ABSTRACT Fusion oncoproteins (FOs) that are formed by chromosomal translocations are the primary drivers of many treatment-resistant cancers that afflict children in particular. A subset of FOs contain intrinsically disordered regions (IDRs) from transcription factors and other transcriptional co-regulators. Recent work has demonstrated that a number of transcription factors and co-regulators undergo liquid-liquid phase separation (PS) through interactions mediated by IDRs within their sequences and form transcriptional condensates. Genes at loci associated with these condensates are significantly upregulated, implicating PS in gene regulation and cell fate. We hypothesize that a subset of FOs enriched in IDRs form aberrant transcriptional condensates through PS, which results in aberrant gene expression and oncogenesis. Furthermore, I hypothesize that these FOs that form aberrant transcriptional condensates are enriched in IDR sequences that were not previously demonstrated to phase separate, and that these sequence features determine both the conformational and material properties of the FOs and the resulting condensates. Understanding the biophysical properties of FO-derived IDRs is important so that others can design therapeutics targeting the resulting condensates. To identify FOs that form condensates under physiological conditions, I selected 100 FOs based on patient-derived transcripts to transfect into mammalian cells as eGFP-tagged proteins. I will screen these 100 FOs for the formation of puncta that are indicative of PS using a semi-automated fluorescence confocal microscopy assay. To determine the IDR sequences responsible for PS, I will subclone IDRs derived from puncta-forming FOs into Escherichia coli expression vectors for overexpression and purification. I will use turbidity assays and confocal microscopy to assess whether these IDRs undergo PS and under what conditions. To probe the specific intermolecular interactions and identify potentially novel motifs, or “stickers”, that drive PS, I will use nuclear magnetic resonance (NMR) spectroscopy to measure changes in dynamics and intermolecular contacts for the IDRs upon PS. Additionally, I will relate sequence/sticker motif composition with the conformational and material properties of FO-derived IDRs. I will use NMR and fluorescence correlation spectroscopy to derive diffusion coefficients that report on the hydrodynamic radius of the IDRs inside and outside of condensates. I will further use SAXS to measure the radius of gyration outside of condensates and the intermolecular spacing within condensates. Finally, I will use fluorescence recovery after photobleaching to assess the fluid dynamics and material state of condensates. These complementary, multi-disciplinary studies will enable me to test the hypotheses discussed above and advance our understanding of the role of PS in the in vitro and cellular behavior of cancer-driving fusion oncoproteins.

PROJECT NARRATIVE The work described by this proposal will benefit the public by helping the biomedical community to understand the interactions driven by so-called fusion oncoproteins responsible for the formation of aberrant biomolecular condensates in cancer cells through the process of phase separation. This work will significantly increase the number of fusion oncoprotein sequences known to undergo phase separation and, more fundamentally, establish how amino acid sequence features determine their conformational behavior and interactions within condensates and how these behaviors influence the fluid properties of the condensates.