Abstract PD7-12: Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells
Authors: Guarducci C , Nardone A , Feiglin A , Migliaccio I , Malorni L , Bonechi M , Benelli M , Di Leo A , Hodgson G , Shapiro G , Brown M , Jeselsohn R 
Published: May 2019

Abstract
Background: Despite the efficacy of the combinations of endocrine therapy (ET) and CDK4/6 inhibitors in the hormone receptor positive (HR+) metastatic breast cancer (BC) setting, the majority of patients eventually acquire resistance to these treatments. The loss of Rb activity is an important mechanism of acquired resistance to CDK4/6 inhibitors. Preclinical and clinical data support the RB1 genetic aberrations, including mutations and deletions with loss of expression, as mechanism of palbociclib (palbo) resistance. To study vulnerabilities and potential treatment targets to overcome resistance to CDK4/6 inhibitors associated with Rb loss we studied a T47D palbo-resistant (T47D PDR) HR+ BC cell model obtained by exposing palbo-sensitive cells to increasing concentrations of palbo. These cells harbour deletion of RB1, and show increased cyclin E1 and decreased ER expression.
Methods: To identify genes that are essential for cell growth and genes whose loss enhances cell growth in palbo-resistant cells with loss of Rb, we performed genome-wide CRISPR-Cas9 knockout (KO) screens in T47D palbo-sensitive (T47D PDS) and T47D PDR cells. We searched for significantly essential genes that are targets with available drugs and tested the efficacy of these compounds in T47D PDS and T47D PDR cells.
Results: By employing a genome wide CRISPR KO screen, we identified 29 genes that were significantly positively selected in the T47D PDR cells. Among these genes were known tumor suppressor genes, such as TSC2 (β score: 0.89) and PTEN (β score: 0.68). We identified close to 600 genes that were significantly essential (negatively selected) for T47D PDR growth. CDK4, CDK6 and CCND1 were essential in the T47D PDS parental cells but lost essentiality with the acquisition of palbo resistance (FDR&gt; 0.05). CDK7 (β score: -1.21) and CDK2 (β score: -1.67) were among the top ranked essential genes (FDR: 0).
CDK7 is a transcriptional CDK and has CDK activating kinase (CAK) activity. The CAK activity includes phosphorylation of CDK2 and CDK9. To follow up on our CRISPR screen results we tested the activity of the selective CDK7 inhibitors SY-1365 (currently in phase 1 clinical development) and THZ1 in the T47D PDS and T47D PDR cells. We saw dose dependent activity in both cell line models. Moreover, the IC50 values were comparable in T47D PDS and T47D PDR cells (THZ1; PDS: 9.93 nM, PDR with Palbo: 32.8 nM, PDR without Palbo: 1.08 nM) (SY1365; PDS: 1.60 nM, PDR with Palbo: 6.70 nM, PDR without Palbo: 1.87 nM). Combination studies of SY-1365 and fulvestrant in T47D PDR showed synergistic activity at lower concentrations.
Conclusions: In a model of palbo resistance with loss of Rb we found that cyclin D1, CDK4 and CDK6 are not essential, suggesting cross-resistance to other CDK4/6 inhibitors in this setting. We identified CDK7 as a significant essential gene and potential therapeutic target. We validated these findings with the selective CDK7 inhibitors, THZ1 and SY-1365. Our results offer a new therapeutic strategy for the treatment of palbo-resistant HR+ BC.
Citation Format: Guarducci C, Nardone A, Feiglin A, Migliaccio I, Malorni L, Bonechi M, Benelli M, Di Leo A, Hodgson G, Shapiro G, Brown M, Jeselsohn R. Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-12.