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Originally published online as doi:10.1189/jlb.1102581 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:302-305.)
© 2003 by Society for Leukocyte Biology

DLX genes as targets of ALL-1: DLX 2,3,4 down-regulation in t(4;11) acute lymphoblastic leukemias

Nicoletta Ferrari*, Giulio L. Palmisano*, Laura Paleari*, Giuseppe Basso{dagger}, Manuela Mangioni{ddagger}, Vincenzo Fidanza§, Adriana Albini*, Carlo M. Croce§, Giovanni Levi and Claudio Brigati*

* Molecular Biology Laboratory, Istituto Nazionale per la Ricerca sul Cancro IST, Genova, Italy;
{dagger} Department of Pediatrics, University of Padova, Italy;
{ddagger} CBA, Genova, Italy;
§ Department of Microbiology and Immunology, Kimmel Cancer Center, Philadelphia, Pennsylvania; and
Evolution des Regulations Endocriennes, Paris, France

Correspondence: Claudio Brigati, Istituto Nazionale per la Ricerca sul Cancro IST, Largo Rosanna Benzi, 10, 16132, Genova, Italy. E-mail: claudio.brigati{at}istge.it


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ABSTRACT
 
Dlx genes constitute a gene family thought to be essential in morphogenesis and development. We show here that in vertebrate cells, Dlx genes appear to be part of a regulatory cascade initiated by acute lymphoblastic leukemia (ALL)-1, a master regulator gene whose disruption is implicated in several human acute leukemias. The expression of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 was absent in All-1 -/- mouse embryonic stem cells and reduced in All-1 +/- cells. In leukemic patients affected by the t(4;11)(q21;q23) chromosomal abnormality, the expression of DLX2, DLX3, and DLX4 was virtually abrogated. Our data indicate that Dlx genes are downstream targets of ALL-1 and could be considered as important tools for the study of the early leukemic cell phenotype.

Key Words: trithorax • RT-PCR • HOM-C • ES cells

The Drosophila gene Trithorax (trx) is a master trancriptional regulator that activates some members of the HOM-C genes group. In turn, the HOM-C genes modulate distal-less (Dll) genes, which are essential for proper development of limbs and other structures of Drosophila [1 ]. In the human system, the trx homologue is the acute lymphoblastic leukemia (ALL)-1 gene, a recurring partner of translocations involving chromosome band 11q23 in human biphenotipic leukemias [2 , 3 ]. ALL-1 is also known to be a potent activator of HOX genes (the homologues of Drosophila HOM-C) [4 5 6 ]. These analogies prompted us to investigate if loss or disruption of ALL-1 could ultimately affect the expression of human Dll homologues, called DLX, themselves homeobox genes. We first examined mouse embryonic stem (ES) cells in which All-1 was inactivated by homologous recombination on one or both alleles [7 ]. By applying semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that the expression of the Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 (identical to Dlx4) genes was reduced in All-1 +/- ES cells by 40%, 69%, 40%, 36%, and 65%, respectively, as determined by densitometric analysis and was virtually absent in All-1 -/- ES cells (Fig. 1 , Table 1 ). These data indicate a direct correlation between All-1 allele dosage and Dlx gene expression and suggest that the Drosophila cascade of activation, Trx-HOM-C-Dll, could be paralleled in humans by the homologues ALL-1-HOX-DLX.



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  		Figure 1. Expression of DLX genes in murine ES cells in which one (+/-) or both alleles (-/-) of All-1 have been inactivated by homologous recombination. Agarose gel migration pattern of RT-PCR products of Dlx2, Dlx3, Dlx5, Dlx6, and Dlx7 genes. The optimum PCR cycle number for the exponential amplification region was determined in preliminary experiments. The signal intensity of the products was analyzed by densitometry and normalized to the actin signal obtained from the same sample. The annealing temperature was 60°C in all cases, except for DLX5 and DLX6, where the annealing temperatures were 65°C and 56°C, respectively.


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  		Table 1. Murine primers and gene accession numbers used for gene expression analysis

HOX genes are known to be essential for hemopoiesis [8 ]; this implies that ALL-1, being a HOX activator [4 5 6 ] could act as an upstream regulator in myeloid/lymphoid development. We therefore compared the level of expression of DLX gene expression in bone marrow (BM) aspirates from ALL patients with or without the t(4;11) chromosomal translocation with the two groups matched for cellular immunophenotype, according to a panel of standard markers (see Fig. 2 and Table 2 ). RT-PCR analysis for DLX2, DLX3, DLX4, DLX5, DLX6, and DLX7 (DLX4 and DLX7 are splice variants of the same transcript, ref. [10 ]; the sequence of DLX1 was not available) showed that the expression levels of the six genes varied dramatically between the two groups (Fig. 2) . In samples from patients with the t(4;11) translocation, we detected virtually no expression of DLX2, DLX3, and DLX4 genes compared with the substantial expression in the controls (P=0.001). The splicing process giving rise to DLX4 seems to follow a complex behavior, as this gene has been found silent in normal, mature B cells [11 ]; similarly, we found it expressed at very low levels in t(4;11) cells and highly expressed in nontranslocated ALL material. Indeed, developmental variables could concur to define DLX expression that could reflect the degree of maturation of these two groups of cells, despite their immunophenotypic similarity. All samples expressed the DLX7 gene but at higher levels by the control group (P=0.05). The DLX5 and DLX6 genes showed a quite distinct behavior: A low but detectable expression was present in both groups with no significant differences. Thus, deletions or rearrangements of ALL-1 are paralleled by reduced Dlx/DLX expression in the mouse and human systems, respectively. One explanation for these results could be that the fusion protein ALL-1/AF4 causes a loss, or partial loss, of an activator function present in ALL-1; alternatively, the fusion could act, directly or indirectly, as a DLX repressor, subverting the normal activation pathway [12 ]. It is interesting that the translocation of ALL-1 appears to have divergent effects on DLX5 and -6 as compared with the loss of All-1 in the ES cell system. Here, we have identified the DLX genes as downstream targets of these putative functions.



