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Molecular Cancer Therapeutics

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Cancer Biology and Translational Studies

ALK Inhibitor Response in Melanomas Expressing EML4-ALK Fusions and Alternate ALK Isoforms

Kasey L. Couts, Judson Bemis, Jacqueline A. Turner, Stacey M. Bagby, Danielle Murphy, Jason Christiansen, Jennifer D. Hintzsche, Anh Le, Todd M. Pitts, Keith Wells, Allison Applegate, Carol Amato, Pratik Multani, Edna Chow-Maneval, John J. Tentler, Yiqun G. Shellman, Matthew J. Rioth, Aik-Choon Tan, Rene Gonzalez, Theresa Medina, Robert C. Doebele and William A. Robinson
Kasey L. Couts
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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  • For correspondence: kasey.couts@ucdenver.edu
Judson Bemis
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Jacqueline A. Turner
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Stacey M. Bagby
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Danielle Murphy
Ignyta, Inc, San Diego, California.
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Jason Christiansen
Ignyta, Inc, San Diego, California.
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Jennifer D. Hintzsche
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Anh Le
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Todd M. Pitts
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Keith Wells
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Allison Applegate
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Carol Amato
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Pratik Multani
Ignyta, Inc, San Diego, California.
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Edna Chow-Maneval
Ignyta, Inc, San Diego, California.
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John J. Tentler
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Yiqun G. Shellman
Department of Dermatology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Matthew J. Rioth
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.Division of Biomedical Informatics and Personalized Medicine, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Aik-Choon Tan
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Rene Gonzalez
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Theresa Medina
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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Robert C. Doebele
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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William A. Robinson
Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
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DOI: 10.1158/1535-7163.MCT-17-0472 Published January 2018
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    Figure 1.

    Identification of melanoma tumors expressing wt ALK, ALKATI, and an EML4-ALK fusion. A, ALK mRNA expression was quantified in melanoma PDX tumors by qRT-PCR analysis with primers located in early exons (E) or exons within the kinase domain (K1, K2) of ALK. Expression was normalized to GAPDH and error bars represent SEM of triplicate reactions. The 12 ALK expression–positive tumors out of 45 total PDX tumors analyzed are shown. B, Immunoblot analysis of ALK-positive PDX tumor lysates. C, Expression of ALK intron 19 was quantified by qRT-PCR. Expression was normalized to GAPDH and error bars represent SEM of triplicate reactions. D, FISH analysis of the MB 2141 PDX tumor using ALK specific 5′ (green) and 3′ (red) break-apart probes. E, Sanger sequencing analysis of EML4-ALK fusion variant transcripts. PCR products were generated from MB 2141 PDX cDNA using an EML4-exon 4 forward primer and ALK exon 17 or 20 reverse primers. PCR products were subcloned and analyzed by Sanger sequencing.

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    Figure 2.

    ALK inhibitors reduce viability in cells expressing EML4-ALK fusions, but not in cells expressing ALKATI or wt ALK. A, Immunoblot analysis of lysates from H3122 NSCLC cells and melanoma cells. B, Cells were treated with increasing concentrations of ALK inhibitors crizotinib or ceritinib. Viability was analyzed after 72 hours and normalized to DMSO-treated controls. C, Cells were treated with increasing concentrations of crizotinib for 2 hours and analyzed by immunoblotting.

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    Figure 3.

    Melanoma cells respond to targeted MEK inhibition against other oncogenic drivers. A, MB 1692, MB 3443, and MB 3429 cells were treated with increasing concentrations of a MEK inhibitor (trametinib) or mutant BRAF specific inhibitor (vemurafenib). Viability was analyzed after 72 hours and normalized to DMSO-treated controls. B, Cells were treated with increasing concentrations of trametinib for 2 hours and analyzed by immunoblotting. C, MB 1692 cells were treated with increasing concentrations of ceritinib alone or in combination with 1 nmol/L or 2 nmol/L trametinib. Viability was analyzed after 72 hours and normalized to nontreated controls. D, MB 1692 cells were treated with the indicated concentrations of trametinib and/or ceritinib for 2 hours and analyzed by immunoblotting. For all viability results, error bars represent SEM of three technical triplicates.

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    Figure 4.

