Research ArticleCancer

Targeting MYCN-expressing triple-negative breast cancer with BET and MEK inhibitors

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Science Translational Medicine  11 Mar 2020:
Vol. 12, Issue 534, eaaw8275
DOI: 10.1126/scitranslmed.aaw8275
  • Fig. 1 MYCN RNA and MYCN protein expression in primary, treatment-naïve TNBC.

    (A) MYCN transcript (TPM) from 197 primary, treatment-naïve TNBCs (source: TCGA, BRCA). μ, mean. (B) Violin plot showing MYCN expression in TNBC (source: TCGA, BRCA; n = 197) compared to neuroblastoma (NB; n = 161) (23), acute myeloid leukemia (AML) (source: TCGA, LAML; n = 173), glioblastoma multiforme (GBM) (source: TCGA, GBM; n = 156), and castration-resistant prostate cancer (CRPC), including neuroendocrine (NE; n = 15) and adenocarcinoma (Adeno; n = 123) (24, 25). Wilcoxon rank sum test comparing TNBC to the other cancer types. P values were adjusted by false discovery rate (FDR), ****P < 0.0001. ns, not significant. (C) MYCN protein quantities (H-scores) from 191 primary, treatment-naïve TNBCs [source: Vanderbilt University Medical Center (VUMC) and US Biomax]. Int., intermediate. (D) Representative MYCN IHC images in TNBC specimens devoid of MYCN protein expression (H-score = 0), which contain intermediate amounts of MYCN (H-score between >0 and ≤30) or have high MYCN (H-score > 30). Scale bars, 20 μm.

  • Fig. 2 Increased percentage of MYCN-expressing cells in residual disease after NAC.

    (A) MYCN H-scores in residual disease from 115 primary, NAC-treated TNBCs (source: VUMC). Null, H-score = 0; Int., H-score >0 to ≤30; High, H-score > 30. (B) Box plot showing MYCN H-scores in primary, treatment-naïve TNBC cases (n = 191; see Fig. 1C) compared to residual disease from primary, NAC-treated TNBC cases [n = 115; see (A)]. Wilcoxon rank sum test, ***P = 0.0001. (C) MYCN H-scores in patient-matched TNBC cases before and after NAC (n = 6; see table S2C for treatments and patient characteristics).

  • Fig. 3 Intratumoral heterogeneity of MYCN and MYC expression in TNBC.

    (A) MYCN H-scores from 38 recurrent TNBC cases with quantification of percent positive cases (H-score > 0) for each site of recurrence, labeled by color [lung (magenta), skin (blue), chest wall (orange), and brain (black)]. (B) MYCN and MYC H-scores for each of the 88 primary, treatment-naïve; 114 primary, NAC-treated; and 38 recurrent TNBC cases. Stacked bar graphs represent the percentages of total cases expressing (H-score) each MYC family isoform [alone (MYCN only and MYC only), both isoforms (MYCN and MYC), or neither isoform (None)]. (C) Representative hematoxylin and eosin (H&E), IHC, and TSA-IF stains of MYCN and MYC in primary and recurrent TNBC. Individual fluorescence images for cell nuclei (blue), MYCN (magenta), and MYC (green) can be found in fig. S5. The dashed lines separating a MYCN-amplified NB-positive control from TNBC cases represent the same exposure times for all samples but a decreased brightness adjustment for MYCN in the NB control due to overexpression of MYCN. Tumor images do not represent serial sections. Scale bars, 50 μm (top four rows) and 20 μm (bottom row).

  • Fig. 4 Evaluation of MYCN-expressing TNBC clonal cell line drug sensitivity.

