High Frequency of C-Terminal Frame-Shift Mutations of RUNX1 Gene in De Novo AML with Partial Tandem Duplication of MLL.

Transcription factor RUNX1 is essential for normal hematopoiesis. RUNX1 mutations, mainly at Runt homology domain (RHD), have been described in patients with AML-M0 and were rarely found in non-M0 AML.

We aimed to analyze the RUNX1 mutations in AML patients with partial tandem duplication of MLL (MLL-PTD) and to investigate the biological functions of the mutants detected.

Bone marrow samples from 93 patients with MLL-PTD were analyzed for RUNX1 mutations. MLL-PTD was screened by Southern-blot analysis followed by RT-PCR or detected by real-time quantitative PCR. Mutational analysis of RUNX1 gene was performed by sequencing of all RT-PCR products amplified from exon 3 through exon 8. Each mutation was reconfirmed with alternative primers. The wild-type, all mutants of RUNX1 (except those truncated at RHD or silent) and pcDNA3.1 were transiently transfected into Cos-7 cells. Immunoblot analysis after immunoprecipitation with anti-FLAG RUNX1 antibody and electrophoretic mobility shift assay were used to determine the interaction with CBFβ and DNA-binding ability of the RUNX1 mutants. Dual luciferase assay system was used to analyze the transactivation potential of RUNX1 mutants in K562 cells.

RUNX1 mutations were detected in 23 patients (24.7%) at diagnosis, with 3 patients carrying double mutations; 14 mutations were located in RHD (exons 3-5) and 12 at C-terminal region (exons 6-8). In addition, one patient acquired a C-terminal mutation at relapse. The patterns of 27 mutations consisted of 6 missense mutations, 3 nonsense mutations, 17 frame-shift mutations, and 1 silent mutation; all were heterozygous. Of the 3 patients with double mutations, clonal analysis showed that one patient had combined missense and frame-shift mutations on the same allele, the other patient had two missense mutations on different alleles, and another patient had a missense mutation and a silent mutation on the same allele. Functional analyses revealed significant difference among mutants. Two missense RUNX1 mutants at RHD (G108D and R174L) and all of the frame-shift mutants in the transactivation domain (TAD) ( S287fsX571, S295fsX571, L300fsX570, V333fsX574, I339fsX569 and P355fsX572) exhibited lack of DNA-binding ability and inhibited transactivation activity of wild-type RUNX1 in a dominant-negative effect. All frame-shift mutants distal to the TAD generated termination codons within the 3'-untranslated region (H377fsX565, Q388fsX572, L414fsX569, L414fsX567 and V425fsX576), they all retained the normal transactivation activity as the wild-type. R177X and R205W retained the ability of heterodimerization with CBFβ but they had markedly reduced DNA-binding and no transactivation potential without inhibitory effect on wild-type RUNX1. L183fsX185 could bind DNA but lacked transactivation activity. S114P and Q370R had normal transactivation activity.

Our results showed that patients with de novo AML with MLL-PTD had a high frequency of frame-shift mutations at C-terminal region of RUNX1; those within TAD had dominant-negative effects whereas those distal to TAD retained the normal transactivation potential.

Supported by grants NHRI-EX96-9434SI, NSC97-2314-B-182 -011-MY3 and MMH-E-96009.

No relevant conflicts of interest to declare.