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/ROS1 inhibitor, overcomes de novo resistance and induces complete and sustained tumor regressions in patient-derived xenograft 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 lorlatinib and crizotinib in clinically relevant ALK-driven neuroblastoma patient-derived xenograft models. Lorlatinib was a markedly more potent inhibitor of ALK and preferentially downregulated several kinases implicated in G2/M cell-cycle transition compared with crizotinib. Lorlatinib treatment also led to the repression of MYCN expression and its occupancy at promoters of the same G2/M kinases. These data provide mechanistic insight into the superior efficacy of lorlatinib over crizotinib for the treatment of ALK-driven neuroblastoma.

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©2025 The Authors; Published by the American Association for Cancer Research
2025
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
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Supplementary data

Supplementary Figure S1

Supplementary Figure S1 shows kinome remodeling in response to ALK inhibition is robust and heterogenous.

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Supplementary Figure S2

Supplementary Figure S2 shows MIB/MS profiling in neuroblastoma models upon treatment with lorlatinib.

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Supplementary Figure S3

Supplementary Figure S3 shows immunoblot validation of MIB/MS data.

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Supplementary Figure S4

Supplementary Figure S4 shows that COG-N-424x, a wild type ALK PDX, does not downregulate G2/M kinases upon ALK inhibition.

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Supplementary Figure S5

Supplementary Figure S5 shows COG-N-424x harboring wild-type ALK does not respond to lorlatinib treatment.

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Supplementary Figure S6

Supplementary Figure S6 shows MIB/MS kinome data for COG-N-453x tumor lysates treated with crizotinib for 30 minutes.

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Supplementary Figure S7

Supplementary Figure S7 shows MIB/MS kinome data for COG-N-453x tumor lysates treated with lorlatinib for 30 minutes.

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Supplementary Figure S8

Supplementary Figure S8 show inhibition of FAK and FER does not increase sensitivity of crizotinib to neuroblastoma cell lines in 2D and 3D culture.

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Supplementary Figure S9

Supplementary Figure S9 shows Lorlatinib potently downregulates multiple kinases involved in the G2/M cell cycle transition.

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Supplementary Figure S10

Supplementary Figure S10 shows Lorlatinib downregulates transcripts of several G2/M kinases to a greater extent than crizotinib.

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Supplementary Figure S11

Supplementary Figure S11 shows Lorlatinib downregulates AURKA expression.

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Supplementary Figure S12

Supplementary Figure S12 shows RNA-seq fold changes of differentially expressed genes shared between crizotinib vs vehicle and lorlatinib vs vehicle.

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Supplementary Figure S13

Supplementary Figure S13 shows cell cycle analysis upon treatment with lorlatinib arrests cells in G2/M phase and induces apoptosis.

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Supplementary Figure S14

Supplementary Figure S14 shows lorlatinib downregulates MYCN expression.

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Supplementary Figure S15

Supplementary Figure S15 shows chromatin immunoprecipitation sequencing assay whereby MYCN binds to promoters of several G2/M kinases.

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Supplementary Figure S16

Supplementary Figure S16 shows chromatin immunoprecipitation sequencing assay whereby c-Myc binds to promoters of several G2/M kinases.

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Supplementary Figure S17

Supplementary Figure S17 shows lorlatinib displaces MYCN and pol II from promoters of G2/M kinases.

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Supplementary Table S1

Supplementary Table S1 shows catalogue numbers of Taqman probes.

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Supplementary Table S2

Supplementary Table S2 shows oligonucleotide sequences of guide RNAs.

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Supplementary Table S3

Supplementary Table S3 shows oligonucleotide sequences of ChIP primers.

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Supplementary Table S4

Supplementary Table S4 shows RNA-seq fold changes of differentially expressed genes shared between crizotinib vs vehicle and lorlatinib vs vehicle contrasts for non kinase genes.

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Supplementary Table S5

Supplementary Table S5 shows RNA-seq p adjusted values of differentially expressed genes in COG-N-453x and NB-1643 tumor models comparing crizotinib vs lorlatinib treatment.

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Supplementary Methods

Supplementary Methods includes supplementary material and methods and references.

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Supplementary Data 1

Supplementary Data 1 includes the raw, unproccessed mass spectrometry label free quantification results of MaxQuant/Andromeda searches for "proteingroups."

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