CLONING AND CHARACTERIZATION OF AFX, THE GENE THAT FUSES TO MLL IN ACUTE LEUKEMIAS WITH A T(X-11)(Q13-Q23)

We report the cloning and characterization of the entire AFX gene whic h fuses to MLL in acute leukemias with a t(X;11)(q13;q23). AFX consist s of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are presen t in the cell line and the ANLL sample with a t(X;11)(q13;q23), The si ngle intron of the AFX gene consists of 3706 nucleotides, It contains five simple sequence repeats with lengths of at least 12 bps, a chi-li ke octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer -like sequences (GATAGTG) that are distributed throughout the entire A FX intron sequence. In the KARPAS 45 cell line the breakpoints occur a t nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene, The AFX protein bel ongs to the forkhead protein family. It is highly homologous to the hu man FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q1 4), a chromosome rearrangement characteristic of alveolar rhabdomyosar comas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cel l line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, th e 5'-part of the AFX protein contains a conserved hexapeptide motif (Q IYEWM) that is homologous to the functionally important conserved hexa peptide QIYPWM upstream of the homeobox domain in Hox proteins. This m otif mediates the co-operative DNA binding of Pbx family members and H ox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with t(X;11)(q13;q23), this h exapeptide motif is separated from the remaining forkhead domain withi n the AFX protein. The predicted amino acid sequence of AFX differs si gnificantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy ca n be explained by the occurrence of two sequencing errors in the earli er work at nucleotide number 783 and 844 (loss of a cytosine residue o r guanosine residue, respectively) that lead to two reading frame shif ts.