A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment
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PROVIDER: PRJNA434649 | ENA |
REPOSITORIES: ENA
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Project description:The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6μM PCI-34051 or 10μM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05μM (ALK-amplified) to 0.8μM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n=88) and the German Neuroblastoma Trial cohort (n=649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.
2018-02-21 | GSE110817 | GEO
Project description:The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3-6?µM PCI-34051 or 10?µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05?µM (ALK-amplified) to 0.8?µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n?=?88) and the German Neuroblastoma Trial cohort (n?=?649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.
| S-EPMC6261943 | biostudies-literature
Project description:Mutations in the tyrosine kinase domain of the Anaplastic Lymphoma Kinase (ALK) oncogene in neuroblastoma occur most frequently at one of three hotspot amino acid residues, with the F1174* and F1245* variants conferring de novo resistance to first and second generation ALK inhibitors including crizotinib and ceritinib. Lorlatinib, a third generation ALK/ROS inhibitor, overcomes de novo resistance and induces complete tumor regressions in patient-derived xenograft (PDX) models unresponsive to crizotinib. Lorlatinib has now completed Phase 1 testing in children and adults with relapsed/refractory ALK-driven neuroblastoma, and entered pivotal Phase 3 testing within the Children’s Oncology Group. To define mechanisms underlying the superior activity of lorlatinib, we utilized a chemical proteomics approach to quantitatively measure functional kinome dynamics in response to the ALK inhibitors lorlatinib and crizotinib, in clinically relevant ALK-driven neuroblastoma PDX models. Lorlatinib was a markedly more potent inhibitor of ALK and preferentially downregulated several kinases implicated in G2/M cell cycle transition compared to crizotinib. Lorlatinib treatment also led to the repression of MYCN expression and its occupancy at promoters of the same G2/M kinases. These data providing mechanistic insight in neuroblastoma into the far improved efficacy of lorlatinib over crizotinib for the treatment of ALK-driven neuroblastoma.
2024-07-29 | GSE273349 | GEO
Project description:Anaplastic Lymphoma Kinase (ALK) most frequently mutated in neuroblastoma (NB) and is atractive molecular target for therapy. However, efficacy of the ALK inhibitor against ALK-amplified NB is unclear. To elucidate genetic alterations induced by treatment of the ALK inhibitor, we compared expression profile between ALK inhibitor-treated and DMSO-treated NB39nu cells using Agilent SurePrint G3 Human GE 8x60K V2 Microarray Kit
2017-11-28 | GSE107354 | GEO
Project description:High anaplastic lymphoma kinase (ALK) protein levels may be correlated with an unfavorable prognosis in neuroblastoma (NBL) patients, regardless of ALK mutation status. We therefore examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated (MUT) and wild type (WT) NBL cell lines. Six of the nineteen NBL cell lines had a point mutation and four an amplification of the ALK gene. ALK amplified cell lines showed similar ALK levels and ALK inhibitor sensitivity as WT cell lines and were therefore co-analyzed. The ALK mRNA (p=0.043), ALK 220 kDa (p=0.009) and ALK 140 kDa (p=0.025) protein levels were higher in ALK mutant (n=6) than WT cell lines (n=13). ALK mRNA and protein levels significantly correlated with ERK1 and ERK2 protein levels, and also with PHOX2B mRNA levels, a neural differentiation marker which is mutated in NBL. Response to ALK inhibitor TAE684 was also significantly correlated with ALK levels. ALK mutant cell lines (n=4) demonstrated a higher sensitivity towards ALK inhibitor TAE684 (14.9 fold more sensitive, p=0.004) than eight WT cell lines. These results underline the importance of ALK mutations but also ALK levels for response to ALK inhibitors in NBL cell lines. Furthermore, the strong correlation of PHOX2B and ALK suggests that neural differentiation stage may be correlated with ALK levels in neuroblastoma. These data will enhance understanding of ALK inhibitor response in future patient trials. To study differentially expressed genes in the neuroblastoma cell lines described in the protein analysis study above, 15 cell lines and 2 derivative cell lines were used.
