Frequent mutation of genes in the PI3K/AKT/mTOR signaling pathway in human cancers has stimulated large investments in over 40 small molecule therapeutic drugs but most have low efficacy in patients. As a result, cancers with high PI3K pathway activity such as triple-negative breast cancer (TNBC) are still treated primarily with conventional chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors with varying degrees of polyselectivity, we find that the preclinical compound Torin2 and related analogs are unusually effective because they inhibit both mTORC1/2 and structurally related PI3K-like kinases (PIKKs). The activity of Torin2 in TNBC cells is most closely mimicked by combining selective inhibitors of mTORC1/2 and Chk1, a kinase that acts downstream of the PIKKs ATR and DNA-PK in the signaling response to replication stress. Torin2 is more cytotoxic in TNBC/basal-like cell lines (N=19) than clinical grade mTORC1/2 inhibitors such as sapanisertib, but produces no cytotoxicity in non-transformed cells with basal-like gene expression (N=2). The high activity of Torin2 in TNBC is associated with greater activation of caspase-dependent apoptosis and stronger inhibition of cell proliferation than conventional PI3K/AKT/mTOR inhibitors, which do not inhibit PIKKs. Pulse-labeling studies with thymidine analogs and live-cell imaging of cells expressing fluorescent cell cycle reporters show that unlike other PI3K pathway drugs, Torin2 blocks progression of S phase, thereby causing accumulation of single-stranded DNA, DNA damage and death by replication catastrophe or mitotic failure. These phenotypes appear to result from co-targeting distinct pathways required during S phase in TNBC cells: inhibiting mTOR perturbs de novo biosynthesis and salvage of pyrimidines and purines, and inhibiting PIKKs causes high levels of replication stress and DNA damage. Moreover, computational models developed to better understand the complex cell cycle mechanism of action of Torin2 demonstrate the importance of co-targeting S-phase vulnerabilities for cytotoxicity. Thus, Torin2 and related analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC and possibly to other cancer cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.
Citation Format: Sameer S. Chopra, Annie Jenney, Adam Palmer, Mario Niepel, Mirra Chung, Caitlin Mills, Sindhu Carmen Sivakumaren, Qingsong Liu, Jia-Yun Chen, Clarence Yapp, Nathanael S. Gray, Peter K. Sorger. Torin2 and related analogs exploit replicative and checkpoint vulnerabilities to induce death of triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B103. doi:10.1158/1535-7163.TARG-19-B103