Research ArticleCancer therapy

MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)–addicted neuroblastomas

See allHide authors and affiliations

Science Signaling  28 Nov 2017:
Vol. 10, Issue 507, eaam7550
DOI: 10.1126/scisignal.aam7550
  • Fig. 1 Sensitivity of neuroblastoma cell lines to MEK inhibition by trametinib.

    (A and B) Viability assessed over 12 days using the resazurin viability assay in neuroblastoma cell lines [CLB-BAR, CLB-GE, CLB-GA, and Kelly in (A) and SK-N-AS, SK-N-BE, SK-N-DZ, IMR32, and CLB-PE in (B)] treated with either trametinib or lorlatinib, as indicated. (C and D) Resazurin assay–based viability in neuroblastoma cell lines CLB-BAR (C) and CLB-GE (D) treated with trametinib or lorlatinib alone or a combination of trametinib and lorlatinib, as indicated. Data are means ± SE of fold relative fluorescence units (RFU) relative to untreated cells from three independent experiments.

  • Fig. 2 Specificity of AKT signaling core components in ALK-positive neuroblastoma cell lines.

    (A to C) Western blotting for the indicated proteins in lysates from anaplastic lymphoma kinase (ALK)–positive neuroblastoma cell lines CLB-BAR (A), CLB-GAR (B), and CLB-GE (C) treated with trametinib or lorlatinib for the indicated time (h, hours). (D) Immunblotting of lysates from IMR32 cells pretreated with trametinib, lorlatinib, or both for 1 hour and then stimulated with ALKAL1 for 30 min. Tubulin (A to C) or total pan–ERK (extracellular signal–regulated kinase) (D) served as loading controls. Data are means ± SE from ≥3 independent experiments. *P < 0.05; Student’s paired t test. wt, wild type.

  • Fig. 3 Trametinib treatment activates AKT signaling via mTORC2.

    (A) ALK-positive neuroblastoma cell lines CLB-BAR and CLB-GE were treated with trametinib, BEZ235, AZD8055, or everolimus either alone or in combination, as indicated. Cell lysates were immunoblotted for p-Rictor (Thr1135), Rictor, p-p70S6K, S6K, p-AKT (Ser473), AKT, p-ERK1/2, and ERK1/2 antibodies. Actin was used as a loading control. (B) CLB-BAR and CLB-GE cells were transfected with either scrambled control or two independent siRNAs targeting Rictor before treatment with trametinib. Lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed for Rictor, p-Rictor (Thr1135), p-Akt (Ser473), AKT, p-ERK1/2, and ERK1/2 abundance by immunoblotting with actin as a loading control. Data are means ± SE from at least three independent experiments. *P < 0.05; Student’s paired t test.

  • Fig. 4 Knockdown of SIN1 suppresses increased activation of AKT after MEK inhibition.

    (A) Immunoblotting in whole-cell lysates from neuroblastoma cell lines CLB-BAR and CLB-GE grown in complete growth medium and treated with trametinib, SCH772984, BEZ235, or a combination thereof, as indicated. (B) Immunoblotting in whole-cell lysates from CLB-BAR and CLB-GE cells treated with trametinib for 1 hour after transfection with scrambled control (siC) or one of two SIN1-targeted siRNAs (si1 and si3). Lysates were separated by SDS-PAGE and analyzed for Rictor, SIN1, p-AKT (Ser473), AKT, p-ERK1/2, and ERK1/2 expression by immunoblotting with actin as a loading control. Data are means ± SE from at least three independent experiments. *P < 0.05; Student’s paired t test.

  • Fig. 5 Efficacy of trametinib in ALK-positive neuroblastoma and NSCLC xenograft models.

    (A to C) Growth curves of RAS-positive SK-N-AS neuroblastoma (A; P ≤ 0.05), EML4-ALK–positive H3122 non–small cell lung cancer (NSCLC) (B; P ≤ 0.05), or ALK-dependent CLB-BAR neuroblastoma (C; not significantly different by Student’s paired t test) xenografts in vehicle- and trametinib-treated mice. Data are means ± SD from n = 6 mice in each group.

  • Table 1 Mutation data for the cell lines.

    Mutation data for selected critical genes for the nine neuroblastoma cell lines used in this study. MNA, MYCN-amplified; HomZ., homozygote; mut, mutant; amp, amplification; ex, exon; expr., expression.

