Dataset Information

Chemical and phosphoproteomics illuminate the cellular mode of action of AKT inhibitors

ABSTRACT: Due to its key role in dysregulated cellular signaling in cancer, AKT has been subject to intense drug discovery efforts leading to small molecule inhibitors investigated in clinical trials, notably breast cancer. To shed more light on how these drugs exert their therapeutic effects, we integrated chemoproteomic target affinity profiling using the kinobeads approach and phosphoproteomics for the five designated AKT inhibitors AZD5363, GSK2110183, GSK690693, Ipatasertib and MK-2206 in breast cancer cells. Kinobead analysis identified between four and 29 nanomolar targets for these compounds and revealed 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 among which 276 were regulated by all drugs. This analysis expanded the AKT signaling network by 119 phosphoproteins that may represent direct or indirect targets of AKT. Within this network, we found 41 regulated phosphorylation sites bearing the AKT substrate motif and recombinant kinase assays validated 16 as novel AKT substrates. These included CEP170 and FAM83H, which suggest a direct regulatory function of AKT in mitosis and cytoskeleton organization. In addition, a specific phosphorylation pattern on ULK1, FIP200, ATG13 and VAPB was found to represent an active state of ULK1, leading to elevated autophagy in response to AKT inhibition. And last, secretome analysis of AKT inhibitor-treated cells identified STC2 as a putative blood-based drug response marker that may be tested in patients in the future.

ORGANISM(S): Homo Sapiens

SUBMITTER: Julia Mergner

PROVIDER: PXD015188 | panorama | Fri Jan 21 00:00:00 GMT 2022

REPOSITORIES: PanoramaPublic

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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:Estrogen receptor positive breast cancer is the most prevalent form of breast cancer. Although a number of available drugs are highly effective at blocking estrogen mediated receptor activity, thousands of patients die every year from ER positive breast cancers because the disease progresses to a stage at which these drugs are no longer effective. Thus, it is crucial to establish a comprehensive understanding of the biology of the estrogen receptor (ER) in ER:positive breast cancers that progress despite hormone therapy, a gap in knowledge that remains a serious impediment to successful treatment of patients with ER positive breast cancer. A key question that must be answered is how the estrogen receptor retains the capacity to activate transcription in the absence or near absence of estrogen. We have found a partial answer to this question upon investigating the effect of amplification and overexpression of Wolf Hirschhorn Syndrome Candidate 1:Like 1 (WHSC1L1), a gene that is amplified in 15% of breast cancers that codes for a histone:lysine methyltransferase. WHSC1L1 lies in the 8p11:p12 amplicon, a region of gene amplification that is strongly associated with breast cancer. In this study, we performed shRNA knockdown of the catalytically inactive short isoform of WHSC1L1 in SUM44PE breast cancer cells and found that expression of the short isoform of WHSC1L1 is necessary for expression of the estrogen receptor in this highly ER:positive cell line. In addition, we found that the estrogen receptor binds chromatin extensively in the absence of exogenous estrogen, including several actively transcribed canonical ER target genes, indicating that estrogen receptor signaling is active in SUM44 cells in estrogen free conditions. These findings represent a novel model for ER biology in luminal B breast cancers harboring amplification of WHSC1L1 and provide insight into the mechanisms by which ER: positive breast cancers become unresponsive to SERMs or aromatase inhibitors.

Dataset Information

Chemical and phosphoproteomics illuminate the cellular mode of action of AKT inhibitors

Publications

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

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