Pyridine based MYC degraders truncate endogenous MYC proteins and shift the balance of the MYC proteome

MYC is a DNA binding transcription factor whose sustained dysregulation promotes the initiation and maintenance for numerous cancers. Small molecules directly binding and blocking the disordered MYC monomer from forming critical protein interactions have supplied numerous probes highlighting the complexities of inhibiting this monumental target and have helped legitimize MYC as a tractable therapeutic target. Unfortunately, most of the inhibitors that have shown promise in pre-clinical settings have not made significant advances towards practical clinical applications. The rise of proximity induced pharmacology has lent new opportunities to understand complex biological pathways and vulnerabilities to help target proteins like MYC where small molecule inhibition has proved challenging. We demonstrate the application of proximity inducing heterobifunctional PROteolysis TArgeting Chimeras (PROTACs) towards regulating concentrations of oncogenic MYC monomers derived from the pyridine scaffold of MYC inhibitor, KJ-Pyr-9. We found MTP3 depletes endogenous full-length MYC proteins and uniquely exacerbates levels of a functional, N-terminally truncated MYC species, tMYC. MTP3 destabilizes the MYC proteome in favor of a tMYC dominant cell state with a distinct regulatory landscape. Our results highlight the complexities of proximity-induced chemistry against highly regulated and dynamic protein targets like MYC and indicate that PROTACs can regulate alternative outcomes beyond target protein degradation. PC3 cells were routinely cultured in RPMI1640 supplemented with 10% FBS at 37oC, 90% RH, and 5% CO2. Cultures were sub-cultured twice a week (at ~80% confluence) and split 1:5. PC3 cells were seeded in 12 well plates to achieve 70% confluence and incubated over-night. The following day, adherent cells were washed with DPBS and supplemented with fresh media containing indicated treatments in 1.2mL final volume (MTP3 is a MYC Targeting PROTAC that truncates MYC proteins at the N-terminus. HO3395 is a MYC-binding small molecule used as a binding control in this study.). After 24 hours, treatment media was aspirated and cells were washed with DPBS. Cells were treated briefly using 0.05% trypsin*EDTA for 3 minutes at RT. Trypsin was neutralized using complete culture media and cells were harvested by scraping. Harvested cells were transferred to 1.5 mL Eppendorf tubes and pelleted for five minutes at 4oC with 125 x rcf. Resulting supernatants were aspirated by pipette and cells were washed using DPBS. PC3 cell pellets were snap frozen using liquid nitrogen and stored at -80oC until processing. Total RNA was extracted and purified from cells using Qiagen RNeasy Plus Mini Kit (Qiagen Cat#74134) and gDNA eliminated using supplied gDNA eliminator columns as instructed by manufacturer. RNA was quantified by DeNovix DS-11 Spectrophotometer. ~2 μg total RNA was provided to Azenta-Genewiz for library production and sequencing reactions prior to analysis