Development of Rational Combination Therapies for Patients with Metastatic Prostate Cancer and Loss of the PTEN Tumor Suppressor Gene

Abstract

Metastatic castration resistant prostate cancer (mCRPC) is a deadly disease with no cure. The current treatments by blocking androgen signaling eventually fail because of cancer cells that become resistant. One of many ways for prostate cancer cells to become resistant, is by increasing the amount of androgen receptor and its activity. In almost half of mCRPC patients, a tumor suppressor PTEN is lost, leading to increased signaling in PI3K/AKT pathway that promotes tumor growth and survival. Drugs that block PI3K/AKT can be effective in patients with PTEN-loss, but with a small clinical benefit because cancer cells can adapt and rely on other pathways as a compensation to survive. For example, breast cancer cells rely on MAP2K4 pathway to compensate the effect of drugs blocking PI3K and thus become resistant. Researchers also showed that fueling the signaling pathways that are already highly active can kill cancer cells. This strategy is referred to as the overactivation of oncogenic signaling. Since the majority of PTEN-loss prostate cancer cells already exhibit highly active AKT pathway and AR signaling, deliberately activating these pathways even further may kill prostate cancer. Therefore, this study aims to find other drug combination that can improve the effectiveness of the AKT blocker called Ipatasertib. We also focus on developing new combination treatments that works specifically with overactivation of oncogenic signaling in PTEN-loss mCRPC. In our study, we conducted experiments to test the fitness of the cells when treated with potential drug combinations. We showed that Ipatasertib in combination with MAP2K4 blocker called HRX-0233 is synergistic, meaning that the combination is much more effective than the sum of their individual effects in prostate cancer cells especially those that lack androgen receptor. To gain a deeper understanding of this synergy, we investigated the levels of activated proteins in the relevant pathways, namely mTORC1 and MAPK pathway, and found that the combination can cooperatively inhibits both pathways more than the individual drugs. In search of other possible combinations, we performed so-called CRISPR knockout screen experiment where we turned off genes one by one and looked for cells that do not survive the treatments with Ipatasertib or HRX-0233. Our main findings revealed that cells without cyclin D1 are vulnerable to Ipatasertib. We confirmed this finding using Palbociclib, a drug that indirectly block the activity of cyclin D1 in combination with Ipatasertib. We showed that Palbociclib can minimally increase sensitivity to Ipatasertib in PTEN-null prostate cancer cells. Additionally, we performed another CRISPR knockout screen experiment to identify vulnerable targets to the overactivation of oncogenic signaling using the drugs called LB-100 and synthetic androgen R1881. LB-100 can promote activation of AKT pathway, while synthetic androgen R1881 can promote AR signaling. Our results from the screen uncovered many candidate genes that when turned off can sensitize cells LB-100 and R1881. Overall, this research highlights the potential of combining Ipatasertib with HRX-0233 as a new therapy for AR-negative prostate cancer and identifies multiple genes that can be targeted when oncogenic signaling is hyperactivated.