Abstract Tumor microenvironments (TME) produce molecular signals that influence therapeutic penetration, distribution, and metabolism as well as positively or negatively regulate tumor cell growth, migration, and response to therapeutics. TME mainly comprises fibroblasts, immune cells, pericytes, endothelial cells, epithelial cells, and the tumor-specific extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) are active stromal cells in the TME, acting as signaling and remodeling centers and aiding the creation of desmoplastic tumor stroma. To better understand the identities of CAF subtypes promoting tumor or abnormal activation, it is essential to find a master transcription factor (TF) that determines the identity of cancer-associated fibroblast subtypes. RUNX2 is known as a transcription factor and is rarely expressed in normal tissues; however, it is expressed in abnormally activated fibroblastic regions. In this study, we hypothesized that a master transcription factor of RUNX2 determines the phenotype of normal fibroblasts to CAF and defines its super-enhancer landscape. To identify CAF-specific and functionally important TFs, we analyzed single-cell RNA-sequencing (scRNA-seq) from various cancer patients, focusing on CAFs. Specifically, H3K27ac and RUNX2 ChIP-seq were performed on CAFs to confirm their super-enhancer profiles. To examine the biological significance of RUNX2, we established cancer-associated fibroblasts from patients with colorectal cancer and loss of RUNX2 using shRNA-mediated methods. Here, we assessed the effect of indirect cancer cell and CAF interaction models (conditioned media) in addition to direct methods (co-culture) using a 3D spheroid. To determine the significance of RUNX2 in established CAFs and cancer cells in vivo, HCT116 colorectal cancer cells were co-grafted with RUNX2 knockdown CAFs into NOD/SCID mice. Using a combination of a direct CAF-HCT116 3D spheroid model and HCT116 cells with CAF-conditioned media, we confirmed that knockdown of RUNX2 in CAFs suppressed the migration or invasion of cancer cells and inhibited the sphere-forming ability of cancer cells by RUNX2-depleted CAFs. Knockdown of RUNX2 CAFs also failed to aggregate spherical 3D phenotypes in the direct model. Interestingly, based on the ChIP-seq analysis of CAFs with H3K27ac and RUNX2, we confirmed that RUNX2 can remodel the super-enhancer landscape in CAFs and that the gene associated with RUNX2 was enriched in myofibroblastic signatures. Using a gel contraction assay, we showed that fibroblast contractility was significantly decreased by RUNX2 knockdown. To evaluate the importance of tumor formation in vivo, we transplanted RUNX2-knockdown CAFs with cancer cells and found that the tumor burden was lower depending on RUNX2 dysfunction. We further analyzed the correlation between the expression of RUNX2 and clinical outcomes and confirmed that RUNX2 is highly expressed in CAFs and strongly correlates with poor survival across several cancer types. Our findings indicate that RUNX2 is a promising target for myofibroblastic function and pro-tumorigenic features of the cancer microenvironment. Citation Format: So-Young Yeo, Keun-Woo Lee, Insuk Sohn, Seok-Hyung Kim. RUNX2 is a master transcription factor that determines the identity of cancer-associated fibroblast subtypes with abnormal activation in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB282.