    (A) Representative TSA-IF stains of MYCN and MYC in the CAL-51 and MDA-MB-468 TNBC cell lines. Colors represent cell nuclei (blue), MYCN (magenta), and MYC (green). Scale bars, 50 μm [overlay fluorescence images at ×20 magnification (left panel for each cell line)] and 20 μm [individual fluorescence images at ×40 magnification (right panels for each cell line)]. (B) Immunoblot analysis of MYCN, MYC, and β-actin in the indicated 33 clonally derived cell lines established from CAL-51. NB control, MYCN-amplified SK-N-BE(2)C cell lysate. (C) Viability of PI3Ki-resistant (PI3KiR) CAL-51 clonally derived cell lines after treatment with escalating doses of GDC-0032 or GDC-0941 for 72 hours. Black and red dose-response curves represent the indicated MYCNLow and MYCNHigh clonally derived cell lines, respectively. Data shown represent the means ± SEM of three biological replicates. (D) Immunoblot analysis of MYCN, MYC, and β-actin in the 14 indicated CAL-51PI3KiR clonally derived cell lines. (E) IC50 of 40 compounds used in a secondary screen to treat five MYCNLow and MYCNHigh cell lines for 72 hours. Colors associate with drug class [PI3K (purple), ATR (orange), BRD family (blue), Aurora kinase A (brown), and MAPK pathway (green)]. Horizontal red dashed lines represent a separation of compounds that had a greater or less than twofold difference in IC50 between MYCNLow and MYCNHigh cell lines. (F) IC50 of 31 CAL-51 clonally derived cell lines after treatment with escalating doses of INCB054329 for 72 hours. Red lines represent means. Student’s t test, ****P < 0.0001. (G) Quantification of crystal violet–stained colonies compared to control for 10 MYCNLow and 4 MYCNHigh cell lines treated with 0.5 μM INCB054329 for 6 days. Red lines represent means. Student’s t test, ***P < 0.001.

  • Fig. 5 Evaluation of MYC family isoform expression after BETi treatment.

    (A) Top: Viability of MYCNLow and MYCNHigh cell lines after siRNA-mediated knockdown using nontargeting (siNT) or anti-MYCN (siMYCN) siRNAs for 96 hours. Data shown represent the mean of three technical replicates. Bottom: Immunoblot analysis of MYCN, MYC, and β-actin in MYCNLow and MYCNHigh cell lines after the described knockdown with 25 nM siRNAs. (B) Genome viewer showing sequencing alignment tracks of nascent transcript PRO-seq mapping at the MYCN and MYC gene loci for the two indicated MYCNLow and MYCNHigh cell lines after treatment with dimethyl sulfoxide control (Unt, blue) or 0.5 μM INCB054329 (BETi, red) for 15 min. (C) MYCN and MYC expression (TPM) in the two indicated MYCNLow (Cln3 and Cln5) and four MYCNHigh (Cln8, Cln15, Cln37, and Cln39) cell lines after treatment with 0.5 μM INCB054329 for 4 hours. (D) Immunoblot analysis of MYCN, MYC, and β-actin in three of the cell lines described in (C) after treatment with 0.5 and 1.0 μM INCB054329 or JQ1 for 24 hours. (E) MYC family isoform TSA-IF on two MYCN-expressing TNBC cell lines (MDA-MB-468 and CAL-51) after 0, 0.25, 0.5, or 1.0 μM INCB054329 or JQ1 for 24 hours. Colors represent cell nuclei (blue), MYCN (magenta), and MYC (green). Scale bars, 50 μm. (F) Quantification of fluorescence intensity per nucleus for MYCN and MYC after BETi treatments described in (E). Data shown represent the median ± SEM of three biological replicates. Student’s t test between untreated and BETi-treated cells, *P < 0.05, **P < 0.01.

  • Fig. 6 Effect of BETi and MEKi combination treatment on MYC family isoform expression and cell viability of MYCN-expressing TNBC cell lines.

