Mass Spectrometry of T47D cells treated with PTK6 PROTAC
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ABSTRACT: PTK6 PROTAC treatment of T47D breast cancer cells were analyzed with mass spectrometry for determining PTK6 PROTAC specificity and global proteomic changes.
Project description:PROteolysis TArgeting Chimeras (PROTACs) represent a novel therapeutic strategy that leverages the ubiquitin-proteasome system for targeted protein degradation. Aptamers, with their high specificity and binding affinity, have recently been explored as alternative recognition elements in PROTAC design. Here, we developed an aptamer-based PROTAC targeting SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), an epigenetic regulator implicated in breast cancer progression. The SETDB1-specific aptamer identified in our previous work was conjugated to a CRBN E3 ligase ligand via click chemistry, generating a serum-stable PROTAC, designated as P-SETDB1-4. P-SETDB1-4 effectively recruits CRBN to SETDB1, inducing proteasome-dependent degradation of SETDB1 in breast cancer cells. Consequently, P-SETDB1-4 significantly inhibits the proliferation and migration of breast cancer cells. Moreover, P-SETDB1-4 enhances the CD8+ T cells cytotoxicity against breast cancer cells and suppresses tumor growth in vivo. RNA sequencing analysis elucidates the molecular mechanism underlying P-SETDB1-4-mediated tumor suppression and promotion of CD8+ T cell-mediated killing. This study provides a promising therapeutic strategy for breast cancer and highlights the potential of aptamer-CRBNL PROTACs for targeting other challenging oncogenic proteins.
Project description:We identified differential gene expression after treatment with BRD4-PROTAC ARV771 in two ABC-like diffuse large B-cella lymphoma cell lines. We have identified cluster of gene expression regulated after BRD4 inhibition which are criticaly important for DLBCL malignancy.
Project description:The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation, and survival enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAFV600E have shown great efficacy in the clinic but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC termed P4B displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAFV600E cell lines. In addition, P4B displayed utility in two cell lines harboring alternate BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a rationale for optimizing the drug-like properties of P4B to enable proof of concept studies in vivo.
Project description:Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins which undermines the growth and survival of AML cells. However, BETi treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4, which potentially compromises the activity of BETi in AML cells. Unlike BETi, BET-PROTAC (proteolysis-targeting chimera) ARV-825 recruits and utilize an E3-ubiquitin ligase to effectively degrade BETPs in AML cells. BET-PROTACs induce more apoptosis than BETi of mtRUNX1 AML cells. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi. It was noted that treatment with BETi or BET-PROTAC caused significant and sustained depletion of RUNX1 in AML cells. We also determined the effects of global depletion of RUNX1 in mtRUNX1 expressing AML OCI-AML5 cells. We observed an overlap in the signature of RUNX1 knockdown by shRNA with that of OTX015 and ARV-825 in OCI-AML5 cells.
Project description:We developed a new METTL3 PROTAC, WD6305, which, unlike inhibitors, can reduce the expression of METTL3 protein. Here, we tested the gene expression after dosing by RNA-seq.
Project description:To validate the specificity of DT2216, we used the stable isotope labeling with amino acids in cell culture (SILAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics to analyze the changes in proteins in WI-38 normal human diploid fibroblasts after DT2216 and DT2216NC treatment. The results show that DT2216, but not DT2216NC, reduced the levels of BCL-XL, but none of other proteins were significantly affected by either agent, demonstrating that DT2216 is a specific BCL-XL PROTAC.
Project description:The objective of the study was to explore how a new compound (CEP1347-VHL-02 PROTAC) which facilitates the breakdown of mitogen-activated protein kinase kinase kinase 11 (MLK3), affects the protein quantities in the total cell extracts from specific breast cancer cell models. The cell lines used in the study (MCF-7, MDA-MB-468, and HCC1806, the last featuring inducible MLK3 expression) underwent treatment with the PROTAC or matched controls. The data is divided into three subsets, each representing results from a distinct cell line.
Project description:Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce selective protein degradation by linking an E3 ubiquitin ligase enzyme to a target protein. This approach allows scope for targeting “undruggable” proteins and several PROTACs have reached the stage of clinical candidates. However, the roles of cellular transmembrane transporters in PROTAC uptake and efflux remain underexplored. Here, we utilized transporter-focused genetic screens to identify the ATP binding cassette transporter ABCC1/MRP1 as a key PROTAC resistance factor. Unlike the previously identified inducible PROTAC exporter ABCB1/MDR1, ABCC1 is highly expressed among cancers of various origins and constitutively restricts PROTAC bioavailability. Moreover, in a genome-wide PROTAC resistance screen, we identified candidates involved in processes such as ubiquitination, mTOR signaling and apoptosis as genetic factors involved in PROTAC resistance. In summary, our findings reveal ABCC1 as a crucial constitutively active efflux pump limiting PROTAC efficacy in various cancer cells, offering insights for overcoming drug resistance.