Role of the histone modifier MLL3 mutation in breast cancer cell plasticity

Breast cancer remains a leading cause of cancer-related death, with metastatic disease having an especially poor prognosis. Identifying mechanisms driving metastasis is critical for developing targeted therapies. MLL3 (also known as KMT2C) is among the most frequently mutated epigenetic regulators in breast cancer, with lossof-function mutations associated with tumor progression, metastasis, and therapy resistance. Through the work supported by our previous R01 award, we have developed novel CRISPR-engineered mammary stem cell (MaSC)-based tumor models combining MLL3 mutations with other co-occurring breast cancer driver mutations. Using these models, we discovered that MLL3 acts as a potent breast tumor suppressor. Unexpectedly, we found that MLL3 tumor suppressor function is primarily mediated by its adaptor activity rather than its catalytic function–a surprising finding. Furthermore, we showed that MLL3 loss promotes a hybrid epithelial-mesenchymal transition (EMT) state via activation of cell-intrinsic interferon-gamma (IFN-gamma) signaling, leading to the generation of highly metastatic cancer cells capable of colonizing multiple distant organs. Based on these findings, we hypothesize that MLL3 adaptor functions are critical for tumor suppression and that its loss drives metastasis through epigenetic reprogramming and IFN-gamma-mediated EMT plasticity. In this Renewal application, we propose the following aims to address key questions that emerge from our previous findings: 1) We will determine how MLL3 adaptor functions mediate tumor suppression. Using CRISPR-engineered MaSC tumor models, we will identify the MLL3-interacting partners involved in tumor suppression and understand how they control chromatin landscape and transcriptional program. 2) We will investigate how IFN-gamma signaling is activated in MLL3-deficient cells and how it induces hybrid EMT, focusing on the role of the dsRNA-sensing machinery and key IFN-gamma downstream mediator IRF1. 3) We will identify therapeutic vulnerabilities in MLL3-mutant and hybrid EMT breast cancer cells by exploiting their IFN-high, viral mimicry state. Our study will uncover mechanisms by which MLL3 loss promotes tumor initiation and metastasis and will establish novel therapeutic strategies for aggressive breast cancers characterized by MLL3 mutations and hybrid EMT phenotypes. Given the prevalence of MLL3 mutations and hybrid EMT across multiple cancer types, findings from our research will have broad implications for understanding and treating human cancer. Justification of mouse model use: In some of the sub-aims, we will use MaSC-based mouse tumor models or human breast cancer xenografts to investigate the mechanisms mediating MLL3 functions in tumor initiation and metastasis, as well as testing therapeutic strategies. Tumor initiation, metastasis and therapeutic response involve complex interactions between tumor cells and host cells/factors, which currently still require in vivo mouse models to faithfully recapitulate.