Abstract PD3-04: Chromatin remodelling is involved in resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: Despite an undoubted benefit of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in estrogen receptor positive (ER+) breast cancer (BC), resistance eventually occurs. DNA-Sequencing studies identified genomic alterations that did not fully recapitulate the resistance landscape. Therefore, we explored whether changes in three-dimensional (3-D) chromatin landscape, potentially induced by single-nucleotide variants (SNVs) of non-coding regulatory regions, are involved in resistance to CDK4/6i. Methods: We generated ER+/HER2- palbociclib-resistant T47D and MCF7 BC cells (T47D-PR and MCF7-PR), by exposing parental cells to increasing doses of the drug until resistance occurred. In resistant and parental cells, we performed: 1) High-throughput Chromosome Conformation Capture (Hi-C), to investigate 3D chromatin remodelling associated with drug resistance; 2) Whole Genome Sequencing (WGS), to detect SNVs, insertions or deletions (InDels, 50 bp) acquired in PR cells; 3) ATAC-Seq to identify transcriptional factors (TFs) potentially leading to drug resistance and 4) RNA-Seq for gene expression. Results: Hi-C identified 2,189 differential interactions (loops) between T47D and T47D-PR cells (FDR 0.05). In detail, T47D-PR cells showed 844 loops gained and 1,345 loops lost. Similarly, we found 896 gained and 1,086 lost interactions in MCF7-PR cells. We intersected coordinates of differential loops with list of candidate enhancers and promoters from cis-Regulatory Elements by ENCODE. We found that ∼80% of all differential interactions in PR cells were enhancers, suggesting their involvement in transcriptional reprogramming. Consistently, analysis of integrated RNA-Seq and Hi-C data revealed that gained/lost loops were associated with changes in expression of their target genes in PR vs. parental cells. By WGS, in T47D-PR cells, we found 5,465 acquired SNVs and 5,248 InDels. Notably, 61.7% and 29.4% SNVs occurred at intergenic and intron genomic regions, respectively. Next, 2,695 (51.3%) and 2,137 (40.7%) InDels occurred at intergenic and intron regions, respectively. In MCF7-PR cells, we found 7,386 acquired SNVs and 5,206 InDels. More in detail, 4,602 (62.3%) and 2,102 (28.5%) SNVs occurred at intergenic and intron regions. Finally, 2,629 (50.5%) and 2,165 (41.6%) InDels occurred at intergenic and intron loci, respectively. We are investigating enrichment of acquired genomic alterations in anchors of loops, compared to the rest of the genome, in PR cells, thus influencing the 3D chromatin conformation. ATAC-Seq revealed 5,223 (LogFC -1) and 4,361 (LogFC 1) newly closed and open regions in T47D-PR vs. T47D cells, respectively. Next, in MCF7-PR vs. MCF7 cells, we found 948 and 1,625 newly closed and open regions, respectively. De novo HOMER motif analysis of newly open regions in T47D-PR and MCF7-PR cells, revealed enrichment for binding sites of FOS family members (such as Fos, Fosl1, Fosl2) and AP-1 transcription factor subunits among the top enriched motifs in both PR cells. We are testing whether therapeutic inhibition of AP-1 transcriptional complex might overcome resistance to CDK4/6i. Instead, motif analysis of newly closed regions revealed that FOXA1 was the top enriched motif in PR cells. Consistently, intersection of ATAC-Seq data with those from publicly available ChIP-seq datasets (Toolkit for Cistrome Data Browser) revealed enrichment for ERα binding sites and other ERα-interacting TFs, such as FOXA1, GATA3, and GREB1. Indeed, PR cells exhibited reduced sensitivity to fulvestrant and faster estrogen-independent growth, compared to parental cells. Conclusions: SNVs and chromatin remodeling are involved in resistance to CDK4/6i. Results from our study may help to identify novel therapeutic vulnerabilities in ER+ BC. Citation Format: F. Pepe, F. Messina, F. Napolitano, G. Nassa, A. Salvati, D. Memoli, C. Fierro, D. Esposito, S. Belli, C. Ascione, G. Attanasio, A. Vallefuoco, A. Benish, L. Formisano, R. Bianco, A. Servetto. Chromatin remodelling is involved in resistance to CDK4/6 inhibitors in ER+ 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 PD3-04.