Research ArticleCancer

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS

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Science Translational Medicine  14 Jun 2017:
Vol. 9, Issue 394, eaal5253
DOI: 10.1126/scitranslmed.aal5253
  • Fig. 1. Potent and selective down-regulation of KRAS mRNA and protein by AZD4785 in vitro and in vivo.

    (A) Sequence and binding region of AZD4785 to the 3′UTR of KRAS mRNA transcript. ds, unmodified bases; ks, cEt-modified bases; mC, 5-methylcytosine; ORF, open reading frame. (B) Relative expression of RAS mRNA isoforms measured by qRT-PCR in A431 cells after 72 hours of treatment with AZD4785 or CTRL ASO. Expression was normalized to ACTIN and shown relative to phosphate-buffered saline (PBS). Representative data from a minimum of two independent experiments are shown. (C) Western blot analysis of RAS and vinculin protein in A431 cells after 72 hours of treatment with CTRL ASO or AZD4785. (D) Mice bearing A431 tumors were treated with PBS or with 16, 32, or 48 mpk of AZD4785 5× weekly for 3 weeks. RAS isoform expression was measured in tumors at the end of the study by qRT-PCR. Expression was normalized to ACTIN and shown relative to PBS. The graph shows individual tumor data, treatment group mean, and SE. mpk/w, mpk/week. (E) IHC analysis of KRAS protein expression in A431 tumors after AZD4785 treatment. Scale bars, 200 μm.

  • Fig. 2. Effects of AZD4785 on proliferation and MAPK pathway signaling in KRAS mutant and wild-type cancer cells in vitro.

    (A) Correlation between IC50 of KRAS down-regulation and inhibition of colony formation by AZD4785 in KRAS wild-type and mutant cell lines. NCI-H1299, NCI-H1793, and Colo201 have an IC50 of colony formation inhibition by AZD4785 of >10 μM. (B) Correlation between IC50 of KRAS and DUSP6 down-regulation by AZD4785 in KRAS wild-type and mutant cell lines. NCI-H1437, NCI-H1299, and Colo201 have an IC50 of DUSP6 down-regulation by AZD4785 of >10 μM. Mean IC50 is shown and generated from a minimum of two independent experiments. (C) Western blot analysis of cell lysates from NCI-H358 or PC9 cells after treatment for 72 hours with a range of doses of AZD4785.

  • Fig. 3. Differentiation of AZD4785 from MAPK pathway inhibitors in vitro.

    (A) Western blot analysis of MAPK and PI3K signaling in NCI-H358 and PC9 cells treated with AZD4785 and CTRL ASO for 72 hours or with selumetinib, SCH772984, or DMSO for 24 hours. (B) Expression of KRAS, DUSP6, and ETV4 mRNA measured by qRT-PCR in NCI-H358 and PC9 cells treated with a dose range of AZD4785 for 72 hours or with selumetinib or SCH772984 for 24 hours. Expression was normalized to GAPDH and expressed relative to PBS control. Data are from a representative experiment (n = 2) showing mean expression of technical replicates and SD. (C) Western analysis of MAPK pathway signaling in NCI-H358 and PC9 cells after monotherapy or combination treatment with AZD4785, CTRL ASO, and selumetinib. ASO treatment was done for 72 hours, and selumetinib treatment was done for 24 hours. For combination treatments, selumetinib was added 48 hours after dosing with AZD4785 for the final 24 hours of incubation. (D) NCI-H358 and PC9 cells grown in soft agar were treated with selumetinib in combination with CTRL ASO or AZD4785. Data are from a representative experiment from a minimum of two showing mean colony number and SD.

  • Fig. 4. PD and efficacy of AZD4785 in KRAS mutant lung cancer xenograft models.

