Induced proximity approaches to rewire transcription
Keywords: Engineering.
Chemical Engineering.

Gene expression controls cell fate and the development of an organism. Transcription, the process of gene expression, is regulated by diverse proteins that interact with chromatin, the packaging of DNA in the nucleus of a eukaryotic cell. Synthetic control of transcription could help us rewire gene expression to generate specific cellular phenotypes, including cell proliferation or programmed cell death (apoptosis). Molecule-molecule proximity inside a cell has been shown to be sufficient for almost all biological processes, including post-translational modification, membrane receptor activation, epigenetic regulation, and transcription. Cell-permeable small molecules that localize two proteins in proximity, called Chemical Inducers of Proximity (CIPs), can be used to modulate biological processes, but their use has largely relied on the introduction of genetic modifications to the cell or organism, precluding their therapeutic application. In the first part of this work, and what I will focus on during this defense presentation, I develop CIPs that co-localize two endogenous transcriptional regulators, to modulate transcription in genetically unmodified cells or organisms in a locus-specific manner. I apply this technology, called "Transcriptional/epigenetic Chemical Inducers of Proximity" (TCIPs), to recruit an endogenous cancer driver, or downstream activator of oncogenic transcription, to instead activate the expression of genes that promote apoptosis. I focus on diffuse large B cell lymphoma, where pro-apoptotic genes are normally epigenetically repressed by the zinc-finger transcription factor BCL6 (B-Cell lymphoma 6). I develop a cascade of biochemical and cell biological assays to develop bivalent small molecules that covalently link together binders of BCL6 with binders of cancer-driving transcriptional activators, such as BRD4 (Bromodomain containing 4), which normally licenses expression of MYC and other oncogenes. The most potent compound, TCIP1, activates transcriptional elongation at BCL6-target, pro-apoptotic genes within 15 minutes, and kills DLBCL cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50s of 1 - 10 nanomolar within 72 hours, and show on-target efficacy in xenograft models of patient tumors. In measurements in 906 different cancer cell lines, primary human cells, and normal healthy mice, TCIP1 exhibits cell- and tissue-specific effects. The TCIP concept, of modulating endogenous transcription with chemical inducers of proximity, has therapeutic applications in other disorders where gene expression is dysregulated. TCIPs open the possibility of replacing many cell and gene therapies with less expensive, more convenient small molecules. To rewire transcription, a better understanding of the normal molecular grammar of transcriptional regulators is required. In the second part of this work, I explore the context-specific roles of ATP-dependent chromatin remodelers, focusing especially on the BAF complex, which is highly mutated in cancers and developmental disorders. One program of particular interest is synaptic activity in neurons, which is critical for proper circuit development. I map the minute-scale response of the BAF complex in response to synaptic activity in neurons, using proteomics, genomics, and acute chemical perturbations. The findings established for the first time a direct biochemical response of BAF complexes in responding to neural activity and provide a roadmap for further investigation of the synapse-to-nucleus pathway connecting BAF to neural activity. These studies establish how induced proximity can be used to rewire the molecular interactions of transcriptional regulators towards therapeutic ends and lend insight into the normal regulation of one class of transcriptional regulators, ATP-dependent chromatin remodelers.