Blood, 1 December 2002, Vol. 100, No. 12, pp. 4247-4248
CORRESPONDENCE
To the editor:
A novel MLL/AF4 fusion gene lacking the
AF4 transactivating domain in infant acute lymphoblastic
leukemia
In a 5-month-old child affected by acute lymphoblastic leukemia
(ALL) cytogenetic analysis showed a translocation involving chromosomes 4q13, 11q23, and 17q11. Standard reverse
transcriptase-polymerase chain reaction (RT-PCR)
analysis of t(4;11) breakpoints did not amplify any known
MLL/AF4 mRNA junction. Therefore, we performed Southern
analysis of the MLL locus in DNA from bone marrow cells sampled at diagnosis and found a rearrangement at locus MLL
of chromosome 11q23. Panhandle PCR amplification1 of the
fragment comprising the der(11) translocation breakpoints yielded a
4.6-kb fragment corresponding to the
BamHI-rearranged fragment detected by Southern
analysis. The amplified fragment contained sequences corresponding to
MLL exon 9 and, after an AluJ0 sequence, a
unique nonrepetitive sequence corresponding to the 937 bp of exon 11 of
the AF4 gene. These data indicated a hitherto unknown
junction between MLL intron 9 and AF4 intron 10 on der(11).
Because the MLL partner fragment included a sequence homologous to
AF4 exon 11, we designed a new antisense primer on
this exon to amplify the junction sequences of MLL/AF4
cDNA, transcribed from the new MLL/AF4 fusion gene.
RT-PCR analysis, with the primer couple 5'-TTCCCAAAACCACTCCTAGTGA-3'
(sense-MLL exon 9) and 5'-TCAGAATGCTCCTGACTCGTG-3' (antisense-AF4 exon 11), yielded a 380-bp (predicted)
fragment. Sequence analysis of the amplified product confirmed a hybrid mRNA with an in-frame junction between MLL exon 9 and
AF4 exon 11 (Figure 1A).
View larger version (11K):
[in this window]
[in a new window]
| Figure 1.
Structural organization of the novel chimeric
MLL/AF4 transcript and protein.
(A) Analysis of the novel MLL/AF4 transcript. An
RT-PCR assay was performed to evaluate whether the novel
MLL/AF4 fusion gene generates a chimeric mRNA. cDNA prepared
from a bone marrow RNA sample was amplified using the new primers
according to sequence analysis of the panhandle product.
The amplification reactions consisted of 40 cycles: 94°C, 30 seconds;
65°C, 60 seconds; and 72°C, 60 seconds. (B) Structural
organization of the novel chimeric MLL/AF4 protein. The
novel chimeric MLL/AF4 protein retains the DNA binding
domains but lacks the AF4-derived transactivating domain,
which is conserved in the usual type of this fusion protein. The
junctions between the 2 protein sequences are aligned and indicated by
an arrow.
 indicates
MLL AT hooks;
 ,
MLL repression domain;
 ,
AF4 transactivation domain; and
 ,
AF4 nuclear localization.
|
|
The predicted structure of the new MLL/AF4 chimeric protein
lacks the transcription activation MLL motif at residues
2829 to 2883 and the whole AF4 domain spanning residues 480 to 560, which is encoded by exon 10 of the AF4 gene and the
first nucleotides of exon 11 (Figure 1B).
