The combination of CDK4/6 inhibitors and endocrine therapy is now the standard first-line treatment for estrogen receptor-positive (ER+) metastatic breast cancer. Despite this, biomarkers predicting primary resistance to CDK4/6 inhibitors and the underlying mechanisms are still poorly understood. This study aimed to identify key molecular features and potential therapeutic targets associated with primary resistance to these inhibitors. An initial cohort of 36 patients treated with palbociclib plus endocrine therapy served as the discovery group. Next-generation sequencing of circulating tumor DNA (ctDNA) from these individuals was conducted to detect genomic changes linked to primary resistance to palbociclib. The identified potential biomarker was subsequently confirmed in a separate validation cohort of 104 patients, as well as in external public datasets. Functional validation of resistance was performed using parental MCF-7 and T47D cell lines, along with modified versions created via small interfering RNA knockdown and lentiviral overexpression. Underlying mechanisms were explored through RNA sequencing, chromatin immunoprecipitation, and reporter gene assays. The efficacy of targeted combination therapies was assessed in patient-derived organoids and xenograft models. Within the discovery group, amplification of S6K1 (observed in 3/35 cases, approximately 9%) emerged as a significant factor contributing to primary resistance against CDK4/6 inhibition. In the validation cohort, elevated S6K1 expression was detected in 15/104 patients (14%). Among those treated with palbociclib, individuals with high S6K1 levels exhibited markedly shorter progression-free survival compared to those with low expression (hazard ratio = 3.0, P = 0.0072). Analysis of aggregated public datasets indicated that S6K1 amplification occurs in about 12% of breast cancer cases overall. Furthermore, elevated S6K1 expression in breast cancer was associated with reduced relapse-free survival (hazard ratio = 1.31, P < 0.0001). In experimental models, S6K1 overexpression driven by gene amplification alone was capable of inducing palbociclib resistance in breast cancer cells. This effect was mediated primarily by upregulation of proteins involved in G1-to-S phase progression and enhanced Rb phosphorylation, largely via activation of the c-Myc signaling pathway. Importantly, co-treatment with an mTOR inhibitor, which targets the pathway upstream of S6K1, effectively reversed this resistance both in cell-based assays and animal models. Amplification of S6K1 represents a key driver of primary resistance to palbociclib in breast cancer. Patients harboring S6K1-amplified tumors may benefit from dual therapy involving CDK4/6 inhibitors and agents targeting S6K1 or its upstream regulators.
