Dataset Information

Disruption of the ALK tumor cell proteome by concurrent inhibition of ALK and SRC kinases.

ABSTRACT: Precision oncology has revolutionized the treatment of ALK-positive lung cancer with targeted therapies. However, refractory tumors with compound mutations or diverse resistance mechanisms remain an unmet clinical need. In this study, we established mouse tumor-derived cell models representing the most common EML4-ALK variants in human lung adenocarcinomas and characterized their proteomic profiles. We demonstrated that Eml4-Alk variant 3 confers a worse response to ALK inhibitors, suggesting its role in promoting resistance. In addition, proteomic analysis of brigatinib-treated cells revealed the upregulation of SRC kinase, which is frequently activated in cancer. Co-targeting of ALK and SRC showed remarkable inhibitory effects on both ALK-driven murine tumor growth and ALK-patient-derived cells. This death mechanism is attributed to the profound perturbation of the (phospho)proteomic landscape, together with a synergistic suppressive effect on the mTOR pathway. Taken together, our study identifies the inhibition of ALK and SRC cells and may offer a promising strategy to overcome resistance mechanisms and improve clinical outcomes in ALK-positive lung cancer patients.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Marcel Schilling

LAB HEAD: Prof. Ursula Klingmüller

PROVIDER: PXD043012 | Pride | 2024-04-11

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	202_Report_Peptide_Report_Pivot.xls	Xls
	202_Report_Phospho_Report_2_Pivot.xls	Xls
	202_Report_Precursor_Report_Pivot.xls	Xls
	202_Report_Protein_Report_Pivot.xls	Xls
	220830_ConditionSetup.xls	Xls

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Publications

Concurrent inhibition of ALK and SRC kinases disrupts the ALK lung tumor cell proteome.

Diaz-Jimenez Alberto A Ramos Maria M Helm Barbara B Chocarro Sara S Frey Dario Lucas DL Agrawal Shubham S Somogyi Kalman K Klingmüller Ursula U Lu Junyan J Sotillo Rocio R

Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 20240319

Precision oncology has revolutionized the treatment of ALK-positive lung cancer with targeted therapies. However, an unmet clinical need still to address is the treatment of refractory tumors that contain drug-induced resistant mutations in the driver oncogene or exhibit resistance through the activation of diverse mechanisms. In this study, we established mouse tumor-derived cell models representing the two most prevalent EML4-ALK variants in human lung adenocarcinomas and characterized their p ...[more]

PMID: 38521003

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Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK interactome, phosphoproteome, phosphotyrosine interactome and proteome as outlined below. ALK Interactome and phosphoproteome: For each experiment, three SILAC conditions (‘Light’: Lys0,Arg0, ‘Medium’: Lys4,Arg6, ‘Heavy’: Lys8,Arg10) of NB1 cells were treated for 30 minutes with each of the following ALK-targeting small-molecule inhibitors: TAE684 (100 nM), crizotinib (500 nM), and LDK378 (250 nM). The experiments were performed as specified below: Experiment 1: Light: 500 nM crizotinib, Medium: 100 nM TAE684, Heavy: 0.1% DMSO Experiment 2: Light: 100 nM TAE684, Medium: 250 nM LDK378, Heavy: 0.1% DMSO Experiment 3: Light: 250 nM LDK378, Medium: 500 nM crizotinib, Heavy: 0.1% DMSO These three triple-SILAC experiments allowed the effect of each inhibitor to be measured in duplicates. ALK phosphotyrosine interactome: To identify phosphotyrosine-specific interaction partners of ALK we used a label-free mass spectrometry-based proteomics approach. Interacting proteins to the peptide sequences (phosphorylated and non-phosphorylated peptide) indicated in the table below were identified by a peptide pull-down assay and mass spectrometry. Peptide pull-downs were performed on NB1 cell lysate prepared from cells treated for 30 minutes with LDK378 (250 nM) or DMSO. Each peptide pull-down was performed on lysates from two biological replicates. Sample name (raw file) Gene Uniprot Site Peptide sequence A1 ALK Q9UM73 pY1078 ELQSPEpYKLSKLR A2 ALK Q9UM73 pY1092 RTIMTDpYNPNYSF A3 ALK Q9UM73 pY1096 TDYNPNpYSFAGKT A4 ALK Q9UM73 pY1131 GAFGEVpYEGQVSG A5 ALK Q9UM73 pY1278 GMARDIpYRASYYR A6 ALK Q9UM73 pY1282 DIYRASpYYRKGGS A7 ALK Q9UM73 pY1283 IYRASYpYRKGGSA A8 ALK Q9UM73 pY1359 RSPGPVpYRIMTQS A9 ALK Q9UM73 pY1507 RLWNPTpYGSWFTE A10 ALK Q9UM73 pY1584 RSGNVNpYGYQQQG A11 ALK Q9UM73 pY1604 APGAGHpYEDTILK A12 ALK Q9UM73 Y1078 ELQSPEYKLSKLR B1 ALK Q9UM73 Y1092 RTIMTDYNPNYSF B2 ALK Q9UM73 Y1096 TDYNPNYSFAGKT B3 ALK Q9UM73 Y1131 GAFGEVYEGQVSG B4 ALK Q9UM73 Y1278 GMARDIYRASYYR B5 ALK Q9UM73 Y1282 DIYRASYYRKGGS B6 ALK Q9UM73 Y1283 IYRASYYRKGGSA B7 ALK Q9UM73 Y1359 RSPGPVYRIMTQS B8 ALK Q9UM73 Y1507 RLWNPTYGSWFTE B9 ALK Q9UM73 Y1584 RSGNVNYGYQQQG B10 ALK Q9UM73 Y1604 APGAGHYEDTILK C3 IRS2 Q9Y4H2 pY675 SSRSDDpYMPMSPA C4 IRS2 Q9Y4H2 pY742 SPEDSGpYMRMWSG C5 IRS2 Q9Y4H2 pY978 RSPLSDpYMNLDFS C6 IRS2 Q9Y4H2 Y675 SSRSDDYMPMSPA C7 IRS2 Q9Y4H2 Y742 SPEDSGYMRMWSG C8 IRS2 Q9Y4H2 Y978 RSPLSDYMNLDFS Additional explanations to MS raw files: -1 extension indicates Control (DMSO lysate) replicate 1. -2 extension indicates LDK378 (treated lysate) replicate 1. -3 extension indicates Control (DMSO lysate) replicate 2. -4 extension indicates LDK378 (treated lysate) replicate 2. Proteome: The NB1 cell line proteome was measured by a label-free mass spectrometry-based approach. The analysis was performed in two biological replicates.