Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor–positive (ERþ) breast cancers treated with prolonged neoadjuvant letrozole. Experimental Design: We performed targeted DNA and RNA sequencing in 68 ERþ breast cancers from patients treated with preoperative letrozole (median, 7 months). Results: Twenty-four tumors (35%) exhibited a PEPI score 4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P ¼ 2.56E15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ERþ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ERþ tumors in METABRIC. Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ERþ breast cancer cells and in patients' ERþ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ERþ breast cancer who fail to respond to preoperative estrogen deprivation.