Association Between RUNX3 Hypermethylation and Acute Myeloid Leukemia Inv(16) Subtype

The RUNX family of transcription factors forms the DNA binding α-chain partners of the heterodimeric core binding factor (CBF) complex. Each of the RUNX proteins, RUNX1, RUNX2, and RUNX3, can form heterodimers with CBFβ. In the M4Eo subtype of human acute leukemia, the chromosomal translocation resulting in inversion 16 encodes a chimeric protein in which CBFβ is fused to smooth muscle myosin heavy chain (SMMHC). Although the exact mechanism of leukemogenesis by this chimera is unknown, it is thought that CBFβ-SMMHC sequesters RUNX1 in the cytoplasm and antagonizes its normal function. Although the role of RUNX1 in hematopoiesis has been previously well-established, recent data have indicated that the RUNX3 gene may also play a key role in the development of human acute leukemias.

To clarify the role of RUNX3 in acute myeloid leukemia (AML), we investigated its expression and promoter DNA methylation in leukemia cell lines and patient samples. Eleven human leukemia cell lines of myeloid origin and twelve of lymphoid origin were used in this study. Cell suspensions from bone marrow aspirate specimens from patients with AML (69 cases), MDS (19 cases) and ALL (6 cases) were obtained prior to therapy from established tissue blocks. Peripheral blood samples were obtained from four healthy volunteers, and CD34+ cells were obtained from another four individuals. Methylation status of the gene promoters of RUNX1, RUNX2 and RUNX3 were evaluated using the Pyrosequencing Methylation Assay (PMA) method, and expression of RUNX3 was analyzed by quantitative real-time PCR and immunohistochemical staining.

Hypermethylation of RUNX1 and RUNX2 was rare in cell lines; RUNX1 was not hypermethylated in any of the studied samples, and RUNX2 was hypermethylated in only two cell lines. In contrast, we found that the RUNX3 promoter was hypermethylated in 17 of the cell lines (74%). Interestingly, we observed a trend toward higher frequency of hypermethylation of RUNX3 in cell lines of myeloid (90%) compared to lymphoid (57%) origin. In patient samples, RUNX3 promoter methylation was below 15% in normal samples, and hypermethylation was found in 32/69 AML samples (46%), 4/19 MDS samples (21%), and 6/6 ALL samples (100%). Of the 69 AML samples, 19 were classified as AML M4Eo, and 50 were other types of AML. 84% of the human AML M4Eo samples were hypermethylated at the RUNX3 promoter region, whereas only 34% of the other AML subtypes were hypermethylated. We also evaluated DNA methylation of RUNX1 and RUNX2 in a subgroup of these samples (66 samples for RUNX1 and 72 for RUNX2) and found that, as in cell lines, these genes are almost universally unmethylated; with the exception of a single AML case, all studied samples presented no promoter methylation. As support of functional outcome, hypermethylation of RUNX3 was correlated with both lower levels of mRNA and protein, as confirmed by qRT-PCR and immunohistochemistry analysis in cell lines and patient samples, and treatment with the DNA demethylating agent Decitabine resulted in mRNA re-expression of RUNX3 concomitantly with decreased promoter methylation.

Finally, we compared clinicopathological features of patients with and without RUNX3 methylation. In this analysis, only non-M4Eo AML cases were compared because of the small number of non-methylated patients in the M4Eo group. Differences were found neither for blood counts nor for overall survival probability. However, relapse-free survival was significantly better for the unmethylated group (p=0.016).

In summary, we showed that promoter methylation of the RUNX3 gene and down regulation of RUNX3 expression occurs almost universally in M4Eo/inversion 16 AMLs, and that in cell lines, RUNX3 repression can be reversed by treatment with the hypomethylating agent decitabine. These results suggest that silencing of RUNX3 is likely an important target in CBF leukemia and that future studies should be dedicated to further characterize the role of RUNX3 in inversion 16 AML and its predictive value of relapse-free survival in AML.