Abstract Background: Advanced Estrogen receptor-positive (ER+) breast cancers benefit from the inhibitors of cell-cycle kinases CDK4 and CDK6. However, 20% of breast cancers show intrinsic resistance to these inhibitors. Although the mechanisms of acquired resistance to CDK4/6 inhibitors have been studied extensively, little is known about mechanisms of intrinsic resistance. We had recently reported a gene called TONSL, a DNA repair and chromatin organization-associated gene located on chromosome 8q24.3 amplicon, as an immortalizing oncogene and its overexpression in ER+ breast cancer is associated with poor outcome. In preclinical models, TONSL-immortalized breast epithelial cell lines generated ER+ adenocarcinomas upon transformation. Here we investigated whether TONSL amplification confers intrinsic resistance to CDK4/6 inhibitors. Here we compare the survival of TONSL amplified vs not amplified ER+ breast cancer patients treated with CDK4/6 inhibitors. Methods: 10,980 ER+ breast cancer samples (TONSL amplified, n=313; Not amplified, n=10,667) were analyzed by next-generation sequencing (WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Copy number variations were determined using CNVKit. A tissue microarray comprising ∼200 ER+ breast cancers was examined for TONSL and phospho-RB expression levels by immunohistochemistry. The role of TONSL in sensitivity to abemaciclib was examined in vitro in a TONSL amplified ER+ breast cancer cell line (generated from MCF-7 derived tumor in nude mice) with and without shRNA-mediated TONSL knockdown using bromodeoxyuridine (BrdU) incorporation ELISA. Real-world median overall survival (mOS) was derived from insurance claims and calculated from the biopsy to last contact or start of CDK4/6 inhibitor (abemiciclib/palbociclib/ribociclib) to last contact or first to last dose of CDK4/6 inhibitor treatment (time on treatment) using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U and p0.05 was considered significant. Results: MCF-7 derivative, which is intrinsically resistant to CDK4/6 inhibitor abemaciclib, regained drug sensitivity as assessed using cell proliferation assay upon TONSL knockdown (p0.05). TONSL amplification/overexpression correlated with elevated RB-loss and P53-loss gene signatures, but not RB phosphorylation. In real-word database, moderate correlations were observed between TONSL copy number and RNA expression (r=0.48, p0.05). Median age of breast cancer patients with TONSL amplification was lower (62 vs 64, p0.05) compared to TONSL not amplified. TONSL amplification was more common in Black or African American patients (20% vs 14%, p0.05), but less common in White patients (68.6% vs 77.7%, p0.05). TONSL amplified breast cancer patients had worse mOS (28.4 months (m) vs 41.9, HR 1.4, 95% CI 1.2-1.6, p0.0001) compared to TONSL not amplified. Importantly, TONSL amplified breast cancer patients treated with CDK4/6 inhibitors had worse mOS (41.6 m vs 56.3, HR 1.43, 95% CI 1.1-1.8, p=0.002). Moreover, TONSL amplified breast cancer patients had shorter mOS with palbociclib time on treatment (8.9 m vs 11 m, HR 1.31, 95% CI 1-1.6, p=0.01) compared to TONSL not amplified. Conclusions: TONSL amplification in ER+ breast cancer is associated with poor outcome and is potentially a biomarker of intrinsic resistance to CDK4/6 inhibitors. Since activity of TONSL is governed by multiple protein complexes, disruptors of TONSL-protein complexes could potentially be developed as sensitizers to CDK4/6 inhibitors. Citation Format: H. Nakshatri, A. S. Khatpe, U. R. Kamdar, G. Sandusky, S. K. Althouse, S. K. Deshmukh, S. Wu, N. Kulkarni, L. A. Poole, P. Coelho, M. B. Lustberg, G. W. Sledge, K. D. Miller, P. Bhat-Nakshatri. Tonsl, an immortalizing oncogene on chr.8q24.3 amplicon, confers intrinsic resistance to cdk4/6 inhibitors in breast cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-09-19.
Nakshatri et al. (Tue,) studied this question.