Abstract 301: RUNX3 upregulates c-MYC via mR1 – an essential genomic element for p53-deficient osteosarcomagenesis

Abstract

Abstract Osteosarcoma (OS) is the most common malignancy of bone in children and adolescents. The 5-year survival rate of patients with relapse or metastasis remain 20%, underscoring a need for new treatment strategies. The disease is generally characterized by recurrent somatic alterations of p53. An osteoblast-specific p53-knockout mouse line (Sp7-Cre;p53F/F; ‘OS mouse' herein) has a high incidence of osteosarcoma with a close histopathological resemblance to that of human. Thus, the OS mouse serves as a rational animal model to study molecular mechanisms of OS development. RUNX3/Runx3 was proved oncogenic in osteosarcomagenesis. We found dominant upregulation of RUNX3 in both human and mouse OS tissues, analyzing gene expression profiles of human OS cases, most of which possess genetically inactivated p53, and those of OS mice. Both human OS cell lines and mouse OS cells cloned from OS mice showed a strong dependency on RUNX3 for tumorigenicity in nude mice. Heterozygous deletion of Runx3 efficiently extended lifespans of OS mice. c-MYC/c-Myc was identified as a positive target of RUNX3. Combining microarray and ChIP-seq analyses of mouse OS cells, we found that c-Myc is transcriptionally upregulated by Runx3. In human and mouse OS cells, expression levels of RUNX3 and c-MYC correlated well with each other and knockdown of RUNX3 greatly reduced c-MYC expression and tumorigenicity. Heterozygous deletion of c-Myc, as well as that of Runx3, prevented osteosarcomagenesis of OS mice. In OS patients, mRNA levels of RUNX3 and c-MYC showed a positive correlation and high expression of each was a predictor of poor prognosis. Furthermore, we located a requisite genomic element for c-MYC induction by RUNX3. Assessing the ChIP-seq profiles of mouse OS cells, we selected several candidates of genomic elements responsible for upregulation of c-Myc within its 3-megabase topological association domain. Epigenome/genome editing of these candidates in human and mouse OS cells revealed that a genomic element ‘mR1', the 6-bp RUNX consensus binding site most proximal to the c-MYC/c-Myc promoter, is essential for c-MYC induction by RUNX3 and tumorigenicity. Indeed, homozygous disruption of mR1 effectively suppressed osteosarcomagenesis of OS mice and thus improved their survivals. Taken together, we propose RUNX3 as a central oncogenic figure in p53-deficient osteosarcomagenesis: RUNX3 is somehow upregulated upon inactivation of p53 and then starts to aberrantly upregulate c-MYC. Moreover, we propose mR1 as an essential genomic element for overexpression of c-MYC by RUNX3. Importantly, mR1-null mice are viable and fertile whereas c-Myc-null mice result in embryonic lethality. This indicates that mR1 is required not for physiological expression of c-MYC in normal development, but for its aberrant upregulation by RUNX3 in osteosarcomagenesis, providing a rationale for targeting mR1 as a novel therapy of OS. Citation Format: Yuki Date, Shohei Otani, Tomoya Ueno, Kosei Ito. RUNX3 upregulates c-MYC via mR1 – an essential genomic element for p53-deficient osteosarcomagenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 301.