Abstract LB296: Anti-cancer effect of Cyclin A/B RxL inhibitors is mediated in part by disruption of the ATR/Chk1 DNA repair pathway

Abstract Cyclin/CDK (Cyclin-Dependent Kinase) complexes regulate the activity of RB and E2F to drive cell cycle progression and replication fidelity. The binding of a subset of CDK substrates and regulators, including E2F, RB, and Myt1, to Cyclins requires the interaction of their RxL motif with the hydrophobic patch (HP) on the Cyclin. We generated cell-permeable, orally bioavailable macrocycles, that selectively bind to the HP of Cyclins A and B (Cyclin A/B RxLi) achieving at least two key effects in cancer cells: 1) disruption of the Cyclin A: E2F interaction that dampens E2F activity at the end of S phase, and 2) displacement of the negative regulator Myt1 from Cyclin B. Collectively Cyclin A/B RxLi increases E2F activity and activates Cyclin B/CDK, effects that ultimately lead to growth inhibition in vitro and induce tumor regressions in vivo in E2F high cancer models through enhanced DNA damage, spindle assembly checkpoint (SAC) activation and mitotic cell death (AACR 2023 #1559, #1550; and ENA 2024 #PB-515). We report here results that further our understanding of how Cyclin A inhibition by Cyclin A/B RxLi may contribute to replication stress and DNA damage. Small cell lung cancer cells exposed to Cyclin A/B RxLi exhibited induction of markers of replication stress and DNA damage and a significant increase of 4N cells incorporating EdU, a sign they are undergoing DNA damage repair in G2/M phase. Consistently, Cyclin A/B RxLi specifically blocked activation of Chk1 by ssDNA damage causing agents, whereas activation of Chk2 was unaffected, suggesting that a Cyclin A RxL-dependent interaction is required to activate the replication stress response pathway. To identify additional Cyclin A interactions involved in the DNA damage response, we performed co-IP experiments followed by LC/MS/MS in the SCLC cell line, NCI-H1048. Treslin, a dual replication/checkpoint protein that contains an RxL-motif, was displaced from Cyclin A by Cyclin A/B RxLi. Disruption of this interaction likely blocks Treslin phosphorylation, a modification required for TOPBP1 binding and ATR activation. These results provide further insights on the mechanism of Cyclin A/B RxLi. In addition to augmenting replication stress and DNA damage by hyperactivating E2F, disruption of the Cyclin A/Treslin interaction prevents DNA repair by blocking ATR/Chk1 activation. The combined effect of Cyclin A/B RxLi on E2F and Treslin, together with the activation of mitotic CDK by displacement of Myt1 from Cyclin B, drives the anticancer activity in E2F high SCLCs. Circle's Cyclin A/B RxLi, CID-078, has recently entered a multi-center phase 1 clinical trial (NCT06577987) for patients with solid tumors, including SCLC. Citation Format: Catherine E. Gleason, Ranya Odeh, Frances Hamkins-Indik, Iolanda Vendrell, Roman Fischer, Benedikt Kessler, Shilpa Singh, Matthew Oser, David J. Earp, Marie Evangelista, Pablo D. Garcia. Anti-cancer effect of Cyclin A/B RxL inhibitors is mediated in part by disruption of the ATR/Chk1 DNA repair pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB296.