Abstract 1559: Mechanisms responsible for hypersensitivity of small cell lung cancers to novel cyclin A and B RxL macrocyclic peptide inhibitors

Abstract Most cancer-directed targeted therapies are developed using small molecule inhibitors that require a druggable binding pocket on the target of interest. Macrocyclic peptides can selectively block protein-protein interactions required for function and hence can be used to target previously undruggable proteins. Using a structure-guided approach, Circle Pharma developed cell-permeable macrocyclic peptides that block the ability of Cyclin A and/or Cyclin B, to bind RxL motifs on their substrates thereby inhibiting Cyclin activity. A separate study (Gleason et al. AACR 2023) revealed that cancer cell lines with RB/E2F dysregulation were highly sensitive dual Cyclin A/B peptides, which includes most small cell lung cancer (SCLC) cell lines with RB1 and TP53 inactivation. Importantly, some cancer cell lines and all non-transformed cell lines examined were insensitive to Cyclin A/B inhibitors suggesting high potential for a therapeutic window. We found that dual Cyclin A/B inhibitors blocked cellular proliferation, caused accumulation of cells in mitosis, and robustly induced apoptosis in SCLC cell lines. In contrast, selective Cyclin A or Cyclin B inhibitors had significantly lower activity than dual Cyclin A/B inhibitors demonstrating that inhibition of both Cyclin A and Cyclin B is required to induce apoptosis in sensitive cells. Using these Cyclin A/B RxL inhibitors, we performed genome-wide CRISPR/Cas9 positive selection resistance screens and subsequent rigorous validation in SCLC cells. Our screen identified Cyclin B and CDK2 as the targets of Cyclin A/B inhibitors. Our screen also showed that MAD1 and the ROD-Zwilch-Zw10 (RZZ) complex of the mitotic spindle assembly checkpoint (SAC), which is regulated by the kinase Mps1, is required for Cyclin A/B inhibitors to induce apoptosis. In line with this, chemical inhibition of Mps1 caused resistance demonstrating that SAC activation by Mps1 is required for the ability of Cyclin A/B inhibitors to induce apoptosis. Interestingly, Cyclin A/B inhibitors selectively induced DNA damage measured by phospho-H2AX accumulation and SAC activation measured by Mps1 activity in highly sensitive RB1- and TP53-deficient SCLC cell lines while having no effect on DNA damage and SAC activation in resistant RB1- and TP53-proficient cell lines. Moreover, Cyclin A/B inhibitors also significantly induced phospho-H2AX accumulation in RB1- and TP53-proficient normal RPE1 cells and A549 cells only after dual CRISPR inactivation of RB1 and TP53. In summary, we found that novel cell-permeable dual Cyclin A/B RxL macrocyclic peptides selectively induce apoptosis in RB1/TP53-deficient cancer cell lines involving a mechanism where DNA damage induces SAC activation leading to apoptosis. Our data suggests that Cyclin A/B RxL peptide inhibitors should be further explored as a therapeutic strategy in RB1/TP53-deficient cancers such as SCLC. Citation Format: Shilpa Singh, Yavuz T. Durmaz, Catherine E. Gleason, Evelyn W. Wang, Rajinder Singh, David J. Earp, Pablo D. Garcia, Matthew G. Oser. Mechanisms responsible for hypersensitivity of small cell lung cancers to novel cyclin A and B RxL macrocyclic peptide inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1559.