Abstract 5379: Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC)
Authors: Garcia Pablo D. , Gleason Catherine E. , Membreno Miles , Hamkins-Indik Frances , Situ Gavin , Levin Bernard , Wang Evelyn W. , Leung Siegfried , Fraga Breena , Bockus Andrew , Aggen James , Spellmeyer David , Earp David J. , Singh Rajinder 
Published: June 2022

Inhibition of substrate binding to Cyclin A has been postulated to be synthetically lethal in retinoblastoma (Rb) mutated cancers (Chen et al 1999). While compounds that target the cyclin-associated kinases are approved for clinical use, attempts to target the protein-protein interactions between cyclins and their substrates, such as E2F, have so far been unsuccessful. Using structure-guided design we have developed cell-permeable macrocycle compounds that selectively inhibit the RxL-mediated binding of substrates to Cyclin A.

We used NCI-H1048, an Rb-/- SCLC cell line, to study the effects of these Cyclin A targeting macrocyclic compounds on cell proliferation, apoptosis, and cell cycle regulation. Proliferation of H1048 cells was inhibited ~90% at 100nM of compound A whereas no inhibition was observed at up to 10 uM in WI-38 cells, a human non tumor lung fibroblast cell line, or with an inactive isomer of compound A in NCI-H1048 cells. Cyclin A inhibition in H1048 cells resulted in accumulation of cells at G2/M, an increased proportion of cells with 4C DNA content by FACS, hyperphosphorylation of RPA and upregulation of both phosphorylated histone H3 (pHH3) and phosphorylated FOXM1 (pFOXM1). Substantial apoptosis (10 to 20-fold) measured using caspase 3/7 activity was induced after just 4 hours incubation of H1048 cells with Cyclin A inhibitors. In vivo efficacy studies in a murine H1048 xenograft model showed reduction in tumor growth. We analyzed sensitivity to these compounds in tumor cell lines representing multiple indications and observed inhibition of cell proliferation at 1 uM or below in 6 out of 10 SCLC cell lines, where all of the responding cell lines were Rb dysfunctional. Sensitivity to these compounds in a larger panel of SCLC and other cancer cell lines is currently under investigation to further evaluate the broader utility of cyclin inhibition.

Macrocycle inhibitors that inhibit Cyclin A RxL-mediated substrate binding have demonstrated G2/M accumulation and apoptosis in Rb -/- SCLC in vitro consistent with synthetic lethality, and anti-proliferative effects in vitro and in vivo. Inhibiting cyclins is mechanistically distinct from strategies that inhibit cell cycle kinases, and is expected to be clinically useful, hence the compound series is being optimized for clinical development.

Citation Format: Pablo D. Garcia, Catherine E. Gleason, Miles Membreno, Frances Hamkins-Indik, Gavin Situ, Bernard Levin, Evelyn W. Wang, Siegfried Leung, Breena Fraga, Andrew Bockus, James Aggen, David Spellmeyer, David J. Earp, Rajinder Singh. Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5379.