    (A to C) Mice bearing NCI-H358 tumors were treated with PBS, AZD4785, or CTRL ASO at 50 mpk 5× weekly for 4 weeks. (A) KRAS, DUSP4, and DUSP6 mRNA were measured in NCI-H358 tumors at the end of the study by qRT-PCR. The expression was normalized to 18S ribosomal RNA (rRNA) and expressed relative to PBS. Data are shown as individual tumor data, treatment group geometric mean, and SE. (B) AZD4785 significantly inhibited tumor growth of NCI-H358 tumors compared to PBS (TGI, 72%; P = 0.0047) and CTRL ASO (TGI, 75%; P = 0.001). Data are shown as the geometric mean of the tumor volume and SE. (C) Surrogate endpoint survival graphs for the NCI-H358 study. Data are shown as the percentage of remaining animals with tumors <4× the initial starting volume in each treatment group. (D to F) Mice bearing NCI-H1944 tumors were treated with PBS, AZD4785, or CTRL ASO at 50 mpk 5× weekly for 7 weeks. (D) KRAS, DUSP4, and DUSP6 mRNA were measured in NCI-H1944 tumors at the end of the study by qRT-PCR. The expression was normalized to ACTIN and presented relative to PBS. Data are shown as individual tumor data, treatment group mean, and SE. (E) AZD4785 significantly inhibited tumor growth compared to PBS (TGI, 80%; P = 0.001) and CTRL ASO (TGI, 67%; P < 0.001). Data are shown as the geometric mean of the tumor volume and SE. (F) Surrogate endpoint survival graphs for the NCI-H1944 study. Data are shown as the percentage of remaining animals with tumors <4× the initial starting volume in each treatment group.

  • Fig. 5. PD and efficacy of AZD4785 in a KRAS mutant lung cancer PDX model.

    (A) KRAS and DUSP6 mRNA expression was assessed by qRT-PCR in LXFA 983 cells in vitro after 72 hours treatment with AZD4785 or CTRL ASO. Representative data from a minimum of two independent experiments are shown. (B) Western blot analysis of MAPK signaling in LXFA 983 cells after 72 hours treatment with AZD4785 and CTRL ASO or 24 hours treatment with selumetinib. (C to G) Mice bearing LXFA 983 tumors were treated with PBS, AZD4785, or CTRL ASO at 50 mpk 5× weekly. (C) KRAS, DUSP4, and DUSP6 mRNA expression were measured in LXFA 983 tumors by qRT-PCR after 4 weeks of dosing. The expression was normalized to 18S rRNA and shown relative to PBS. Data are shown as individual tumor data, treatment group geometric mean, and SE. (D and E) KRAS protein expression measured by IHC in LXFA 983 tumors after 4 weeks of dosing. (D) Representative images are shown (scale bars, 100 μm) and (E) quantitation of KRAS (membrane H score) determined by Image analysis platform. Data are the mean membrane H score and SE. (F) AZD4785 significantly inhibited LXFA 983 tumor growth at the end of study compared to PBS (TGI, 98% at day 41, P < 0.001) and CTRL ASO (TGI, 95%; P < 0.001). Data are shown as the geometric mean of the tumor volume and SE. The arrow indicates the time point at which half of the animals were removed for PD analysis. (G) Surrogate endpoint survival graphs for the LXFA 983 study. Data are shown as the percentage of remaining animals in each treatment group with tumors <4× the initial starting volume. Two cohorts of animal are shown: data from all animals (n = 15) up to day 27 (filled symbol, unbroken line) and data from animals (n = 8) treated for 41 days (open symbol, broken line).

  • Fig. 6. PD and efficacy of AZD4785 in a KRAS wild-type lung cancer PDX model.

    Mice bearing LXFA 526 tumors were treated with PBS, AZD4785, or CTRL ASO at 50 mpk 5× weekly. (A) KRAS and DUSP6 expression measured by qRT-PCR in the LXFA 526 tumors after 4 weeks of dosing. The expression is normalized to 18S rRNA and expressed relative to PBS. (B and C) KRAS protein expression measured by IHC in LXFA 526 tumors after 4 weeks of dosing. (B) Representative images are shown (scale bars, 100 μm) and (C) quantitation of KRAS (H score) determined by Image analysis platform. Data are the mean H score and SE. (D) AZD4785 showed modest inhibition of tumor growth versus PBS (51%; P = 0.001); however, there was no significant TGI compared to the CTRL ASO. Data shown are treatment group geometric mean and SE. (E) Surrogate endpoint survival graphs for the LXFA 526 study. Data are shown as the percentage of remaining animals with tumors <4× the initial starting volume in each treatment group.

  • Fig. 7. ASO-mediated KRAS knockdown in mouse and monkey to assess tolerability.

