Research ArticleCANCER EPIGENETICS

Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition

See allHide authors and affiliations

Sci. Signal.  05 Feb 2019:
Vol. 12, Issue 567, eaau2922
DOI: 10.1126/scisignal.aau2922
  • Fig. 1 ORY-1001 exhibits antiproliferative activity in a subset of SCLC cell lines.

    (A) Antiproliferative activity of ORY-1001 (in concentrations of up to 10 μM) was screened across a panel of 275 cell lines in a 4-day cell viability assay (CellTiter-Glo). EC50 values in responsive cell lines ranged from 0.1 to 23 nM. NSCLC, nonsmall cell lung cancer. GI, gastrointestinal. (B) RNA-seq analyses performed using DeSeq2 (47) in SCLC cell lines (NCI-H146, NCI-H187, NCI-H510A, and NCI-H1417) treated with 10 nM ORY-1001 for 96 hours. Differentially expressed genes (1400; table S2) with FDR < 0.05 were used for enrichment analysis with the online tool Enrichr (14), assigned according to the PANTHER and Gene Ontology (GO) biological process databases. For the PANTHER pathways listed, all significant pathways with FDR < 0.05 are shown. For GO biological processes, a subset of significant processes with FDR < 0.05 are shown, including Notch signaling (GO:0007219). (C) Heat map showing differentially expressed genes in Notch pathway (GO:0007219), showing log 2 fold change (FC) in ORY-1001–treated relative to vehicle-treated cells.

  • Fig. 2 LSD1 inhibition with ORY-1001 results in the activation of NOTCH signaling.

    (A) Dose-response curves of seven PDX model–derived cells cultured and treated ex vivo with ORY-1001 for 120 hours. Viability was assessed with the CellTiter-Glo assay and calculated relative to the vehicle control. Data are means ± SEM from at least n = 3 biological replicates. (B) Immunoblots (top) and photomicrographs (bottom) for FHSC04 cells treated ex vivo with ORY-1001. Protein abundance was assessed at 48, 72, and 96 hours of 1 nM ORY-1001 treatment, and photomicrographs were taken at 96 hours. Blots are representative of two independent experiments. c-CASP3, cleaved caspase 3. Scale bars, 100 μM. (C and D) As described in (B) for JHU-LX48 (C) and JHU-LX108 (D) cells. As a positive control for cleaved caspase 3 and PARP, lysates from FHSC04 cells treated for 96 hours with 1 nM ORY-1001 [also represented in (B)] was run on the same blot (“+” lane). Blots are representative of two independent experiments. (E) RNA-seq MA plot showing differentially expressed genes across the seven PDX models treated ex vivo with 1 nM ORY-1001. Red, up-regulated genes; green, down-regulated genes; each with log 2 fold change > 0.585 and FDR < 0.05 as identified using edgeR. CPM, counts per million. (F) Gene Ontology analysis using GOseq (19) showing the top 6 significant GO terms in the genes differentially expressed with FDR < 0.05 (see Methods and table S4 for full list). (G) Abundance of NOTCH1, REST, and ASCL1 transcripts in the PDX models ex vivo assessed using real-time PCR, with expression plotted relative to that of GAPDH (n = 3 independent experiments). (H) Immunoblot showing kinetics of changes in NOTCH1, NOTCH2, REST, and ASCL1 protein abundance in the drug-sensitive FHSC04 model treated ex vivo with 1 nM ORY-1001 for 120 hours. Data are means ± SD from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by a two-tailed unpaired Student’s t test. ACTB, beta actin.

  • Fig. 3 Differential histone acetylation of LSD1-regulated genes.

    (A) Read density tracks, visualized using University of California, Santa Cruz (UCSC) genome browser, of normalized ChIP-seq for H3K4me2 (black), H3K27Ac (red), and LSD1 (blue) in NCI-H510A and NCI-H526 cell lines. Cells were treated with vehicle (VEH) or 1 nM ORY-1001 for 96 hours. Shaded area highlights region in NOTCH1 with LSD1 binding and increased H3K27Ac upon ORY-1001 treatment in the NCI-H510A cell line. (B) Read density tracks of normalized ChIP-seq for H3K27Ac in cells derived from FHSC04 PDXs, a markedly ORY-1001–responsive model. Cells were treated ex vivo with vehicle or 1 nM ORY-1001 for 48 hours. Shaded area highlights region in NOTCH1 with increased H3K27Ac. (C and D) ChIP-PCR validation of H3K27Ac changes in NOTCH1, REST, ASCL1, and, as a control, ACTB in vehicle in SCLC cell lines treated for 96 hours (C) or FHSC04 cells treated for 48 hours (D) with vehicle or 1 nM ORY-1001 for 96 hours. Data are means ± SD from n = 3 experiments.

