Abstract P2-03-10: DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer
Authors: Nanda Sarmistha , Shea Martin J. , Schiff Rachel , Osborne C. Kent , Rimawi Mothaffar , Fu Xiaoyong 
Published: March 2023

Abstract
Background: The use of CDK4/6 inhibitors (CDK4/6i) has led to a remarkable progress in the treatment of advanced ER-positive (+) breast cancer (BC). The fact that both CDK4/6i and endocrine therapy (ET) target cell-cycle G1-S transition suggests some overlap of resistance mechanisms. However, clinical variability in response to CDK4/6i in patients progressing on ET suggests the involvement of additional or unique resistance mechanisms. Using bioinformatics analyses of clinical and preclinical data, we now identified genes involved in DNA replication licensing, in particular, excess MCM2 as a new mechanism of resistance to CDK4/6i. Methods: Gene expression data from the neoadjuvant NeoPalAna trial of the CDK4/6i palbociclib (Palbo) and the aromatase inhibitor (AI) anastrozole in ER+ BC was downloaded from Gene Expression Omnibus (GSE93204). Response to AI and Palbo was obtained using the published Ki67 immunohistochemistry data and cutoff on biopsies post treatment. Gene Set Enrichment Analysis comparing tumors with different sensitivity to AI vs. Palbo was performed using the Gene Ontology related to DNA replication, DNA repair, and DNA damage checkpoint. The DNA replication gene set was further sub-assigned by their selective role in origin licensing, firing, elongation, replication repair, and checkpoint regulation. A series of bioinformatics analyses was applied on five ER+/HER2-negative BC cell lines with gene expression and DepMap (from CCLE) and Palbo sensitivity data (from GDSC). Two ER+ BC cell models (MCF7 and T47D) and their estrogen deprivation (mimicking AIs)-resistant (EDR) derivatives were used for further studies including RNA-seq, Palbo sensitivity (data from PMID: 33536276), and chromatin fractionation assays. Results: We found that the DNA replication-associated gene set, comprising &gt; 80% genes outside the Rb-loss gene signature, was significantly enriched in baseline biopsies of Palbo-resistant (PalboR) tumors from patients in the NeoPalAna trial. Of the DNA replication genes, the subset of genes involved in origin licensing were preferentially enriched in PalboR vs. AI-resistant tumors. Similarly, the enrichment of genes involved in replication initiation was also seen in ER+/HER2-negative BC cell lines with a decreased response to Palbo. Notably, these PalboR cell lines showed a reduced vitality to shRNA knockdown of the replication initiation genes compared to randomly selected other genes or the Rb-loss gene set. Based on the modeling of DepMap gene dependency score and Palbo sensitivity, we nominated minichromosome maintenance 2 (MCM2), the key origin licensing factor, as the top gene that is essential for PalboR cell survival. Additionally, using our previously reported MCF7-EDR and T47D-EDR cell models with increased or decreased sensitivity to Palbo compared to their parental lines, respectively, we observed a corresponding decrease or increase in the expression of origin licensing genes and MCM2 in EDR vs. parental lines. Decreased Palbo sensitivity in the EDR cells was associated with sustained MCM2 chromatin loading and reduced expression of genes including the cyclin-dependent kinase inhibitor p21. Ongoing studies investigate whether elevated MCM2 levels confer resistance to CDK4/6i by resolving replication stress. Conclusions: Our strategic bioinformatics analyses reveal that excess DNA replication licensing is associated with CDK4/6i resistance in clinical and preclinical settings upon resistance to estrogen deprivation. Among the replication initiation-associated genes, MCM2 plays a potential role in conferring CDK4/6i resistance via sustaining origin licensing and suppressing p21. Our study provides a new avenue via lens of replication licensing to explore novel mechanisms and therapeutic opportunities in CDK4/6i resistance.
Citation Format: Sarmistha Nanda, Martin J. Shea, Rachel Schiff, C. Kent Osborne, Mothaffar Rimawi, Xiaoyong Fu. DNA replication licensing is associated with resistance to CDK4/6 inhibitors in ER-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-10.