Immune control of triple negative breast cancer by WEE1 inhibition

Background: Triple negative breast cancer (TNBC), i.e. estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative, is the most aggressive type of breast cancer with a high propensity for metastasis, and poor prognosis. Treatment options for TNBCs are limited because they are usually insensitive to most available hormonal or targeted therapeutic agents owing to their triple negative nature. As breast cancer is able to induce immune responses, targeting the immune system is an encouraging strategy for its treatment. In particular, TNBC appears highly immunogenic because of a significant accumulation of tumor-infiltrating lymphocytes (TILs), and a higher mutational burden that can produce immunogenic neoantigens. Exciting clinical results from phase I trials using checkpoint inhibitors directed against PD-1/PD-L1 have been reported. However, only a subset of patients (20%) respond to anti-PD-1/PD-L1 treatment. Therefore, identifying rational combinations is needed to enhance the activity of immune checkpoint blockade in TNBC. WEE1 kinase phosphorylates and inactivates CDK1/Cdc2-bound cyclin B, is a gatekeeper of the G2-checkpoint arrest for premitotic DNA repair. Importantly, through a high throughput screening using a kinase library containing 704 kinases against TNBC cells, it has been shown that WEE1 inhibition sensitizes TNBCs and cisplatin resistant cancer cells to cisplatin-induced lethality, supporting the validity of WEE1 inhibition as a viable therapeutic target in TNBC. Despite the direct genotoxic activity, the extent of WEE1 functions beyond cell-cycle regulation in tumor cells remains largely unknown. Interestingly, our preliminary data show higher expression levels of WEE1 in human TNBC than other BC, and targeting WEE1 kinase activity using MK-1775 (AZD1775), a selective and potent pyrazolo-pyrimidine derivative inhibitor inhibits the growth of established 4T1 TNBC, and has an extra potential to modulate intra-tumoral Treg levels, promoting CD8+ T cell anti-tumor immunity. Further, knockdown of WEE1 expression in cancer cells is sufficient to reduce tumor Treg infiltration while increasing the IFN- production from antitumor CD8+ effector T cells. Notably, WEE1 inhibition directly suppresses the in vitro generation and suppressive function of both human and mouse Tregs. These data suggest a robust potential of WEE1 inhibitors as immunotherapeutic reagents by reversing Treg-mediate immune suppression besides its direct genotoxic effect on tumor cells. Overarching challenge: Chemotherapy is still the primary established treatment for TNBC, while the therapeutic effect is not optimal due to frequently occurred drug resistance. Thus, it is urgent to develop more effective approaches to treatment for TNBC. Given the high immunogenicity of TNBC, we here propose a novel strategy to enhance efficacy of immunotherapy using DNA replication checkpoint WEE1 inhibition. Hypothesis: Based on the above rationale and preliminary data, we hypothesize that WEE1 maintains an immunosuppressive and pro-tumorigenic microenvironment, and suppressing WEE1 activity, including via a clinically relevant WEE1 inhibitor, may be therapeutically beneficial by boosting immune-mediated TNBC clearance in combination with immune checkpoint inhibition. Our specific Aims: Aim 1. To determine the immunomodulatory effects of WEE1 expression in tumor cells. Aim 2. To determine the immunomodulatory effects of WEE1 expression in T cells. Aim 3. To determine the therapeutic efficacy of WEE1 inhibition in combination