Project Summary – Project 2 Whitehead Institute at MIT There have been substantial advances in our understanding of the transcriptional control of cell states and how they are dysregulated in tumor cells. While investigating the transcriptional and epigenetic regulation of multiple myeloma (MM) cells in the previous funding period, our studies led to a new model for transcriptional control of cell state, one where the transcriptional and epigenetic apparatus becomes compartmentalized in biomolecular condensates and is regulated by biomolecules in a manner not anticipated by the canonical models of gene regulation (Figure 1). We discovered that the super-enhancers that drive oncogenes form large condensates that compartmentalize master transcription factors (TFs), epigenetic apparatus, regulatory RNA molecules and large numbers of RNA polymerase molecules to enable high rates of oncogene transcription. Furthermore, we found that newly transcribed RNA molecules make a profound contribution to local gene regulation both by directly binding to TFs and by altering the lifetime of the super-enhancer condensates. Importantly, we also discovered that these transcriptional condensates have chemical properties that concentrate the tested antineoplastic drugs over a hundred-fold, such that the pharmacological properties of the drug are altered in the vicinity of the drug’s target. Further insights into condensate physicochemistry should enable development of therapeutic molecules with improved efficacy and reduced toxicity. Based on these studies we propose to advance our understanding, in MM cells, of the regulation of transcriptional condensates and the biochemical environment in these condensates that influences drug behavior. To achieve these goals, the following Aims will be pursued: 1) To investigate the role of RNA binding by master transcription factors in MM cell state, 2) To investigate the role of RNA in regulation of oncogenic transcriptional condensates and DNA damage repair condensates in MM, and 3) To investigate the features of condensate chemistry that provide a specific chemical environment for enhancer-associated apparatus and that concentrate antineoplastic drugs. These proposed studies will advance our understanding of the regulation of transcriptional condensates and the biochemical environment in these condensates, and may thus enable development of novel therapeutic molecules with improved efficacy and reduced toxicity.