Targeting MNK/GRP78 Pathway to Disrupt Proteostasis Rewiring in Castration-Resistant Prostate Cancer

Technical Abstract: Background: Prostate cancer (PCa) is a significant contributor to cancer-related mortality among men in the United States, with an estimated 299,101 new cases and 35,250 deaths expected in 2024 alone. While initial treatments for localized disease can be effective, the development of castration-resistant prostate cancer (CRPC) poses a formidable challenge. CRPC arises when cancer cells evolve to resist androgen deprivation therapy, including androgen signaling inhibitors (ASI). Current ASI therapies typically extend survival by only 2.5 to 5 months once resistance develops. This limited efficacy is primarily due to adaptive resistance mechanisms that emerge within PCa cells. These mechanisms involve intricate changes in cellular signaling pathways, particularly those governing protein homeostasis (proteostasis). Therapeutic interventions targeting the androgen receptor (AR) disrupt protein homeostasis, causing proteotoxic stress and cell death. However, surviving drug-tolerant cells must adapt to overcome imbalances in the proteostasis network to ensure their survival. The precise mechanisms by which PCa cells rewire their proteostasis networks to evade AR pathway inhibition and acquire resistance to ASI therapies remain poorly understood. By unraveling the intricacies of proteostasis reprogramming in ASI-resistant CRPC, new therapeutic strategies can be devised to target these adaptive pathways and enhance the efficacy of existing treatments. Hypothesis: This study hypothesizes that targeting the MNK/GRP78 signaling pathway can disrupt the rewired protein homeostasis network in ASI-resistant CRPC, thereby overcoming resistance and enhancing patient outcomes. The central hypothesis is that the MNK/GRP78 pathway is selectively reactivated in ASI-resistant tumors, restoring proteostasis. Disrupting this pathway could counter ASI resistance effectively. Specific Aims: 1. Dissect the Role of AR Signaling in Regulating Protein Homeostasis in PCa: Investigate how AR signaling impacts the protein homeostasis network, focusing on the unfolded protein response (UPR) pathway. Examine AR binding to promoter sequences of ATF6 and IRE1 genes and assess the effects of knocking down these genes on protein homeostasis and tumor suppression. 2. Decipher the Role of GRP78 Phosphorylation in Proteostasis Network Rewiring in ASI-Resistant PCa: Explore the mechanisms by which GRP78 phosphorylation at the S311 site activates the IRE1 and ATF6 branches of the UPR pathway, contributing to ASI resistance. Use site-directed mutagenesis, phospho-specific antibodies, and mouse models to evaluate the therapeutic potential of inhibiting these pathways. 3. Elucidate MNK-Mediated Phosphorylation of GRP78 in ASI-Resistant PCa: Identify the specific kinase responsible for GRP78 phosphorylation, focusing on MNK''s role. Conduct kinase array analysis and in vitro kinase assays to confirm MNK''s involvement and evaluate the efficacy of MNK inhibitors in preclinical models. Study Design: The study utilizes a combination of in vitro experiments and in vivo mouse models to explore how the MNK/GRP78 signaling pathway rewires the protein homeostasis network in ASI-resistant CRPC. Initially, luciferase reporter assays will assess AR binding to ATF6 and IRE1 gene promoter sequences. Subsequently, Chromatin Immunoprecipitation (ChIP) analyses will confirm AR binding under dihydrotestosterone (DHT) treatment conditions, with untreated cells and negative controls used to validate specificity. The study will then evaluate the therapeutic potential of inhibiting ATF6 and IRE1 branches in CRPC mouse models, including standard and enzalutamide-resistant models, using tumor growth and biomarker analysis as endpoints. Furthermore, the efficacy of MNK inhibition in overcoming enzalutamide resistance will be assessed in humanized mouse models, with vehicle-treated groups serving as controls to ensure the specificity of the MNK inhibitor effects. Additionally, the study involves employing site-directed mutagenesis to investigate GRP78 phosphorylation, generating phospho-specific antibodies, and utilizing kinase array analysis to identify the kinase responsible for GRP78 phosphorylation. Impact: The proposed research has the potential to significantly improve the treatment of CRPC by introducing new therapeutic strategies targeting the MNK/GRP78 pathway. In the near term, this could enhance the effectiveness of existing treatments and extend patient survival. In the long term, it aims to develop targeted therapies that improve quality of life and reduce mortality rates for CRPC patients. These outcomes align with the FY24 PCRP Overarching Challenges, ultimately benefiting Service members, Veterans, and all men affected by this disease.