    (A and B) BALB/c mice were treated twice weekly with PBS, CTRL ASO, or mouse-selective KRAS (mKRAS) ASOs at 50 mpk for 6 weeks. (A) IHC analysis of KRAS protein in mouse tissues at the end of the study (scale bars, 200 μm). (B) qRT-PCR measuring KRAS mRNA in mouse tissues at the end of the study. mRNA expression was normalized to total RNA and expressed relative to PBS. Individual animal data, mean, and SE are shown. (C) Sequence alignment of monkey and human KRAS mRNA isoforms 3′ of the open reading frame, with the binding site of AZD4785 highlighted in red. (D) qRT-PCR demonstrating KRAS mRNA down-regulation in primary cynomolgus monkey hepatocytes after transfection with AZD4785. mRNA expression was normalized to total RNA and presented relative to PBS. Cynomolgus monkeys were treated for 6 weeks with AZD4785 or vehicle. For the first week, animals were subcutaneously dosed with 40 mpk four times and subsequently once weekly with 40 mpk. (E) IHC analysis of KRAS protein in monkey tissues at the end of the study (scale bars, 200 μm). (F) qRT-PCR measuring KRAS mRNA in monkey tissues at the end of the study. mRNA expression was normalized to total RNA and presented relative to PBS. Individual animal data, mean, and SE are shown.

  • Table 1. AZD4785 PK measurements from liver at the end of the LXFA 983 studies.
    ModelStudyDose
    concentration
    Length of study
    (days)
    Liver PK
    (μg/g)
    MeanSD
    LXFA
    983
    P198U/
    Q610
    250 mpk/week27215.232.8
    LXFA
    983
    P198U/
    Q610
    250 mpk/week41239.029.8
    LXFA
    983
    P198U3/
    Q738
    250 mpk/week35407.853.1
    LXFA
    983
    P198U3/
    Q738
    125 mpk/week35344.449.1

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/394/eaal5253/DC1

    Materials and Methods

    Fig. S1. Effect of AZD4785 on KRAS, DUSP6, and ETV4 mRNA expression in KRAS mutant and KRAS wild-type cell lines.

    Fig. S2. Sensitivity of NSCLC lines to KRAS knockdown by ASO in 2D versus 3D growth assays.

    Fig. S3. Effect of AZD4785 on colony formation in KRAS mutant and KRAS wild-type cell lines.

    Fig. S4. Effect of AZD4785, selumetinib, and SCH772984 on the proliferation of KRAS mutant and KRAS wild-type cell lines.

    Fig. S5. Effect of AZD4785, selumetinib, and SCH772984 on signaling in KRAS mutant and KRAS wild-type cell lines.

    Fig. S6. Effects of AZD4785 and selumetinib combination on signaling and proliferation of NSCLC cells.

    Fig. S7. In vivo study assessing the kinetics of tumor PD and liver PK of AZD4785.

    Fig. S8. Effect of AZD4785 treatment on HRAS and NRAS expression in the NCI-H358 xenograft model.

    Fig. S9. Tolerability of AZD4785 treatment in xenograft studies.

    Fig. S10. Waterfall plots of xenograft and PDX studies.

    Fig. S11. PD and efficacy of AZD4785 and additional cEt KRAS ASOs in the NCI-H358 model.

    Fig. S12. Selectivity of murine KRAS ASOs and impact on MAPK pathway signaling in liver tissue from mice.

    Fig. S13. Summary of the effects of the murine KRAS ASOs on body weight, plasma chemistry, and circulating blood cells in mice.

    Fig. S14. Selectivity of AZD4785 and impact on MAPK pathway signaling in liver tissue from monkeys.

    Fig. S15. Impact of murine KRAS ASOs on plasma concentrations of inorganic phosphates and calcium.

    Table S1. Summary of predicted off-targets for AZD4785.

    Table S2. Details of the cell lines used in the study.

    Table S3. Activity of AZD4785 in KRAS mutant NSCLC xenograft models.

    Table S4. Inhibition of KRAS mRNA across a panel of normal tissues after treatment of mice with murine KRAS ASOs.

    Table S5. Summary of tissue histopathology in the mouse tolerability study after treatment with the murine-selective KRAS ASOs.

    Table S6. Summary of body and organ weights, plasma chemistries, and circulating blood cells in the mice after treatment with the murine-selective KRAS ASOs.

    Table S7. Summary of tissue histopathology in the monkey tolerability study after treatment with AZD4785.

    Table S8. Summary of body and organ weights in the monkey tolerability study after AZD4785 treatment.