    Cell lineGenomic profile*ALK status (amp, mut ex23–25)MYCN statusTP53 status mut ex5–9NRAS statusNF1 expr.
    CLB-BARMNAAmp. whole ALK exc. part
    of i3 (nonamp). Note, exome
    sequencing shows del ex4–11
    MYCN-amplifiedNo mut in TP53No mut in NRASHigh
    CLB-GEMNAALK amp. ALK mut F1174VMYCN-amplifiedNo mut in TP53No mut in NRASHigh
    CLB-GAR11q-del2p gain with break
    within ALK intron 1.
    Mutation R1275Q
    Not MYCN-amplifiedNo mut in TP53No mut in NRASHigh
    KellyMNA + 11q-delNo amplification. 2p-gain.
    Mutation F1174L
    MYCN-amplifiedHomZ. Mut P177TNo mut in NRASHigh
    SK-N-DZMNANo mutation.
    No amplification
    MYCN-amplifiedNo mut in TP53No mut in NRASIntermediate
    CLB-PEMNA + 11q-del2p gain. No mutation.
    No amplification
    MYCN-amplifiedHomZ. mut C176FNo mut in NRASHigh
    IMR32MNAAmp. of ALK ex3–4 only.
    No mutation
    MYCN-amplifiedNo mut in TP53No mut in NRASHigh
    SK-N-AS11q-delNo mutation.
    No amplification
    Not MYCN-amplifiedNo mut in TP53Mut in NRAS: Q61KLow
    SK-N-BEMNA2p gain. No mutation.
    No amplification
    MYCN-amplifiedHomZ. mut C145FNo mut in NRASLow

    *Genomic profile as defined in (74).

    †Genomic profile as defined in (75).

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/10/507/eaam7550/DC1

      Fig. S1. SNP array genome profiles of the nine neuroblastoma cell lines.

      Fig. S2. NF1 abundance in neuroblastoma cell lines.

      Fig. S3. Sensitivity of neuroblastoma cell lines to trametinib.

      Fig. S4. AKT signaling inhibition does not lead to increased activation of MAPK.

      Fig. S5. Sensitivity of EML4-ALK–positive NSCLC cell lines to trametinib.

      Fig. S6. Phospho-RTK array analysis after MEK inhibition.

      Fig. S7. Increased AKT activation in cells upon treatment with trametinib and rapamycin.

      Fig. S8. Increased AKT activation in xenografts upon treatment with trametinib.

      Table S1. Chromosomal profiles of the cell lines.

      Table S2. Trametinib does not act synergistically with lorlatinib to inhibit ALK-positive neuroblastoma cell line proliferation.

      Reference (76)

    • Supplementary Materials for:

      MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)–addicted neuroblastomas

      Ganesh Umapathy, Jikui Guan, Dan E. Gustafsson, Niloufar Javanmardi, Diana Cervantes-Madrid, Anna Djos, Tommy Martinsson, Ruth H. Palmer, Bengt Hallberg*

      *Corresponding author. Email: bengt.hallberg{at}gu.se

      This PDF file includes:

      • Fig. S1. SNP array genome profiles of the nine neuroblastoma cell lines.
      • Fig. S2. NF1 abundance in neuroblastoma cell lines.
      • Fig. S3. Sensitivity of neuroblastoma cell lines to trametinib.
      • Fig. S4. AKT signaling inhibition does not lead to increased activation of MAPK.
      • Fig. S5. Sensitivity of EML4-ALK–positive NSCLC cell lines to trametinib.
      • Fig. S6. Phospho-RTK array analysis after MEK inhibition.
      • Fig. S7. Increased AKT activation in cells upon treatment with trametinib and rapamycin.
      • Fig. S8. Increased AKT activation in xenografts upon treatment with trametinib.
      • Table S1. Chromosomal profiles of the cell lines.
      • Table S2. Trametinib does not act synergistically with lorlatinib to inhibit ALK-positive neuroblastoma cell line proliferation.
      • Reference (76)

      [Download PDF]

      Technical Details

      Format: Adobe Acrobat PDF

      Size: 1.68 MB


      Citation: G. Umapathy, J. Guan, D. E. Gustafsson, N. Javanmardi, D. Cervantes-Madrid, A. Djos, T. Martinsson, R. H. Palmer, B. Hallberg, MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)–addicted neuroblastomas. Sci. Signal. 10, eaam7550 (2017).

      © 2017 American Association for the Advancement of Science

    Stay Connected to Science Signaling

    Navigate This Article