Dataset Information

Chemical phosphoproteomics sheds new light on the targets and modes of action of AKT inhibitors

ABSTRACT: Due to its important roles in oncogenic signaling, AKT has been subject to extensive drug discovery efforts leading to small molecule inhibitors investigated in advanced clinical trials. To better understand how these drugs exert their therapeutic effects at the molecular level, we combined chemoproteomic target affinity profiling using kinobeads and phosphoproteomics to analyses the five clinical AKT inhibitors AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib and MK-2206 in BT-474 breast cancer cells. Kinobead profiling identified between four and 29 nanomolar targets for these compounds and showed that AKT1 and AKT2 were the only common targets. Similarly, measuring the response of the phosphoproteome to the same inhibitors identified ~1,700 regulated phosphorylation sites, 276 of which were perturbed by all five compounds. This analysis expanded the known AKT signaling network by 119 phosphoproteins that may represent direct or indirect targets of AKT. Within this new network, 41 regulated phosphorylation sites harbor the AKT substrate motif and recombinant kinase assays validated 16 as novel AKT substrates. These included CEP170 and FAM83H, suggesting a regulatory function of AKT in mitosis and cytoskeleton organization. In addition, a specific phosphorylation pattern on the ULK1-FIP200-ATG13-VAPB complex was found to determine the active state of ULK1, leading to elevated autophagy in response to AKT inhibition.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Breast Cancer

SUBMITTER: Svenja Wiechmann

LAB HEAD: Bernhard Kuster

PROVIDER: PXD021836 | Pride | 2021-08-05

REPOSITORIES: Pride

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			Action	DRS
	02136_A01_P022305_B00_A00_R1.raw	Raw
	02136_A02_P022313_B00_A00_R1.raw	Raw
	02136_A03_P022321_B00_A00_R1.raw	Raw
	02136_A04_P022329_B00_A00_R1.raw	Raw
	02136_A05_P022337_B00_A00_R1.raw	Raw

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Chemical Phosphoproteomics Sheds New Light on the Targets and Modes of Action of AKT Inhibitors.

Wiechmann Svenja S Ruprecht Benjamin B Siekmann Theresa T Zheng Runsheng R Frejno Martin M Kunold Elena E Bajaj Thomas T Zolg Daniel P DP Sieber Stephan A SA Gassen Nils C NC Kuster Bernhard B

ACS chemical biology 20210323 4

Due to its important roles in oncogenic signaling, AKT has been subjected to extensive drug discovery efforts leading to small molecule inhibitors investigated in advanced clinical trials. To better understand how these drugs exert their therapeutic effects at the molecular level, we combined chemoproteomic target affinity profiling using kinobeads and phosphoproteomics to analyze the five clinical AKT inhibitors AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib, and MK-22 ...[more]

PMID: 33755436

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Project description:Pancreatic ductal adenocarcinoma is one of the most aggressive cancer types and is well-known for its general low intrinsic radiosensitivity. In order to resolve mechanisms of how PDAC cells carry out radioresistance, a combination of proteomics and phosphoproteomics of two radiosensitive as well as radioresistant murine PDAC cell lines upon exposition to radiation was performed. A high depth of (phospho)proteomic identifications with > 13000 confidently identified phosphorylation sites and > 7800 proteins was achieved. Measuring the response of the phosphoproteome upon radiation revealed >700 phosphorylation sites on >400 proteins which were regulated in all four cell lines independently of the sensitivity status. This analysis validated already known radiomarkers but also uncovered novel members of the radiation-dependent signaling network in PDAC cells that were shown to be exclusively based on protein phosphorylation but not protein expression. Among those radioresponsive phosphoproteins were ten novel ATM substrates, which were validated by in vitro kinase assays. Comparison of radiosensitive and -resistant cells revealed a complex regulation of apoptotic processes, while changes in expression of DNA repair proteins seemed to not play a pivotal role. Especially increased expression of NQO1 was found to be a potential mechanism enabling resistance by clearing harmful reactive oxygen species from the PDAC cells. Further analysis of the radioresistance-associated-phosphoproteome, which was especially enriched in phosphoproteins with cytoskeleton organizational function, uncovered increased Actin dynamics and FAK activity in radioresistant cells. Both likely lead to increased survival, migrational capacity and perturbations in chromatin condensation which could oppose radiation. Based on the displayed adaptions in cellular protein expression and signaling in resistant PDAC cells, pharmacological inhibition of NQO1 and FAK could be suggested to sensitize towards radiation.