In vivo CRISPR screening links NFKB1 to endocrine resistance in ER+ breast cancer

Resistance to endocrine therapy (ET) remains a major clinical challenge in the treatment of estrogen receptor-positive (ER+) breast cancer, underscoring the need for novel therapeutic targets. To identify genetic drivers of ET resistance, we conducted an in vivo genome-wide CRISPR-Cas9 screen in MCF7 cells implanted into ovariectomized nude mice under estrogen-deprived conditions. Despite the bottlenecks inherent to in vivo pooled screening, recurrent enrichment analysis identified NFKB1 as a candidate regulator of estrogen-independent tumor progression. Functional studies confirmed that NFKB1 deficiency enhanced tumorigenicity and conferred resistance to tamoxifen and fulvestrant both in vitro and in vivo. Mechanistically, transcriptomic and biochemical analyses revealed that NFKB1 deficiency activated canonical NF-κB signaling, leading to inflammatory gene induction and enhanced ER signaling. Furthermore, pharmacologic inhibition of NF-κB signaling restored ET sensitivity in NFKB1-deficient cells. Analysis of TCGA breast cancer datasets revealed reduced NFKB1 expression in luminal breast tumors, whereas expression of other NF-κB family members was largely unchanged. Further analysis showed that low NFKB1 expression was associated with poorer clinical outcomes in patients with ER+ breast cancer. Collectively, these findings identify NFKB1 as a negative regulator of NF-κB signaling and endocrine resistance in ER+ breast cancer and provide mechanistic evidence linking NF-κB activation to ligand-independent ER signaling. Our results support further investigation of NFKB1 as a candidate biomarker and of NF-κB pathway inhibition as a potential therapeutic strategy in endocrine therapy-resistant breast cancer. These findings also illustrate the utility of in vivo CRISPR screening for identifying candidate regulators of endocrine resistance in breast cancer.