Immunoassays for the quantification of <scp>ALK</scp> and phosphorylated <scp>ALK</scp> support the evaluation of on‐target <scp>ALK</scp> inhibitors in neuroblastoma

Tucker, Elizabeth R.; Tall, Jennifer R.; Danielson, Laura S.; Gowan, Sharon; Jamin, Yann; Robinson, Simon P.; Banerji, Udai; Chesler, Louis

doi:10.1002/1878-0261.12069

Cited by 8 publications

(10 citation statements)

References 19 publications

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“…Importantly, differences in sensitivity to various ALK small-molecule inhibitors have been reported for different ALK mutants [8, 27]. The ALK inhibitor alectinib (CH5424802) has been shown to be able to block the resistant gatekeeper mutation L1196M [24].…”

Section: Resultsmentioning

confidence: 99%

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations

et al. 2019

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The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

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Section: Resultsmentioning

confidence: 99%

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations

et al. 2019

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“…For ALK immunoassay analysis, 15 μg of protein per well was added in duplicate for each sample to measure either total or pY1586 ALK, and for immunoblotting, 5 μg of denatured lysates were loaded into 4–12% Bis/Tris gels, both according to previously published methods. 37 …”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was transferred to a fresh tube, and the protein content was quantified using the Direct Detect system (Millipore). For ALK immunoassay analysis, 15 μg of protein per well was added in duplicate for each sample to measure either total or pY1586 ALK, and for immunoblotting, 5 μg of denatured lysates were loaded into 4–12% Bis/Tris gels, both according to previously published methods …”

Section: Methodsmentioning

confidence: 99%

Designing Dual Inhibitors of Anaplastic Lymphoma Kinase (ALK) and Bromodomain-4 (BRD4) by Tuning Kinase Selectivity

et al. 2019

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Concomitant inhibition of anaplastic lymphoma kinase (ALK) and bromodomain-4 (BRD4) is a potential therapeutic strategy for targeting two key oncogenic drivers that co-segregate in a significant fraction of high-risk neuroblastoma patients, mutation of ALK and amplification of MYCN. Starting from known dual polo-like kinase (PLK)-1–BRD4 inhibitor BI-2536, we employed structure-based design to redesign this series toward compounds with a dual ALK–BRD4 profile. These efforts led to compound (R)-2-((2-ethoxy-4-(1-methylpiperidin-4-yl)phenyl)amino)-7-ethyl-5-methyl-8-((4-methylthiophen-2-yl)methyl)-7,8-dihydropteridin-6(5H)-one (16k) demonstrating improved ALK activity and significantly reduced PLK-1 activity, while maintaining BRD4 activity and overall kinome selectivity. We demonstrate the compounds’ on-target engagement with ALK and BRD4 in cells as well as favorable broad kinase and bromodomain selectivity.

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“…There are now open trials for pediatric patients with either Ceritinib or Lorlatinib. Ceritinib, which received USA Food and Drug Administration approval in 2014 for treatment of patients with ALK -rearranged metastatic non-small cell lung cancer (NSCLC), has shown efficacy above that of Crizotinib in neuroblastoma models [ 37 ] . However, its success in neuroblastoma patients is yet not fully evaluated, apart from a favorable case report for a tumor harboring an ALK I1171T mutation [ 38 ] .…”

Section: Direct and Combinatorial Therapeutics For Alk ...mentioning

confidence: 99%

Targeting MYCN and ALK in resistant and relapsing neuroblastoma

Tucker¹,

Poon²,

Chesler³

2019

CDR

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Neuroblastoma, a tumor of peripheral nerve, is the most common solid tumor of young children. In high-risk disease, which comprises approximately half of patients, death from chemotherapy-resistant, metastatic relapse is very frequent. Children who relapse exhibit clonal enrichment of two genomic alterations: high-level amplification of the MYCN oncogene, and kinase domain mutations of the anaplastic lymphoma kinase (ALK) gene. Overall survival in this patient cohort is less than 15% at 3 years, and there are few options for rationally targeted therapy. Neuroblastoma patients exhibit de novo resistance to many existing ALK inhibitors, and no clinical therapeutics to target MYCN have yet been developed. This review outlines the international efforts to uncover mechanisms of oncogenic action that are therapeutically targetable using small-molecule inhibitors. We describe a mechanistic interaction in which ALK upregulates MYCN transcription, and discuss clinical trials emerging to develop transcriptional inhibitors of MYCN, and to identify effective inhibitors of ALK in neuroblastoma patients.

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Immunoassays for the quantification of ALK and phosphorylated ALK support the evaluation of on‐target ALK inhibitors in neuroblastoma

Cited by 8 publications

References 19 publications

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations

Designing Dual Inhibitors of Anaplastic Lymphoma Kinase (ALK) and Bromodomain-4 (BRD4) by Tuning Kinase Selectivity

Targeting MYCN and ALK in resistant and relapsing neuroblastoma

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