2011-07-07 | E-GEOD-22771 | biostudies-arrayexpress
Project description:High anaplastic lymphoma kinase (ALK) protein levels may be correlated with an unfavorable prognosis in neuroblastoma (NBL) patients, regardless of ALK mutation status. We therefore examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated (MUT) and wild type (WT) NBL cell lines. Six of the nineteen NBL cell lines had a point mutation and four an amplification of the ALK gene. ALK amplified cell lines showed similar ALK levels and ALK inhibitor sensitivity as WT cell lines and were therefore co-analyzed. The ALK mRNA (p=0.043), ALK 220 kDa (p=0.009) and ALK 140 kDa (p=0.025) protein levels were higher in ALK mutant (n=6) than WT cell lines (n=13). ALK mRNA and protein levels significantly correlated with ERK1 and ERK2 protein levels, and also with PHOX2B mRNA levels, a neural differentiation marker which is mutated in NBL. Response to ALK inhibitor TAE684 was also significantly correlated with ALK levels. ALK mutant cell lines (n=4) demonstrated a higher sensitivity towards ALK inhibitor TAE684 (14.9 fold more sensitive, p=0.004) than eight WT cell lines. These results underline the importance of ALK mutations but also ALK levels for response to ALK inhibitors in NBL cell lines. Furthermore, the strong correlation of PHOX2B and ALK suggests that neural differentiation stage may be correlated with ALK levels in neuroblastoma. These data will enhance understanding of ALK inhibitor response in future patient trials.
2011-07-07 | GSE22771 | GEO
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.
2018-11-21 | PXD006404 | Pride
Project description:Neuroblastoma is an embryonal neoplasm that remains of dramatic prognosis in its aggressive forms. Activating mutations of the ALK tyrosine kinase receptor have been identified in sporadic and familial cases of this cancer. We generated knock-in mice carrying the two most frequent Alk mutations observed in neuroblastoma patients. We used microarrays to detail the global programme of gene expression underlying the impact of ALK mutations on neuroblastoma formation in a MYCN amplified background. We selected several murine neuroblastoma tumors for RNA extraction and hybridization on Affymetrix microarrays. We generated three groups of tumors: 10 MYCN amplified tumors, 11 MYCN amplified/ALK F1174L tumors and 10 MYCN amplified/ALK R1275Q tumors.
2014-06-24 | E-GEOD-46583 | biostudies-arrayexpress
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 phosphoproteome upon siRNA depletion of ALK and upon ALK inhibition using the ALK-targeting small-molecule inhibitor lorlatinib. For quantitative phosphoproteomics we used a tandem mass tag (TMT)-based approach. Conditions for the TMT 11-plex setup is specified below. For each siRNA depletion experiment, NB1 cells were treated with siRNA (80 nM; as specified below) for 48 hours prior to stimulation with 0.1% DMSO for 30 minutes. For inhibitor treatment, NB1 cells were treated for 30 minutes with either 10 microM or 10 nM lorlatinib. The experimental treatment conditions and TMT11-plex labeling are specified below: 126: siControl replicate 1, 0.1% DMSO 127N: siControl replicate 2, 0.1% DMSO 127C: siControl replicate 3, 0.1% DMSO 128N: siALK sequence 1, 0.1% DMSO 128C: siALK sequence 2, 0.1% DMSO 129N: siALK mix of sequence 1 and 2, 0.1% DMSO 129C: 10 microM lorlatinib replicate 1 130N: 10 microM lorlatinib replicate 2 130C: 10 microM lorlatinib replicate 3 131N: 10 nM lorlatinib replicate 1 131C: 10 nM lorlatinib replicate 2
2018-11-21 | PXD009477 | Pride
Project description:The goal of this study is to identify the mechanism for how ALK promotes tumorigenesis in neuroblastoma. We developed a human stem cell model of neuroblastoma and generated isogenic tumors using MYCN and MYCN/ALK. RNAseq analysis of both tumors revealed an enrichment in focal adhesion signaling and allowed us to find POSTN as a key mediator of ALK-mediated tumor growth.
2024-03-21 | GSE228194 | GEO