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  		Figure 2. Expression levels of DLX and BCLxL in leukemia cells with or without the t(4;11) aberration. BM samples were obtained from eight t(4;11)(q21; q23) infant ALL cases (clinical and general hematological parameters are available upon request). The leukemia cells showed the following membrane markers: CD19+, CD7–, SmIg–, CD10–, CD13–, CD33–, Cdw65–, CyIgM–. Eighteen pre-B ALL patients not bearing the translocation but exhibiting a comparable cell immunophenotype were chosen as controls. Chromosome preparations were obtained from BM cultures, and standard cytogenetic analysis was used. In addition to assessing the t(4;11) rearrangement, the presence of the fusion mRNA was confirmed by RT-PCR [9 ]. RT-PCR analysis of DLX2, -3, -4, -5, -6, and -7 and BCLxL gene expression (right-most columns) in t(4;11) and control ALL patients’ BM expressed in arbitrary units. Values represent the mean densitometric analysis of the two groups relative to the glyceraldehdye-3-phosphate dehydrogenase (G3PDH) gene internal standard for each RNA sample. PCR number cycles were determined in the region of exponential amplification. Bars represent standard deviations. The annealing temperature was 60°C in all cases.


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  		Table 2. The primers used for DLX gene expression analysis in ALL patients

To investigate whether the functional link between ALL-1 and DLX could reflect a role for DLX genes in the differentiation program of hematopoietic cells, we examined three normal BM samples and found that DLX2, -3, and -4 were preferentially expressed in the CD34+ fraction, and DLX5 and -6 were preferentially expressed in the CD34– fraction (data not shown). This is in apparent contrast with our patients’ results, which showed a lack of DLX2, -3, and -4 expression in the presumably more "immature" cells t(4;11). However, it has recently been argued that the CD34– compartment could harbor more primitive progenitors [13 , 14 ], which could partially skew DLX expression levels. Of note, a third isoform of DLX4, BP1, is expressed in CD34– and not in CD34+ cells [11 ]. This may imply that they have different functions in hematopoiesis. It is interesting that the DLX gene expression pattern supports the hypothesis of a DLX 2, -3, and -4 common regulatory cascade with the HOX genes. This is consistent with the data from normal BM, where we show that CD34+ cells, known to express several HOX genes [15 ], also express DLX2, -3, -4, and -7 genes, and the CD34– compartment shows down-regulation of HOX [16 ] and DLX genes.

In acute leukemia, resistance to apoptosis has been described as a key feature resulting from an altered balance between pro- and antiapoptotic genes [17 , 18 ]. We thus measured the expression levels of the BCLxL gene (Fig. 2 , right-most columns) as an internal indicator of a cellular "antiapoptotic" state. The samples from t(4;11) ALL patients showed significantly higher levels of BCLxL transcripts with respect to controls (P<0.01). It is interesting that the only sample of our patients’ group showing 50% cells harboring the t(4;11) translocation showed an intermediate BCLxL level and an increase in DLX2, -3, and -4 (single-value data are available on request). These results showed an inverse correlation in the expression level of the DLX2, DLX3, DLX4, and DLX7 genes and BCLxL. No differences in BCLxL were noted in the different All-1 knockout ES cell lines (data not shown). These results suggest that the lack of expression of DLX genes in t(4;11) leukemia cells could lead toward a resistance to apoptosis, hence, higher malignancy. This possibility is corroborated by the lack of DLX2, -3, and -4 expression in cell lines resistant to fenretinide-induced apoptosis [19 ] and further stressed by our data on BCLxL expression. Thus, low DLX2, -3, and -4 and high BCLxL levels in t(4;11) patients would indicate antiapoptotic activity in these cells and explain their documented resistance to stress-induced cell death [20 ]. As the expression profile of BCLxL has been used as a prognostic factor in acute myeloid leukemia [21 ], the relationship between BCLxL and DLX gene expression could be an additional, useful tool at diagnosis or post-treatment to monitor the radiation or chemotherapy-induced apoptosis in t(4;11)-affected patients.


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ACKNOWLEDGEMENTS
 
The support of Telethon Italy to this research is gratefully acknowledged. This work was partially supported by grants from AIRC (to N. F.), C.N.R., European Community (to G. L.), and Istituto Superiore di Sanità-Progetto Italia-USA: terapia dei tumori. L. P. was supported by a FIRC fellowship. G. L. and C. B. are considered senior coauthors. We are grateful to Dr. Douglas Noonan (IST, Genova, Italy) for helpful suggestions in preparing this work. We also thank Dr. M. Spinelli, Department of Pediatrics, University of Padova, Italy, for help with collection of the clinical samples.

Received November 27, 2002; revised May 2, 2003; accepted May 8, 2003.


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