Epigenetic Silencing of the RUNX3 Gene by Promoter Hypermethylation in Patients with Acute Myeloid Leukemia.
Authors: Maddipoti Sirisha C , Bueso-Ramos Carlos , Yang Hui , Fernandez Michael , Kuang Shaoquing , Fang Zihong , Stevenson William , Wei Yue , Pierce Sherry , Garcia-Manero Guillermo 
Published: October 2019

Abstract
The RUNX family of transcription factors forms the DNA binding α-chain partner of the heterodimeric core binding factor (CBF) complex. Each of the RUNX proteins, RUNX1 (AML1), RUNX2, and RUNX3 (AML2), can form heterodimers with CBFβ. While the role of RUNX1 in hematopoiesis has previously been well established, recent data have indicated that the RUNX3 gene may also play a key role in the development of human acute leukemias. RUNX3 promoter hypermethylation and downregulation of gene expression have been shown in human gastric and lung cancers, indicative of its function as a tumor suppressor gene. Prior cDNA gene expression arrays of acute myeloid leukemia have noted a downregulation of RUNX3 gene expression in blast cells of inversion 16 AML M4 Eo, with no evidence for somatic mutations in this gene. We therefore wanted to analyze the promoter methylation status of RUNX3 in patients with inversion 16 AML. Using bisulfite treatment of DNA, PCR amplification of the RUNX3 promoter, and pyrosequencing analysis, we initially studied 23 leukemia cell lines. We found that the RUNX3 promoter was hypermethylated at 17 of 23 cell lines, using a cutoff of >15% for hypermethylation, with a mean methylation percentage of 43 and a range of 4–97 (median 31%). We subsequently analyzed RUNX3 gene expression levels in eight of the leukemia cell lines by real-time PCR and were able to demonstrate low baseline expression, with reexpression after treatment with the hypomethylating agent decitabine. We also showed a decrease in percentage methylation of the RUNX3 promoter after treating three of the cell lines with decitabine. We then determined the methylation profile of 81 patients with acute myeloid leukemia (median age 65 [20–84], median WBC at presentation 10 [0.7–114], median percent of marrow blasts 52 [8–94], cytogenetics: inv16 22 (25%), t(8;21) 4 (4%), diploid 23 (27%), the rest abnormal). We observed that 21 of 22 AML M4 Eo samples (95%) were hypermethylated at RUNX3, with a mean methylation percentage of 50 and a range of 4.5–98 (median 49%). Of the other AML subtypes, 20 of 59 patient samples (33%) were hypermethylated, with a mean methylation of 23%, and range of 1–79 (median 12.5%). The RUNX3 promoter was unmethylated in four CD34+ normal controls, and six peripheral blood controls. No correlation between RUNX3 methylation and prognosis was detected in the non inv16 AML cases. Immunohistochemistry performed on the AML M4 Eo bone marrow specimens confirmed the presence of the core-binding factor chimeric protein. We also studied six ALL patient samples and all six were hypermethylated at the RUNX3 promoter, with a mean methylation of 30%, and a range of 21–39 (median 31%). Finally, 19 MDS samples were studied: only four were hypermethylated with an average of 10.5%, and a range of 2.5–47 (median 6.1%). We also analyzed the methylation profile of the RUNX1 and RUNX2 genes on the leukemia cell lines, AML, ALL, and MDS patient samples, and normal controls. The RUNX1 and RUNX2 promoters were universally unmethylated. Our results indicate that epigenetic dysregulation of RUNX3 is likely an important target in the molecular pathway of leukemogenesis in core binding factor leukemia.