    ALK inhibitors decrease growth of melanoma xenograft tumors expressing an EML4-ALK fusion, but not ALKATI only. A, MB 2141 and MB 1692 subcutaneous xenograft tumors were generated in nude mice and treated with vehicle, crizotinib (100 mg/kg), or ceritinib (50 mg/kg). Growth was measured biweekly for 25 days, at which time all tumors were harvested. B, Immunoblot analysis for total ALK and β-actin in tumor lysates from vehicle tumors harvested at day 25. Lysates from two independent tumors were analyzed for each condition. C, Immunoblot analysis for AKT/p-AKT, ERK1/2/p-ERK/1/2, and β-actin in MB 2141 tumors harvested after 7 days of treatment and MB 3429 tumors harvested after 25 days of treatment. Lysates from two independent tumors were analyzed for each condition for MB 3429 tumors. (Veh, vehicle; Criz, crizotinib; Cerit, ceritinib).

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    Figure 5.

    A patient with melanoma expressing ALKATI does not respond to treatment with entrectinib. A, The patient specimen was analyzed for ALK gene expression using targeted RNA next-generation sequencing. The average number of sequencing reads for exons 18–19 (upstream of the kinase domain, not included in ALKATI) and exons 20–25 (kinase domain included in ALKATI) of the ALK gene are shown. B, Radiographic images from CT scans performed prior to treatment (baseline) and 2 weeks after the start of treatment are shown for a right hilar pulmonary tumor nodule (top) and a left lower lobe pulmonary tumor nodule (bottom).

Tables

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  • Table 1.

    Driver gene mutations in ALK expressing melanomas

    SampleALK statusSubtypeBRAFNRASKITGNAQGNA11NF1
    MB 2141EML4-ALK + ALKATIMucosal——————
    MB 3443ALKATIMucosal—Q61H————
    MB 2724ALKATIAcralP402Ha—————
    MB 3429ALKATINodularG596C———R183C—
    MB 1692ALKATISSAGK-BRAF—————
    MB 2661ALKATI+ wt ALKNodularG466E—————
    MB 2223ALKATI+ wt ALKNodular—————Q1255Xa
    MB 2040wt ALKAcralV600E—————
    MB 1245wt ALKSSV600E—————
    MB 1374wt ALKUnk PriARMC10-BRAF—————
    MB 1588wt ALKUnk Pri—————E844Xa
    • NOTE: “—” indicates no mutation detected.

    • Abbreviations: SS, superficial spreading; Unk Pri, unknown primary.

    • ↵aSpecific mutation has not been functionally characterized.

Additional Files

  • Figures
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  • Supplementary Data

    • Supplementary Table 1 - Primer sequences
    • Figure S1 - ALK qRT-PCR screen in melanoma PDX models
    • Figure S2 - Characterization of EML4-ALK fusion variants in MB 2141
    • Figure S3 - Non-specific immunoblot bands in MB 1692 and MB 1374
    • Figure S4 - Overexpression of EML4-ALK, ALKATI, and wtALK in NIH3T3
    • Figure S5 - Additional ALK inhibitor testing and IC50 values
    • Figure S6 - siRNA knockdown of ALKATI
    • Figure S7 - Expression of ALKATI and EML4-ALK isoforms in PDX vs xenografts
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Molecular Cancer Therapeutics: 17 (1)
January 2018
Volume 17, Issue 1
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ALK Inhibitor Response in Melanomas Expressing EML4-ALK Fusions and Alternate ALK Isoforms
Kasey L. Couts, Judson Bemis, Jacqueline A. Turner, Stacey M. Bagby, Danielle Murphy, Jason Christiansen, Jennifer D. Hintzsche, Anh Le, Todd M. Pitts, Keith Wells, Allison Applegate, Carol Amato, Pratik Multani, Edna Chow-Maneval, John J. Tentler, Yiqun G. Shellman, Matthew J. Rioth, Aik-Choon Tan, Rene Gonzalez, Theresa Medina, Robert C. Doebele and William A. Robinson
Mol Cancer Ther January 1 2018 (17) (1) 222-231; DOI: 10.1158/1535-7163.MCT-17-0472

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ALK Inhibitor Response in Melanomas Expressing EML4-ALK Fusions and Alternate ALK Isoforms
Kasey L. Couts, Judson Bemis, Jacqueline A. Turner, Stacey M. Bagby, Danielle Murphy, Jason Christiansen, Jennifer D. Hintzsche, Anh Le, Todd M. Pitts, Keith Wells, Allison Applegate, Carol Amato, Pratik Multani, Edna Chow-Maneval, John J. Tentler, Yiqun G. Shellman, Matthew J. Rioth, Aik-Choon Tan, Rene Gonzalez, Theresa Medina, Robert C. Doebele and William A. Robinson
Mol Cancer Ther January 1 2018 (17) (1) 222-231; DOI: 10.1158/1535-7163.MCT-17-0472
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