Kindlin-2 Loss Rewires Breast Cell and Tumor Epigenomes to Reveal Novel Methylation-Dependent Drivers

Abstract Metastatic breast cancer (BC) remains one of the leading causes of female cancer-related mortality worldwide. BC disease progression is driven by complex oncogenic signaling networks that promote invasion, immune evasion, and therapeutic resistance. The integrin-binding adaptor protein oncogene Kindlin-2 (K2) is a critical regulator of BC metastasis and survival. While previous studies have characterized the transcriptomic changes associated with K2 activity, the role of K2 in shaping the tumor epigenome remains poorly defined. This study aims to identify methylation changes attributable to K2 knockout (K2-KO) and thus addressing a gap of therapeutic potential by exploring novel methylation-dependent targets and pathways. We conducted genome-wide DNA methylation profiling in both cultured 4T1 control cells, 4T1-derived tumors, K2-KO cells and K2-KO tumors (n=3 per group) in a syngeneic mouse model. We evaluated 280,190 CpG sites on Illumina Infinium Mouse Methylation BeadChip across 12 samples. Differential methylation gene (DMG) analysis (fold change |FC| > 2.5; FDR < 5%) was conducted by comparing K2-KO vs control in both cell lines and tumors. Gene set enrichment analysis (GSEA) was implemented for overlapping DMGs between K2-KO cells and tumors to identify significantly dysregulated pathways. Statistically significant DMGs were examined in TCGA BC cohort for clinical significance where a β-value cutoff of 0.5 defined methylation status (hyper- vs hypomethylated) and BC strata was grouped by early (I-II) and advanced (III) stage. Overall survival was assessed using Kaplan-Meier (KM) and log-rank test. Our analysis identified 10815 DMGs in K2-KO cells and 71 DMGs in K2-KO tumors, with 57 DMGs altered across both models. Among these significant associations, we identified DMGs previously linked to BC progression, including PDLIM2 and ZMIZ1. Notably, the novel transcriptional corepressor gene BCOR, an epigenetic regulator, exhibited divergent methylation states in our cell (hyper; p = 4.88 × 10-6, log2FC = 1.98) and tumor (hypo; p = 0.04, log2FC = -2.7) model, suggesting tumor-specific selective pressures may influence metastasis-related gene. GSEA identified 27 significant pathways, including canonical cancer signaling, cAMP/cGMP-PKG signaling, oxytocin signaling, adrenergic and dopaminergic synapses. KM analysis in TCGA indicated women with stage 3 BC and BCOR hypermethylation (β > 0.5) had poorer overall survival (p=0.002). Collectively, this study identifies K2 as likely playing a key role in epigenetic processes underlying metastatic BC and highlighting the potential of methylation-regulated pathways as therapeutic targets. Our findings identified several novel DMGs, including clinical significance of BCOR with overall BC survival, and highlighted previously unrecognized pathways. Future work will define the functional and clinical impact of these alterations. Citation Format: Yanning Wu, Khalid Sossey-Alaoui, Fredrick Schumacher. Kindlin-2 loss rewires breast cell and tumor epigenomes to reveal novel methylation-dependent drivers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3195.