Treating breast cancer by novel WEE1 inhibitors

Clinical development of new therapeutics for breast cancer to dampen the tumor-induced immune suppression and help "wake up" the immune system to fight cancer is warranted. Indeed, antibodies blocking the inhibitory signal triggered by the CTLA-4 and PD-1 on T-cells have emerged as potent means to fight the immunosuppressive milieu (1-4). Despite the impressive objective responses in cancer patients by targeting these immune checkpoint inhibitors, clinical benefits are yet modest. One potential explanation is that tumor cells employ a number of different mechanisms to promote immune escape in the context of breast cancer (5, 6), including the soluble factors (e.g. cytokines and metabolites) produced from the tumor, and infiltration of different immunosuppressive cells in the tumor microenvironment such as myeloidderived suppressor cells (MDSC) and T-regulatory cells (Treg) (7, 8). Novel therapeutic opportunity thus exists in targeting these immunosuppressive pathways. CD4+CD25+FoxP3+ Tregs, or other types of suppressive cells accumulating in the blood, and at tumor sites in most patients and animals with cancer suppress antitumor immunity, seem to be a major mechanism of tumor immune escape (8-11) and are particularly a crucial impediment to breast cancer immunotherapy (12). Several cancer immunotherapeutics targeting Tregs, including Treg depletion, are currently being tested in the clinic. Further, therapeutic benefit of immune-checkpoint blockade, such as anti-CTLA-4 therapy, could be attributed at least in part to depletion of tumor-infiltrating Tregs (13-15). Thus, optimal strategies need for establishment of reducing Tregs or impairing their suppressive activity in tumor, together with mobilizing and expanding antitumor effector T cells. WEE1 kinase phosphorylates and inactivates Cdk1/Cdc2-bound cyclin B, is a gatekeeper of the G2-checkpoint arrest for premitotic DNA repair (16). WEE1 is overexpressed in various cancer types (17, 18). Notably, preclinical studies (19-23) with breast cancer cell lines and animal models demonstrated that WEE1 inhibition using siRNA or small molecule inhibitors reduced cancer cell viability, decreased tumor burden, and increased survival, supporting the validity of WEE1 inhibition as a viable therapeutic target in breast cancer. Combination of DNA-damaging cancer therapy (such as radiotherapy and/or cytostatics) with WEE1 inhibition is a rational approach to push cancer cells in mitotic catastrophe resulting from premature entry into mitosis with unrepaired lethal DNA damage (17, 18). Despite the direct cytotoxic activity, little is known about the effect of WEE1 inhibition on host immune system. Interestingly, our preliminary data show that MK-1775 (AZD1775), a selective and potent pyrazolo-pyrimidine derivative inhibitor of WEE1 (24), blocks the generation and suppressive activity of Tregs while increasing the infiltration of antitumor CD8+ effector T cells in addition to its direct cytotoxic effect on cancer cells. These results have led to a novel hypothesis that MK-1775 administration may augment breast cancer immunotherapy by suppressing Tregs. In this proposal, we will characterize the phenotype and function of Tregs in the tumor microenvironment following MK-1775 treatment, and explore the clinical potential of WEE1 inhibitors using the MK-1775 combined with PD-1 blockade in our established breast tumor models. Our work will identify an unappreciated role of WEE1 inhibition in reversing tumor-induced immune suppression and points to a novel and feasible bre