Cancer 2011; 117: 2898–2909. cell lymphoma subclasses with different clinical behavior. 152 Jin B, Yao B, Li JL, Fields CR, Delmas AL, Liu C et al. Leukemia 2006; 20: 1855–1862. DNMT1 and DNMT3B modulate distinct polycomb-mediated 164 Wu X, Rauch TA, Zhong X, Bennett WP, Latif F, Krex D et al. CpG histone modifications in colon cancer. Cancer Res 2009; 69: island hypermethylation in human astrocytomas. Cancer Res 7412–7421. 2010; 70: 2718–2727. 153 Dunwell TL, Hesson LB, Pavlova T, Zabarovska V, Kashuba V, 165 Wang Y, Hayakawa J, Long F, Yu Q, Cho AH, Rondeau G et al. Catchpoole D et al. Epigenetic analysis of childhood acute ‘Promoter array’ studies identify cohorts of genes directly lymphoblastic leukemia. Epigenetics 2009; 4: 185–193. regulated by methylation, copy number change, or transcription 154 Shames DS, Girard L, Gao B, Sato M, Lewis CM, Shivapurkar N factor binding in human cancer cells. Ann NY Acad Sci 2005; et al. A genome-wide screen for promoter methylation in lung 1058: 162–185.

Leukemia