Photoproximity Chemoproteomic Platforms to Study Dynamic Protein Complexes In Situ

c-MYC is reported to be aberrantly expressed in over 70% of all cancers and is known to be a master transcriptional regulator in normal as well as diseased cellular states. Because c-MYC is difficult to target with high specificity using biologics or small molecule probes, proximity labeling platforms, such as PhotoPPI, offer a means to target and interrogate such proteins of interest (POI) with high precision. In this thesis, we discuss the development of the second-generation PhotoPPI platform (Spatial-PhotoPPI) for the tunable high-resolution mapping of dynamic PPIs of transcriptional regulator c-MYC proto-oncogene protein in live cells. Through novel modulation of the photoreactive properties of the o-nitroveratryl (NV) linker, we have developed next generation PhotoPPI probes which demonstrate a high or low degree of inner sphere labeling depending on the degree of linker reactivity. We have demonstrated more specific labeling preferences in-vitro and in live cells for the lead probe (AC1), making it well suited for the monitoring of nuclear processes. We used this platform to generate an interaction network of c-MYC under native conditions in live cells, revealing previously reported high confidence interactions such as protein MAX and HDAC3, as well as novel members of the bromodomain (BD) protein family (BRD3, BRD2 and PBRM1). In addition, previously undiscovered interactions such as MTA1, MTA2, TRIM33 and MCMBP, among others, were discovered. Because c-MYC is canonically viewed as 'undruggable', we have profiled the dynamic changes that occur to the c-MYC interaction network through inhibition of BD dependent transcriptional complexes using small molecule BD inhibitor JQ-1. This revealed expected and novel rewiring events with short and long treatments, thus validating indirect c-MYC inhibition through pharmacological targeting of BD transcriptional hubs. Interestingly, we observed several JQ1 independent BAF complex interactions (PBRM1 and SMARCC1) that persist in the presence of inhibitor, suggesting BD independent MYC transcriptional regulation that is explored further. Finally, we show that MYC-BAF complex interactions in SCLC are pharmacologically targetable using reported BAF inhibitors.