Abstract GS3-10: Partitioning of cancer therapeutics in nuclear condensates
Authors: Klein Isaac , Boija Ann , Afeyan Lena , Hawken Susana Wilson , Fan Mengyang , Dall'Agnese Alessandra , Oksuz Ozgur , Henninger Jonathan , Shrinivas Krishna , Sabari Benjamin , Sagi Ido , Clark Victoria , Platt Jesse , Kar Mrityunjoy , McCall Patrick , Zamudio Alicia , Mantiega John , Coffey Eliot , Li Charles , Hannett Nancy , Guo Yang , Decker Tim-Michael , Lee Tong , Zhang Tinghu , Weng Jing-Ke , Taatjes Dylan , Chakraborty Arup , Sharp Phillip , Chang Young Tae , Hyman Anthony , Gray Nathanael , Young Richard 
Published: February 2021

Abstract
The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. To test this idea, we focused our study on nuclear condensates reported in cell lines, demonstrated they occur in normal human and malignant breast cancer, and developed assays to test clinically active antineoplastic small molecule drugs relative to these condensates.To study the behavior of drugs within condensates, these were modeled in vitro with purified proteins and visualized by fluorescent confocal microscopy. We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. For each drug, the small molecule partitioned into the same condensate in vitro in which its established target resides in vivo. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration.Alkylating agents are a class of commonly used antineoplastic compounds, of which cisplatin is a prominent example. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Thus, we conclude that cisplatin preferentially modifies transcriptional condensate-associated DNA in cells, and that this causes selective condensate disruption. The mechanisms that produce drug resistance can provide clues to drug activity in the clinical setting. Investigating the behavior of tamoxifen within ER transcriptional condensates demonstrated that it disrupts these condensates in vitro and on oncogenes in cells; hormonal therapy resistant ESR1 mutations render these condensates resistant. MED1 overexpression, a poorly understood mechanism of tamoxifen resistance, increased the size of ER-MED1 condensates, thereby rending tamoxifen more dilute and ineffective when concentrated therein. This suggest that altering the size and nature of transcription condensates in breast cancer can mediate drug resistance in the clinical setting.Our results show that antineoplastic drugs partition selectively into condensates, that this can occur through physicochemical properties independent of their molecular targets, and that resistance to drugs may occur through condensate altering mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment.
Citation Format: Isaac Klein, Ann Boija, Lena Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan Henninger, Krishna Shrinivas, Benjamin Sabari, Ido Sagi, Victoria Clark, Jesse Platt, Mrityunjoy Kar, Patrick McCall, Alicia Zamudio, John Mantiega, Eliot Coffey, Charles Li, Nancy Hannett, Yang Guo, Tim-Michael Decker, Tong Lee, Tinghu Zhang, Jing-Ke Weng, Dylan Taatjes, Arup Chakraborty, Phillip Sharp, Young Tae Chang, Anthony Hyman, Nathanael Gray, Richard Young. Partitioning of cancer therapeutics in nuclear condensates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-10.