    (A) Immunoblot analysis for pERK1/2, total ERK1/2, MYCN, MYC, and β-actin in MYCNLow and MYCNHigh cell lines after treatment with MAPK pathway inhibitors at 0.25 μM for 24 hours. (B) Immunoblot analysis of pERK1/2, total ERK1/2, MYCN, MYC, and β-actin in four MYCNHigh CAL-51 cell lines after treatment with 0.25 μM trametinib (Tram), 0.5 μM INCB054329 (INCB), 0.5 μM JQ1, or the combination of trametinib with either BETi for 48 hours. All immunoblot experiments shown are representative of at least two biological replicates. (C) MYC family isoform TSA-IF on two MYCN-expressing TNBC cell lines (MDA-MB-468 and CAL-51) after 0.25 μM trametinib, 0.5 μM INCB054329, 0.5 μM JQ1, or the combination of trametinib with either BETi for 48 hours. Colors represent cell nuclei (blue), MYCN (magenta), and MYC (green). Scale bars, 50 μm. (D) Violin plots showing quantification of fluorescence intensity for MYCN and MYC per nucleus after BETi treatments described in (C). TSA-IF images and quantification are representative of three biological replicates. (E) Crystal violet colony formation assays for MDA-MB-468 and CAL-51 after treatment with the indicated concentrations of trametinib, INCB054329, or JQ1 alone, or either BETi in combination with trametinib, for 6 days. Values in red represent mean delta Bliss synergy for combination treatments of three biological replicates.

  • Fig. 7 Evaluation of tumor growth after BETi and MEKi combination treatment in vivo.

    (A) MYCN and MYC expression (TPM) in three TNBC PDX models (TM00096, TM01273, and TM00090). (B) Representative IHC and quantification of percent positive cells for MYCN and MYC in TM00096, TM01273, and TM00090 sections. Scale bars, 20 μm. (C) Tumor volume (in cubic millimeters) of TM00096, TM01273, and TM00090 treated with trametinib [0.1 mg/kg, once daily (QD)] or INCB054329 [50 mg/kg, twice daily (BID)] alone or in combination for 14 days. Red bars represent means. (D) Representative IHC and quantification of percent positive cells for MYCN and MYC in HBCx1 and BCM-2147 sections. Scale bars, 20 μm. (E) Tumor volume (in cubic millimeters) of TM00096, HBCx1, and BCM-2147 treated with trametinib, INCB054329, or JQ1 (50 mg/kg, BID) alone, or either BETi in combination with trametinib for 22 days. The dashed lines represent the mean initial tumor volume at time of first treatment. Avg, average; TGI, tumor growth inhibition. Data shown represent the means ± SEM. Student’s t test between vehicle, BETi, and corresponding combination-treated tissue, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 8 Evaluation of MYC family isoform expression after BETi and MEKi combination treatment in vivo.

    (A) Representative TSA-IF of MYCN and MYC in TM00096 after 2 or 22 days of treatment with trametinib (0.1 mg/kg, QD), INCB054329 (50 mg/kg, BID), or JQ1 (50 mg/kg, BID) alone, or either BETi in combination with trametinib. Colors represent cell nuclei (blue), MYCN (magenta), and MYC (green). Scale bars, 20 μm. (B) Quantification of IHC (percent positive cells) and (C) violin plots showing the distribution of TSA-IF intensity per nucleus for MYCN and MYC in TM00096, HBCx1, and BCM-2147 sections after treatments described in (A) for 22 days. Student’s t test between vehicle and treatment arms and between single-agent BETi and corresponding combination-treated tissue, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/534/eaaw8275/DC1

    Materials and Methods

    Fig. S1. Identification of TNBC breast cancers in TCGA and METABRIC datasets.

    Fig. S2. Distribution of MYCN expression across TNBC.

    Fig. S3. IHC detection of MYCN in CDX and PDX tissue.

    Fig. S4. MYCN expression in primary TNBC and patient-matched metastases.

    Fig. S5. MYCN and MYC intratumoral heterogeneity in primary and recurrent TNBC.

    Fig. S6. MYCN and MYC expression in TNBC cell populations and CAL-51 clonally derived cell lines.

    Fig. S7. BETi sensitivity of CAL-51 MYCNHigh cell lines.