    Table S9. Summary of circulating blood cells in the monkey tolerability study after AZD4785 treatment.

    Table S10. Summary of plasma chemistries in the monkey tolerability study after AZD4785 treatment.

    Table S11. Summary of urinalysis in the monkey tolerability study after AZD4785 treatment.

    Table S12. Details of the parameters used for analyzing colony formation across the cell line panel.

    Table S13. Summary of the individual animal tumor volumes in the xenograft and PDX studies.

    References (52, 53)

  • Supplementary Material for:

    Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS

    Sarah J. Ross, Alexey S. Revenko, Lyndsey L. Hanson, Rebecca Ellston, Anna Staniszewska, Nicky Whalley, Sanjay K. Pandey, Mitchell Revill, Claire Rooney, Linda K. Buckett, Stephanie K. Klein, Kevin Hudson, Brett P. Monia, Michael Zinda, David C. Blakey, Paul D. Lyne,* A. Robert Macleod*

    *Corresponding author. Email: paul.lyne{at}astrazeneca.com (P.D.L.); rmacleod{at}ionisph.com (A.R.M.)

    Published 14 June 2017, Sci. Transl. Med. 9, eaal5253 (2017)
    DOI: 10.1126/scitranslmed.aal5253

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Effect of AZD4785 on KRAS, DUSP6, and ETV4 mRNA expression in KRAS mutant and KRAS wild-type cell lines.
    • Fig. S2. Sensitivity of NSCLC lines to KRAS knockdown by ASO in 2D versus 3D growth assays.
    • Fig. S3. Effect of AZD4785 on colony formation in KRAS mutant and KRAS wild-type cell lines.
    • Fig. S4. Effect of AZD4785, selumetinib, and SCH772984 on the proliferation of KRAS mutant and KRAS wild-type cell lines.
    • Fig. S5. Effect of AZD4785, selumetinib, and SCH772984 on signaling in KRAS mutant and KRAS wild-type cell lines.
    • Fig. S6. Effects of AZD4785 and selumetinib combination on signaling and proliferation of NSCLC cells.
    • Fig. S7. In vivo study assessing the kinetics of tumor PD and liver PK of AZD4785.
    • Fig. S8. Effect of AZD4785 treatment on HRAS and NRAS expression in the NCI-H358 xenograft model.
    • Fig. S9. Tolerability of AZD4785 treatment in xenograft studies.
    • Fig. S10. Waterfall plots of xenograft and PDX studies.
    • Fig. S11. PD and efficacy of AZD4785 and additional cEt KRAS ASOs in the NCI-H358 model.
    • Fig. S12. Selectivity of murine KRAS ASOs and impact on MAPK pathway signaling in liver tissue from mice.
    • Fig. S13. Summary of the effects of the murine KRAS ASOs on body weight, plasma chemistry, and circulating blood cells in mice.
    • Fig. S14. Selectivity of AZD4785 and impact on MAPK pathway signaling in liver tissue from monkeys.
    • Fig. S15. Impact of murine KRAS ASOs on plasma concentrations of inorganic phosphates and calcium.
    • Table S1. Summary of predicted off-targets for AZD4785.
    • Table S2. Details of the cell lines used in the study.
    • Table S3. Activity of AZD4785 in KRAS mutant NSCLC xenograft models.
    • Table S4. Inhibition of KRAS mRNA across a panel of normal tissues after treatment of mice with murine KRAS ASOs.
    • Table S5. Summary of tissue histopathology in the mouse tolerability study after treatment with the murine-selective KRAS ASOs.
    • Table S6. Summary of body and organ weights, plasma chemistries, and circulating blood cells in the mice after treatment with the murine-selective KRAS ASOs.
    • Table S7. Summary of tissue histopathology in the monkey tolerability study after treatment with AZD4785.
    • Table S8. Summary of body and organ weights in the monkey tolerability study after AZD4785 treatment.
    • Table S9. Summary of circulating blood cells in the monkey tolerability study after AZD4785 treatment.
    • Table S10. Summary of plasma chemistries in the monkey tolerability study after AZD4785 treatment.
    • Table S11. Summary of urinalysis in the monkey tolerability study after AZD4785 treatment.
    • Table S12. Details of the parameters used for analyzing colony formation across the cell line panel.
    • Table S13. Summary of the individual animal tumor volumes in the xenograft and PDX studies.
    • References (52, 53)

    [Download PDF]

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