Targeting lineage plasticity to inhibit basal-like breast cancer progression

Project Summary Basal-like breast cancer (BLBC) represents one of the most aggressive breast cancer subtypes, occurring disproportionately in younger women and individuals with inherited BRCA1 mutations. It substantially overlaps with triple-negative breast cancer (TNBC) and is currently treated primarily with chemotherapy, which often results in significant toxicity and frequent drug resistance. The poor prognosis associated with BLBC is linked to its pronounced lineage plasticity and intratumoral heterogeneity, both of which complicate treatment. Recent findings indicate that a key event in BLBC initiation is the reacquisition of embryonic multipotency. Oncogenic mutations can drive committed luminal progenitor cells (LPCs) to revert into embryonic multipotent- like (EMP-like) cells, which share features with embryonic mammary stem cells and display hybrid basal-luminal characteristics. Importantly, such EMP-like cells have also been observed in the premalignant breast tissue of women carrying germline BRCA1 mutations and in TNBC tumors, underscoring the clinical relevance of EMP- like cells. Our studies in murine BLBC models demonstrate that EMP-like cells express elevated levels of SOX9, a transcription factor critical for stem and progenitor cell identity. Loss of SOX9 blocks this de-differentiation process and prevents the transition from premalignant lesions to invasive BLBC, pointing to dedifferentiation into multipotency as a potential therapeutic target. This aligns with a recent large-scale single-cell proteomic analysis of TNBC tumors, which showed that SOX9-high basal-luminal cancer cells strongly correlate with early recurrence and poor survival, further emphasizing their clinical significance. Despite the important role of these cells, the underlying mechanisms driving de-differentiation to EMP-like states remain largely unknown. Defining these mechanisms will be essential for developing new strategies to intercept BLBC progression and ultimately improve patient outcomes. In this R01 Revision proposal in response to NOFO RFA-CA-25-005, we will utilize the Pro-Code technology based in vivo CRISPR screen and spatial perturbation screen (Perturb-map), technologies developed by Dr. Brian Brown (Co-Inv) with the support of the NCI IMAT program, to uncover tissue/tumor microenvironment signals that promote the de-differentiation to SOX9-high EMP-like state. Building on our existing transcriptomics data of EMP-like cells, we will perform Pro-Code-based in vivo CRISPR screen to identify receptors and signaling molecules that promote the acquisition of SOX9-high EMP-like state (Aim 1). In parallel, we will use the Perturb- map to define microenvironment niches that facilitate the induction of EMP-like cells (Aim 2). These studies will uncover key microenvironmental drivers for the formation of stem-like tumor cells and identify potential targets for eliminating these key precancerous cells that are critical for malignant transformation.