One in eight men in the United States are estimated to develop prostate cancer (PCa) during their lifetime. The main oncogenic driver of PCa is the androgen receptor (AR), a ligand activated transcription factor that contains three domains: a large intrinsically disordered N-terminal domain (NTD), a DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). We recently showed that the full-length AR forms nuclear condensates upon androgen stimulation in PCa models. These condensates colocalize with players of the transcriptional machinery and promote the expression of oncogenic programs. We also discovered that in androgen-dependent models, the treatment-resistant AR splice variant V7 (AR-V7), which lacks the LBD, does not form condensates. Nevertheless, in castration-resistant models, AR-V7 condensates form independently of androgens. In this study, we explore the impact of mutating or truncating critical residues or regions associated with AR's transcriptional activity on its capacity to form nuclear condensates in LNCaP cells. We also employ various bioinformatics tools to predict additional AR residues and sequences with high likelihood of undergoing phase separation. Consequently, we generate these predicted truncations and mutations, assessing their potential to form condensates in LNCaP cells. Additionally, we examined the effects of MED1 phosphorylation and treatment with various inhibitors targeting distinct AR domains on the ability of the full-length and AR-V7 proteins to form droplets in vitro and in PCa cells. Our objective is to deepen our comprehension of the factors driving AR condensate formation in PCa. By doing so, we aim to elucidate this novel mechanism of transcriptional regulation and identify novel therapeutic options for patients with advanced stages of PCa disease.

Abstract In The United States of America 1 in 8 men will develop prostate cancer (PCa) in their lifetime. The assembly of phase-separated condensates containing players of the transcriptional machinery such as transcription factors, coactivators (e.g. MED1), and RNA polymerase II enhances the transcription of key oncogenes in various cancer including PCa. Many proteins undergoing phase-separation possess intrinsically disordered regions (IDR) that mediate multivalent intra- and inter-molecular interactions, essential for condensate formation. We recently demonstrated that full-length AR, the main oncogenic driver in PCa, is more prone to form nuclear transcriptional condensates upon androgen stimulation in PCa models than in benign epithelial prostate models. In this study, we investigated the effects of mutating or truncating key residues or regions involved in AR transcriptional activity on its ability to form nuclear condensates in LNCaP cells as visualized by confocal microscopy. We also used various bioinformatics tools to predict additional AR residues and sequences with high propensity for phase-separation. We thus made various truncations and mutations and evaluated their ability to form condensates in LNCaP cells. We also tested the effect MED1 phosphorylation and treatment with various inhibitors targeting different domains of the AR on the ability of full-length protein to form droplets in vitro. We hope by better understanding what drive AR condensates formation in PCa, to elucidate this new mechanism of transcriptional regulation and to identify new therapeutic avenues for patients with advanced forms of the disease. Citation Format: Nicholas C. Pinette, Shabnam Massah, Sofia Kochkina, Fan Zhang, Maitree Biswas, Joseph Lee, Jörg Gsponer, Nada Lallous. Characterization of androgen receptor properties and interactome in mediating transcriptional condensates in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5292.

ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins in prokaryotic organisms. Much is now understood about the structure of these transporters and many reviews have been written on that subject. In contrast, less has been written on the assembly of ABC transporter complexes and this will be a major focus of this book chapter. The complexes are formed from two cytoplasmic subunits that are highly conserved (in terms of their primary and three-dimensional structures) across the whole family. These ATP-binding subunits give rise to the name of the family. They must assemble with two transmembrane subunits that will typically form the permease component of the transporter. The transmembrane subunits have been found to be surprisingly diverse in structure when the whole family is examined, with seven distinct folds identified so far. Hence nucleotide-binding subunits appear to have been bolted on to a variety of transmembrane platforms during evolution, leading to a greater variety in function. Furthermore, many importers within the family utilise a further external substrate-binding component to trap scarce substrates and deliver them to the correct permease components. In this chapter, we will discuss whether assembly of the various ABC transporter subunits occurs with high fidelity within the crowded cellular environment and whether promiscuity in assembly of transmembrane and cytoplasmic components can occur. We also discuss the new AlphaFold protein structure prediction tool which predicts a new type of transmembrane domain fold within the ABC transporters that is associated with cation exporters of bacteria and plants.