  • Fig. 4 Genetic perturbation of LSD1, ASCL1, and NOTCH1 in SCLC cells.

    (A) Immunoblot analysis of the indicated proteins in response to lentiviral LSD1 knockdown in the NCI-H510A SCLC cell line and the FHSC04 PDX model ex vivo. ACTB is used as a loading control. Blots are representative of two independent experiments. (B) Proliferation analysis using CellTiter-Glo in response to LSD1 knockdown, analyzed as summarized below. (C) Immunoblot analysis of the indicated proteins in response to ASCL1 knockdown in the NCI-H510A SCLC cell line and the FHSC04 PDX model ex vivo. An ORY-1001–treated FHSC04 protein extract is used as a positive control for NOTCH1 and REST. Blots are representative of two independent experiments. (D) Proliferation analysis in response to ASCL1 knockdown, as in (B). RLU, relative light units. (E) Immunoblot analysis of the indicated proteins in FHSC04TetR cells in response to an inducible expression of N1ICD [doxycycline (Dox; 1 μg/μl) for 96 hours]. (F) Growth curve analysis of FHSC04TetR in response to N1ICD expression. All data are means ± SD from n = 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.005 by a two-tailed unpaired Student’s t test.

  • Fig. 5 Pharmacological inhibition of NOTCH signaling partially rescues effects of ORY-1001.

    (A) Immunoblot analysis of the indicated proteins in response to ORY-1001 (1 nM) with or without the GSIs RO4929097 or DBZ (0.5 μM) for 48 hours in SCLC cell lines NCI-H1417 and NCI-H69 and ex vivo PDX models MSK-LX227C and FHSC04. ACTB is used as a loading control. Blots are representative of two independent experiments. (B) Immunoblot analysis of HEY1 and HES1 abundance in adherent FHSC04 cells treated as in (A). Data are means ± SD from n = 3 experiments. **P < 0.01 and ***P < 0.005 by a two-tailed unpaired Student’s t test. (C) Dose-response curves in adherent FHSC04 cells treated ex vivo for 120 hours. Viability was quantified relative to vehicle control using the CellTiter-Glo assay. Data are means ± SD from n = 4 biological replicates, each with three technical replicates. (D) An extension of (C), showing the viability of FHSC04 cells in response to ORY-1001 at the 10 nM dose, alone and in the presence of RO4929097 or DBZ (0.5 μM), with comparison to each single GSI alone and vehicle. Data are means ± SD from n = 4 experiments, each with three technical replicates. **P < 0.01 and ****P < 0.0001 by a two-tailed unpaired Student’s t test. (E) Immunoblot analysis of cleaved PARP in FHSC04 cells in response to 96-hour treatment with ORY-1001 (10 nM) alone or with RO4929097 or DBZ (0.5 μM). Blots are representative of two independent experiments.

  • Fig. 6 ORY-1001 efficacy in PDX models of SCLC.

    (A) Tumor growth inhibition curves over the treatment period (left) and Kaplan-Meier curves (right) showing time to reach 6× initial tumor volume (ITV) across six PDX models of SCLC treated with ORY-1001 (400 μg/kg once weekly) or with saline. Mice were treated with saline or ORY-1001 once tumors reached 150 mm3. Data are means ± SEM; n = 10 animals total per treatment group for FHSC04 (pooled from two experiments, each with n = 5 per group); for all others, n = 5 to 6 per group. Tumor growth curves: *P < 0.05, **P < 0.005, and ***P < 0.0005 by two-way analysis of variance (ANOVA) with Sidak’s posttest. Kaplan-Meier curves show P value from log-rank test. (B) Tumor volume plots for mice in FHSC04 model treated with saline or ORY-1001 depicted in (A) extending beyond treatment end at 21 days (solid line). Mouse tumor volumes are individually plotted for response and durability of tumor regressions. (C) Repeat of ORY-1001 treatment in FHSC04 with comparison to a CIS-ETO combination therapy (left), n = 5 mice per treatment group. Change in body weight is shown (right) as CIS-ETO treatment led to reductions in body weight not seen with ORY-1001. Cisplatin was dosed at 5 mg/kg once per week and etoposide at 10 mg/kg every 3 days. (D) TUNEL and PH3 immunohistochemistry analysis to assess proliferation and cell death in FHSC04 PDXs collected 10 days after treatment initiation. n = 5 tumors per group; P value was assessed by Student’s t test. n.s., not significant. Scale bars, 6.5 μm. (E) Volcano plot showing differentially expressed genes in FHSC04 PDXs after 10 days’ treatment with ORY-1001 as identified using edgeR. n = 5 control and 5 ORY-1001–treated mice. Red, up-regulated genes; green, down-regulated genes; FDR < 0.01. (F) Western blot analyses and quantifications (bottom) of ASCL1 and NOTCH pathway protein abundance in FHSC04 PDX in vivo. Data are means ± SEM from n = 3 mice per group; tumors harvested 10 days after treatment initiation. ***P < 0.005 by a two-tailed unpaired Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/567/eaau2922/DC1

