Plk1 in Prostate Cancer Lineage Plasticity

Technical Abstract: Background: Since androgen receptor (AR) is essential for development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC), androgen signaling inhibitors (ASI) such as enzalutamide are becoming the first line treatment for CRPC. Unfortunately, enzalutamide treatment causes the transdifferentiation of luminal cells into basal cells, stem-like cells and neuroendocrine cancer (NEPC), resulting in enzalutamide resistance. Therefore, understanding PCa lineage plasticity will identify targets/pathways whose inhibition might potentially reverse treatment-induced transdifferentiation back to AR-dependent adenocarcinoma, rendering cells to be sensitive to enzalutamide again. The progression from CRPC to NEPC is mainly driven by transcription / epigenetic regulation. NEPC can be categorized by two subtypes based on overexpression of two neuronal transcription factors: ASCL1 (Achaete-Scute Family BHLH Transcription Factor 1) and Neurod1 (Neurogenic Differentiation 1). ASCL1, directly involved in neuronal lineage differentiation, is a pioneer transcription factor that binds to closed chromatin regions. Neurod1 drives activation of brain development pathways with the help from its co-transcription factors EBF3 and LHX8. EZH2 (enhancer of zeste homolog 2), the catalytic subunit of the Polycomb Repressive Complex (PRC2) that drives H3K27me3 formation and gene silencing, has a documented rule in NEPC as well. However, how ASCL1, Neurod1 and EZH2 are regulated to promote lineage plasticity is largely unknown. Hypothesis/Objective: We hypothesize that polo-like kinase 1 (Plk1)-mediated phosphorylation of ASCL1, Neurod1 and EZH2 drives lineage plasticity in PCa, and that inhibition of Plk1 reverses the neuronal transdifferentiation process back to AR-dependent adenocarcinoma, rendering cancer cells to be sensitive to enzalutamide again. Specific Aims: We plan to test our central hypothesis and, thereby, accomplish the objective of this application by pursuing the three aims. In Aim 1, we will examine how Plk1 regulates PCa lineage plasticity via transcription factors. The working hypothesis is that Plk1 phosphorylation of ASCL1 and Neurod1 leads to their activation, thus increased chromatin accessibility, elevation of transcription of the genes that drive stemness and neuronal differentiation, eventually promoting lineage plasticity. Specifically, we will 1) dissect how Plk1 phosphorylation of ASCL1 at T233 regulates lineage plasticity in PCa, and 2) examine whether lineage plasticity is enhanced by Plk1-associated Neurod1-S52 phosphorylation. In Aim 2, we will dissect how Plk1 phosphorylation of EZH2 contributes to lineage plasticity in PCa. We postulate that Plk1 also contributes to PCa lineage plasticity epigenetically via phosphorylation of EZH2 at T144, which likely due to either inhibition of tumor suppressors or activation of oncogenes involved in lineage plasticity. In Aim 3, we will determine whether Plk1 inhibition renders NEPC to be sensitive to enzalutamide. The working hypothesis is that inhibition of Plk1 plus enzalutamide is a novel approach to treat NEPC, as Plk1 inhibition reverses neuronal transdifferentiation back to AR-dependent adenocarcinoma. Study Design: For Aim 1, we will analyze how Plk1 phosphorylation of ASCL1 and Neurod1 regulates lineage plasticity with both cell culture model (LNCaP/AR/tet-sh-p53&Rb1) and a prostate reconstitution model. The state-of-art ATAC-seq, ChIP-seq and RNA-seq will be used to compare chromatin accessibility and gene expression of cells expressing different forms of ASCL1 (WT, T233A, T233D) and Neurod1 (WT, S52A, S52D). For Aim 2, we will compare phenotypes of LNCaP/AR/tet-sh-p53&Rb1 cells expressing different forms of EZH21 (WT, T144A, T144D) and perform single cell RNA-seq with prostates from genetically engineered mice (GEM) (TRAMP vs TRAMP; EZH2-T144A). For Aim 3, we will test if NMS-P937 plus enzalutamide can inhibit NEPC synergistically in both GEM and xenograft models. Impact: The proposed study is of high significance, because, if positive, the proposed research will suggest an immediate clinical trial to use NMS-P937 plus enzalutamide to treat NEPC patients. The study is highly innovative, as it provides a novel mechanism to explain how Plk1-associated activity regulates lineage plasticity, thus t-NEPC. With respect to expected outcomes, the work proposed is expected to demonstrate that Plk1- mediated phosphorylation of ASCL1, Neurod1 and EZH2 results in enhanced lineage plasticity, consequently promoting NEPC. Further, inhibition of Plk1 with NMS-P937 is expected to be render t-NEPC back to AR- dependent adenocarcinoma, thus be sensitive to enzalutamide again. The proposed research directly addresses two of the PCRP Overarching Challenges: develop new treatments or improve upon existing therapies to improve outcomes for men with lethal PCa; define the biology of PCa progression to lethal PCa to reduce death.