New Macrocyclic Peptides Selectively Kill Chemotherapy-Resistant Lung Cancer Cells

Small-cell lung cancer (SCLC) is defined by near-universal loss-of-function mutations in RB1 and TP53, eliminating the G1-S cell cycle checkpoint and generating abnormally high E2F transcription factor activity. While E2F drives cell proliferation at normal levels, its hyperactivation paradoxically triggers apoptosis — a therapeutic vulnerability that has been difficult to exploit pharmacologically. This study, published in Nature, describes the development of first-in-class orally bioavailable macrocyclic peptides that block RxL-motif-mediated interactions between cyclins A and B and their substrates, providing a novel mechanism to selectively kill E2F-hyperactive cancer cells.

The research team used structural models based on cyclin A crystal structures (PDB: 1URC and 1JSU) to design macrocyclic peptides targeting the conserved hydrophobic patch on cyclins A and B that binds short linear RxL motifs on substrate proteins. Through iterative medicinal chemistry, the team developed lead compound cyclin A/Bi, a cell-permeable, passively absorbed macrocycle. Biochemical assays confirmed nanomolar-range inhibition of both cyclin A–E2F1 and cyclin B–MYT1 RxL interactions, with selectivity profiling confirming minimal off-target cyclin D or CDK kinase inhibition.

In viability screens across more than 50 cancer cell lines, cyclin A/Bi selectively killed SCLC lines and other cancer lines with high E2F transcriptional activity while showing substantially less toxicity to cancer lines with intact RB1 or low E2F activity, and minimal effect on normal lung epithelial cells. Genome-wide CRISPR genetic screens identified that cyclin A/Bi-induced apoptosis required cyclin B and CDK2 — but not CDK1 — as well as intact spindle assembly checkpoint (SAC) components including BUB1B and MAD2L1. Loss of these SAC genes strongly rescued cells from cyclin A/Bi-induced death, pointing to SAC activation as the critical killing mechanism.

Mechanistically, cyclin A/Bi was found to operate through two complementary gain-of-function effects. First, by blocking cyclin A's RxL-dependent phosphorylation and suppression of E2F1, the compound hyperactivated E2F target gene expression — including upregulation of cyclin B itself. Second, by blocking cyclin B's RxL interaction with its negative regulator MYT1 (which normally keeps cyclin B–CDK1 inactive at the G2-M checkpoint), free cyclin B accumulated and, strikingly, formed neomorphic cyclin B–CDK2 complexes not normally observed at physiological levels. These ectopic cyclin B–CDK2 complexes were shown biochemically and through proteomics to drive aberrant SAC activation, mitotic entry dysregulation, and ultimately apoptotic cell death. Importantly, this neomorphic complex formation was specific to cells with pre-existing high cyclin B levels driven by elevated E2F activity, explaining tumor selectivity.

In vivo, orally administered cyclin A/Bi demonstrated significant tumor growth inhibition in multiple chemotherapy-resistant SCLC patient-derived xenograft (PDX) models, with well-tolerated dosing regimens. These PDX models included tumors resistant to standard etoposide/platinum regimens, representing the clinical scenario of relapsed SCLC where no effective targeted therapy currently exists. The findings establish a proof-of-concept for RxL inhibition as a viable oncology strategy and identify a synthetic lethal interaction between G1-S checkpoint loss and cyclin A/B RxL inhibition across multiple cancer types beyond SCLC, including other RB1-deleted or CDKN2A-lost malignancies.