Cryptic MLL, AF10, ARMC3 Rearrangement in Secondary AML Creates ARMC3-MLL Fusion That Disrupts Gene Encoding Armadillo Repeats Similar to Major Cancer Genes.

Introduction: Complex rearrangements are required to form 5′-MLL-AF10-3′ translocation breakpoint junctions because the 5′ to 3′ orientation of AF10 at band 10p12 is telomere to centromere. Previously we discovered a cryptic MLL translocation and 5′-MLL-AF10-3′ fusion transcripts in a case of secondary FAB M4 AML with a normal karyotype, which occurred after rhadomyosarcoma treatment with etoposide (Megonigal 2000). Here we characterized the genomic breakpoint junctions of the translocation, which not only proved to be a 3-way rearrangement, but also to involve a novel partner gene.

Methods: BamHI reverse panhandle PCR was used to clone the unknown 5′-partner gene-MLL-3′ genomic breakpoint junction. The corresponding fusion transcript was obtained by RT-PCR. The 5′-MLL-AF10-3′ genomic breakpoint junction was obtained by PCR with gene specific primers.

Results: Consistent with the smaller of two BamHI MLL bcr rearrangements on the Southern blot, BamHI reverse panhandle PCR gave a 4090 bp product, with the ARMC3 (armadillo repeat containing 3) gene from band 10p12 at the 5′ side fused to the 3′ portion of the MLL bcr. The ARMC3 breakpoint was position 1901 or 1902 relative to the start of intron 17. The MLL bcr breakpoint at position 6473 or 6474 in intron 8, was 5′ to the secondary leukemia MLL translocation breakpoint hotspot. Identical C residues at the ARMC3 and MLL breakpoints precluded more precise breakpoint determinations and suggested NHEJ DNA repair typical of other MLL genomic breakpoint junctions. The novel rearrangement produced an in-frame 5′-ARMC3-MLL-3′ transcript with ARMC3 exon 17 and MLL exon 9 at the point of fusion, in addition to two alternatively spliced 5′-MLL-AF10-3′ transcripts joining MLL exon 7 or MLL exon 8 to AF10 exon 10. Southern blot analysis, restriction mapping, the 5′-MLL-AF10-3′ fusion transcripts and the 5′-ARMC3-MLL-3′ genomic breakpoint junction informed gene-specific primers for PCR-based cloning of the 5′-MLL-AF10-3′ genomic breakpoint junction. MLL bcr position 6202, 6203, 6204, 6205, 6206 or 6207 in intron 8 was fused to position 2737, 2738, 2739, 2740, 2741 or 2742 relative to the start of AF10 intron 9, and 5′-ATTAG-3′ sequences were present at the breakpoints in both genes. Comparison of the 5′-MLL-AF10-3′ and 5′-ARMC3-MLL-3′ genomic breakpoint junctions indicated that 265 to 271 bases from MLL were deleted during the translocation.

Conclusions: Since ARMC3 is adjacent and centromeric to AF10 at band 10p12, and has the same 5′ to 3′ orientation from telomere to centromere, these results suggest that the complex 3-way rearrangement occurred by splitting of band 11q23 and insertion of band 10p12 material containing the 3′ portion of AF10 through to the 5′ portion of ARMC3 into the MLL bcr. Formation of the 5′-MLL-AF10-3′ and 5′-ARMC3-MLL-3′ rearrangements on the der(11) chromosome in this manner is consistent with the formation of 5′-MLL-AF10-3′ breakpoint junctions via complex translocations. Additional studies are in progress to determine whether a 5′-ARMC3-AF10-3′ genomic breakpoint junction was created on the der(10) chromosome. The uncharacterized ARMC3 protein is of interest because catenin proteins (eg. β-catenin), plakophilins and the tumor suppressor APC contain Arm repeats. This raises the possibility of a potential contribution of the predicted ARMC3-MLL fusion protein in the genesis of the AML.