The AF4 gene partners the MLL gene in 11q23
translocations in about 50% of childhood and adult ALL
cases.2 The MLL gene is fused to at least 25 different gene partners. The sequence and the predicted structure of
the proteins encoded by these genes do not appear to have any unifying
characteristic that would clarify their role in the
leukemogenic process. In addition, in some acute leukemias
MLL may show exon duplications. These issues raised the
possibility that an alteration of MLL alone is sufficient to
transform hemopoietic precursors and that the fusion partner has no
role. However, evidence that partner genes play a role in the
leukemogenic process comes from knock-out and knock-in experiments.3 Indeed, an important role for AF4
is emerging not only from the epidemiologic restriction of
MLL/AF4 to ALL4 but also from the recent finding
that lymphoid development is severely impaired in AF4
/
mice.3
The normal function of the AF4 gene is not known, but
a domain with transcriptional activity at nucleotides 480 to
560 and the nuclear localization of the AF4
protein5,6 suggests it regulates transcription (ie, the
AF4 gene seems to encode a transcription factor whose
expression is relevant for committed lymphoid precursors to complete
differentiation). This suggests that the MLL/AF4
gene arrests the lymphoid differentiation program by altering an early multipotential progenitor cell, either through a gain- or
loss-of-function of the MLL/AF4 protein. Therefore the
protein might induce aberrant expression of target genes so impairing
lymphoid differentiation,7 or alternatively it might lose
the capacity to induce the expression of genes important for
differentiation.2 The AF4 breakpoints in this
novel MLL/AF4 fusion gene lie at least 4 introns further downstream compared with all other MLL/AF4 known
translocations. Therefore, the new fusion gene lacks both the
AF4-derived and the MLL-derived
(nucleotides 2772-3579) transactivating domain5 (Figure 1B). The resulting fusion protein is hence able to bind DNA
through the MLL-derived AT hook domain, and through
the AF4-derived nuclear targeting sequence
domains8 but could lose its capacity to activate the
expression of these genes. Consequently, it is not inconceivable that
loss-of-function, through transcription block, could be the mechanism
of leukemogenesis in our ALL patient.
It is not clear whether transcription block could apply to other
MLL/AF4 fusion genes that retain the transactivating domain at residues 480 to 560.
Fabrizio Pane, Mariano Intrieri, Barbara Izzo, Concetta Quintarelli, Domenico Vitale, Roberta Migliorati, Lucia Sebastio, and Francesco Salvatore
Correspondence: Fabrizio Pane, CEINGE-Biotecnologie Avanzate and
Dipartimento di Biochimica e Biotecnologie Mediche, Università di
Napoli Federico II, Via S Pansini 5, 80131 Naples, Italy; e-mail:
fabpane{at}unina.it
Acknowledgments
Supported by grants from AIRC (Associazione Italiana per la
Ricerca sul Cancro, Milan), Consiglio Nazionale Delce Ricerche (CNR)-Progetto Strategico & Progetto Finalizzato (PF) Biotecnologie (Rome), Biogem (Naples), MIUR (Ministero Dell' Istruzione, Dell' Università e Della Ricerca, Rome), AIL (Associazione Italiana Leucemie, Rome), and Regione Campania (Naples).
References
1.
Felix CA, Kim CS, Megonigal MD, et al.
Panhandle polymerase chain reaction amplifies MLL genomic translocation breakpoint involving unknown partner gene.
Blood.
1997;90:4679-4686[Abstract/Free Full Text].
2.
Look AT.
Oncogenic transcription factors in the human acute leukemias.
Science.
1997;278:1059-1064[Abstract/Free Full Text].
3.
Isnard P, Core N, Naquet P, Djabali M.
Altered lymphoid development in mice deficient for the mAF4 proto-oncogene.
Blood.
2000;96:705-710[Abstract/Free Full Text].
4.
Biondi A, Cimino G, Pieters R, Pui CH.
Biological and therapeutic aspects of infant leukemia.
Blood.
2000;96:24-33[Free Full Text].
5.
Prasad R, Yano T, Sorio C, et al.
Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia.
Proc Natl Acad Sci U S A.
1995;92:12160-12164[Abstract/Free Full Text].
6.
Li Q, Frestedt JL, Kersey JH.
AF4 encodes a ubiquitous protein that in both native and MLL-AF4 fusion types localizes to subnuclear compartments.
Blood.
1998;92:3841-3847[Abstract/Free Full Text].
7.
Ma C, Staudt LM.
LAF-4 encodes a lymphoid nuclear protein with transactivation potential that is homologous to AF-4, the gene fused to MLL in t(4;11) leukemias.
Blood.
1996;87:734-745[Abstract/Free Full Text].
8.
Nakamura T, Alder H, Gu Y, et al.
Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs.
Proc Natl Acad Sci U S A.
1993;90:4631-4635[Abstract/Free Full Text].