    Fig. S8. Changes in MYC target gene expression in CAL-51 MYCNHigh cell lines after BETi treatment.

    Fig. S9. Differential gene expression analyses between MYCNRatioHigh and MYCRatioHigh TNBC.

    Fig. S10. Effect of BETi and MEKi combination treatments on weight of treated mice.

    Fig. S11. Evaluation of apoptosis and proliferation after BETi and MEKi treatment in TNBC PDX models.

    Fig. S12. Evaluation of MYC family isoform expression after BETi and MEKi combination treatment in vivo.

    Table S1. MYCN and MYC expression in breast cancer PDX models.

    Table S2. Characteristics of patients with treatment-naïve and NAC-treated primary TNBC.

    Data file S1. Tabular data points for experiments with a sample size of n < 20.

    Data file S2. IHC results for MYCN and MYC in primary, treatment-naïve; primary, NAC-treated; and recurrent TNBC cases.

    Data file S3. Primary drug screen results using CAL-51 MYCNLow and MYCNHigh cell lines.

    Data file S4. Secondary drug screen results using CAL-51 MYCNLow and MYCNHigh cell lines.

    Data file S5. Tabular data points for MYC family isoform TSA-IF in CAL-51 after single-agent BETi treatment.

    Data file S6. Tabular data points for MYC family isoform TSA-IF in MDA-MB-468 after single-agent BETi treatment.

    Data file S7. Tabular data points for MYC family isoform TSA-IF in TNBC cell lines and PDX tissue after BETi and MEKi single-agent and combination treatment.

    References (6679)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification of TNBC breast cancers in TCGA and METABRIC datasets.
    • Fig. S2. Distribution of MYCN expression across TNBC.
    • Fig. S3. IHC detection of MYCN in CDX and PDX tissue.
    • Fig. S4. MYCN expression in primary TNBC and patient-matched metastases.
    • Fig. S5. MYCN and MYC intratumoral heterogeneity in primary and recurrent TNBC.
    • Fig. S6. MYCN and MYC expression in TNBC cell populations and CAL-51 clonally derived cell lines.
    • Fig. S7. BETi sensitivity of CAL-51 MYCNHigh cell lines.
    • Fig. S8. Changes in MYC target gene expression in CAL-51 MYCNHigh cell lines after BETi treatment.
    • Fig. S9. Differential gene expression analyses between MYCNRatioHigh and MYCRatioHigh TNBC.
    • Fig. S10. Effect of BETi and MEKi combination treatments on weight of treated mice.
    • Fig. S11. Evaluation of apoptosis and proliferation after BETi and MEKi treatment in TNBC PDX models.
    • Fig. S12. Evaluation of MYC family isoform expression after BETi and MEKi combination treatment in vivo.
    • Table S1. MYCN and MYC expression in breast cancer PDX models.
    • Table S2. Characteristics of patients with treatment-naïve and NAC-treated primary TNBC.
    • References (6679)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Tabular data points for experiments with a sample size of n < 20.
    • Data file S2 (Microsoft Excel format). IHC results for MYCN and MYC in primary, treatment-naïve; primary, NAC-treated; and recurrent TNBC cases.
    • Data file S3 (Microsoft Excel format). Primary drug screen results using CAL-51 MYCNLow and MYCNHigh cell lines.
    • Data file S4 (Microsoft Excel format). Secondary drug screen results using CAL-51 MYCNLow and MYCNHigh cell lines.
    • Data file S5 (Microsoft Excel format). Tabular data points for MYC family isoform TSA-IF in CAL-51 after single-agent BETi treatment.
    • Data file S6 (Microsoft Excel format). Tabular data points for MYC family isoform TSA-IF in MDA-MB-468 after single-agent BETi treatment.
    • Data file S7 (Microsoft Excel format). Tabular data points for MYC family isoform TSA-IF in TNBC cell lines and PDX tissue after BETi and MEKi single-agent and combination treatment.

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