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. Overall design: 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

MYC是一种DNA结合转录因子，其持续失调会促进多种癌症的发生与维持。能够直接结合并阻断紊乱状态的MYC单体形成关键蛋白质相互作用的小分子，已为解析该重大靶点的抑制复杂性提供了诸多探针，同时也证实了MYC是一个可靶向的治疗靶点。遗憾的是，多数在临床前研究中显示出潜力的抑制剂，在迈向实际临床应用的过程中并未取得显著进展。邻近诱导药理学（proximity induced pharmacology）的兴起，为理解复杂生物通路及易感位点提供了新机遇，助力靶向那些小分子抑制已被证实极具挑战性的蛋白质（如MYC）。本研究验证了邻近诱导型双功能蛋白降解嵌合体（PROteolysis TArgeting Chimeras，PROTACs）在调节源自MYC抑制剂KJ-Pyr-9吡啶骨架的致癌性MYC单体浓度方面的应用。我们发现MTP3可耗尽内源性全长MYC蛋白，且能特异性上调一种具有功能的N端截短型MYC亚型（tMYC）的水平。MTP3会扰乱MYC蛋白质组，促使细胞转向以tMYC为主导的细胞状态，并伴随独特的调控图谱。本研究结果凸显了针对MYC这类高度调控且动态变化的蛋白质靶点进行邻近诱导化学干预的复杂性，同时表明PROTACs可在靶标蛋白降解之外调控其他结局。
实验整体设计：
PC3细胞常规培养于添加10%胎牛血清（FBS）的RPMI1640培养基中，培养条件为37℃、90%相对湿度及5%二氧化碳。细胞每周传代两次（当汇合度约为80%时），按1:5的比例分瓶。将PC3细胞接种于12孔板中，使其汇合度达到70%，随后过夜培养。次日，贴壁细胞用磷酸盐缓冲液（DPBS）洗涤后，更换为含指定处理因素的新鲜培养基，终体积为1.2mL（注：MTP3是靶向MYC的PROTAC，可在N端截短MYC蛋白；HO3395是一种可结合MYC的小分子，本研究中将其用作结合对照）。处理24小时后，吸去处理培养基，用DPBS洗涤细胞。用0.05%胰蛋白酶-EDTA在室温下短暂处理细胞3分钟，用完全培养基中和胰蛋白酶后，通过刮取收集细胞。将收集的细胞转移至1.5mL离心管中，在4℃下以125×相对离心力（rcf）离心5分钟以沉淀细胞。吸去上清液后，用DPBS重悬洗涤细胞。将PC3细胞沉淀用液氮快速冷冻后，保存于-80℃冰箱直至后续处理。使用Qiagen RNeasy Plus Mini试剂盒（货号：Qiagen Cat#74134）从细胞中提取并纯化总RNA，并按照制造商说明书使用配套的基因组DNA（gDNA）去除柱去除gDNA。使用DeNovix DS-11分光光度计对RNA进行定量。取约2 μg总RNA送至Azenta-Genewiz公司进行文库构建及测序，后续再进行数据分析。