ID4 maintains undifferentiated and immune-evasive states in aggressive triple-negative breast cancer and represents a novel druggable vulnerability

Background: Inhibitor of Differentiation 4 (ID4) is overexpressed in triple-negative breast cancer (TNBC) and is essential for proliferation, anchorage-independent growth, and tumorigenicity of MDA-MB-231 cells. These findings position ID4 as a candidate driver of undifferentiated, aggressive TNBC phenotypes. However, its expression pattern across molecularly heterogeneous TNBC subtypes and its therapeutic potential remain undefined. Methods: We integrated in silico analyses of clinically annotated TNBC cohorts with functional studies in ID4-high TNBC models. ID4 expression and co-expression patterns across TNBC subtypes were evaluated using bc-GenExMiner v5.2. ID4 was silenced by CRISPR-Cas9 or shRNA in three representative TNBC cell lines: MDA-MB-231 (mesenchymal stem-like, MSL), MDA-MB-468 (basal-like 1), and BT-549 (mesenchymal-like). Pharmacologic ID4 degradation was achieved using AGX51, a first-in-class pan-ID degrader. Proliferation was monitored by MTT assay and IncuCyte live-cell imaging. Drug response was further assessed in five patient-derived basal-like breast cancer stem cell (BCSC) cultures, with proliferation evaluated in all five and mammosphere formation in two. Results: In silico analysis revealed that ID4 expression is significantly enriched in the two most aggressive, immune-suppressed TNBC subtypes: basal-like immune-suppressed (BLIS) and mesenchymal-like immune-altered/immunomodulatory (MLIA; formerly mesenchymal stem-like/MSL). In MLIA tumors, ID4 strongly co-expresses with mesenchymal, myoepithelial, and stemness programs (YAP/TAZ-Hippo, FOXC1, SOX10, ACTA2, and basal cytokeratins). Both genetic silencing and pharmacologic degradation of ID4 markedly impaired proliferation across all tested cell lines and BCSC cultures. Notably, AGX51 completely disrupted mammosphere formation and 3D structural integrity in the two BCSC models examined. Stable ID4-knockout and knockdown TNBC cell lines were successfully generated for ongoing RNA-seq and DNA-methylation profiling. Conclusions: These data establish ID4 as a subtype-enriched, druggable vulnerability in the most aggressive and immune-suppressed TNBC subsets. Ongoing transcriptomic and epigenomic studies will elucidate the molecular circuits governed by ID4 and identify rational combination strategies for BLIS, MLIA, and mesenchymal stem-like TNBC. This abstract incorporates text revised with the assistance of artificial intelligence for language clarity and conciseness