    Fig. S1. Generation and characterization of an SCLC PDX model derived from isolated CTCs.

    Fig. S2. NOTCH pathway marker changes in PDXs treated ex vivo with ORY-1001.

    Fig. S3. Lack of NOTCH1 activation in NCI-H526 cells in response to ORY-1001.

    Fig. S4. LSD1 binding and histone modification at REST locus in response to ORY-1001 in SCLC.

    Fig. S5. Effects of ORY-1001 in nonadherent and adherent populations of FHSC04 cells.

    Fig. S6. ORY-1001 efficacy and ASCL1 abundance in SCLC PDX model FHSC36.

    Fig. S7. Toxicity analysis of in vivo responses to ORY-1001.

    Fig. S8. Genomic alterations in PDX model FHSC04.

    Fig. S9. Gene expression changes in FHSC04 in vivo in response to ORY-1001.

    Table S1. ORY-1001 inhibits proliferation in a subset of SCLC cell lines.

    Table S2. DEseq2 analysis of differentially expressed transcripts in ORY-1001–treated SCLC cell lines.

    Table S3. Characteristics of the PDX models.

    Table S4. edgeR analysis of differentially expressed transcripts in ORY-1001–treated PDX SCLCs.

    Table S5. GO analysis of ORY-1001–treated PDXs.

    Table S6. edgeR analysis of ORY-1001–treated FHSC04 PDX tumors.

    Table S7. GO analysis of ORY-1001–treated FHSC04 PDX tumors.

    Table S8. shRNA sequences.

    Table S9. Primer sequences for qPCR.

    Table S10. Antibodies.

  • The PDF file includes:

    • Fig. S1. Generation and characterization of an SCLC PDX model derived from isolated CTCs.
    • Fig. S2. NOTCH pathway marker changes in PDXs treated ex vivo with ORY-1001.
    • Fig. S3. Lack of NOTCH1 activation in NCI-H526 cells in response to ORY-1001.
    • Fig. S4. LSD1 binding and histone modification at REST locus in response to ORY-1001 in SCLC.
    • Fig. S5. Effects of ORY-1001 in nonadherent and adherent populations of FHSC04 cells.
    • Fig. S6. ORY-1001 efficacy and ASCL1 abundance in SCLC PDX model FHSC36.
    • Fig. S7. Toxicity analysis of in vivo responses to ORY-1001.
    • Fig. S8. Genomic alterations in PDX model FHSC04.
    • Fig. S9. Gene expression changes in FHSC04 in vivo in response to ORY-1001.
    • Legends for tables S1 to S10

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel file). ORY-1001 inhibits proliferation in a subset of SCLC cell lines.
    • Table S2 (Microsoft Excel file). DEseq2 analysis of differentially expressed transcripts in ORY-1001–treated SCLC cell lines.
    • Table S3 (Microsoft Excel file). Characteristics of the PDX models.
    • Table S4 (Microsoft Excel file). edgeR analysis of differentially expressed transcripts in ORY-1001–treated PDX SCLCs.
    • Table S5 (Microsoft Excel file). GO analysis of ORY-1001–treated PDXs.
    • Table S6 (Microsoft Excel file). edgeR analysis of ORY-1001–treated FHSC04 PDX tumors.
    • Table S7 (Microsoft Excel file). GO analysis of ORY-1001–treated FHSC04 PDX tumors.
    • Table S8 (Microsoft Excel file). shRNA sequences.
    • Table S9 (Microsoft Excel file). Primer sequences for qPCR.
    • Table S10 (Microsoft Excel file). Antibodies.

Navigate This Article