Research ArticleCancer

Development of combination therapies to maximize the impact of KRAS-G12C inhibitors in lung cancer

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Science Translational Medicine  18 Sep 2019:
Vol. 11, Issue 510, eaaw7999
DOI: 10.1126/scitranslmed.aaw7999
  • Fig. 1 A whole-genome shRNA screen identifies linsitinib and trametinib sensitizers.

    (A) Schematic of the pooled whole-genome shRNA screen to identify sensitizers to trametinib (Tram) and/or linsitinib (Lins) in KRAS-mutant NSCLC cells. (B) Genes of the mTOR pathway identified in the shRNA screen. Genes that increased sensitivity to linsitinib when knocked down are shown in green, and genes that produced resistance are shown in orange. (C) Fold change (log2) of the number of reads in drug-treated cells versus vehicle-treated cells. Each bar represents one shRNA targeting the same gene. For MTOR and TSC2, the best five scoring shRNAs have been plotted. For RRAGC, all the shRNAs in the screen have been plotted. (D to E) H23 cells were infected with the indicated shRNAs. After puromycin selection, cells were treated for 6 days with either serial dilutions of linsitinib (MTOR and RRAGC, D) or serial dilutions of linsitinib and 1.5 nM trametinib (TSC2, E), and cell viability was measured. Scrambled shRNA (SCR) was used as control. The arrow in (E) indicates the viability when cells were treated only with 1.5 nM trametinib. Mean ± SD of biological replicates and representative of three independent experiments. (F) H23 cells were infected with the indicated shRNAs. After puromycin selection, cells were treated for 24 hours with DMSO or 1 μM linsitinib, and cell lysates were probed with the indicated antibodies. Right panels show the Western blot quantification. The numbers in each shRNA indicate the last two numbers in the shRNA name (data file S1).

  • Fig. 2 Combination of IGF1R, mTOR, and MEK inhibitors decreases the viability of KRAS-mutant NSCLC cells.

    (A and B) KRAS-mutant cells were treated with serial dilutions of everolimus (Ever) (A) or AZD8055 (AZD) (B) together with 1 μM linsitinib, 1 or 5 nM trametinib (1 nM for H23 cells and 5 nM for H358 and H1792), or the combination of both, and cell viability was measured after 6 days. Mean ± SD of two to three independent experiments. (C) Apoptosis induction (caspase-3 cleavage) in H1792 cells treated for 48 hours with serial dilutions of everolimus or AZD8055 in the presence or absence of 1 μM linsitinib, 5 nM trametinib, or the combination of both. Mean ± SD of biological replicates and representative of two independent experiments. (D and E) KRAS-mutant (D) or nontransformed AT2 lung (E) cells were treated with several drug combinations (40 nM everolimus, 1 μM linsitinib, and 1 or 5 nM trametinib) and stained with crystal violet at various time points. Veh, vehicle. (F and G) Viability data of 10 KRAS-mutant (F) and 7 KRAS wild-type (G) cells treated for 6 days with 40 nM everolimus or 20 nM AZD8055 in the presence or absence of 1 μM linsitinib and 5 nM trametinib. Mean ± SD, unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

  • Fig. 3 Combination of IGF1R with mTOR inhibitors results in a strong inhibition of PI3K/AKT and mTOR pathways.

    (A) KRAS-mutant (MUT) cells were treated for 24 hours with 40 nM everolimus, 20 nM AZD8055, 1 μM linsitinib, or the combinations. Cell lysates were probed with the indicated antibodies. (B) Quantification of the Western blots shown in (A). (C) NSCLC cell lines were treated for 24 hours with 40 nM everolimus, 20 nM AZD8055, 1 μM linsitinib, or the combinations. Cell lysates were probed with the indicated antibodies. For all Western blots, see fig. S4C. (D) Quantification of the Western blots shown in (C). Data have been normalized to the DMSO-treated cells (Ctrl). (E) Relative phosphorylation of AKT in cells treated with mTOR and IGF1R inhibitors compared with cells treated with mTOR inhibitor alone. Data are represented as mean ± SD, unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. (F and G) KRAS-mutant (MUT) (F) and KRAS wild-type (WT) (G) cells were treated for 24 hours with different drug combinations (1 μM linsitinib, 40 nM everolimus, and/or 5 nM trametinib). Cell lysates were probed with the indicated antibodies. PARP, poly(ADP-ribose) polymerase.

  • Fig. 4 mTOR inhibition causes activation of the IGF1R pathway in KRAS-mutant cells.

    (A) H1792 cells were treated with DMSO (vehicle), 100 nM everolimus, or 80 nM AZD8055 for 24 hours. Cell lysates were assayed using a phospho-RTK array kit. Phosphorylated RTKs that change their amounts with the drug treatment are highlighted with boxes. InsR, insulin receptor. (B) KRAS-mutant cells were treated for 24 hours with various concentrations (nM) of everolimus or AZD8055, and cell lysates were probed with the indicated antibodies. (C) KRAS wild-type cells were treated with DMSO, 100 nM everolimus, or 80 nM AZD8055 for 24 hours. Cell lysates were assayed using a phospho-RTK array kit. Phosphorylated RTKs that change their amounts with the drug treatment are highlighted with boxes. Shorter exposure of phospho-arrays for H2170 cells is shown in fig. S5B. (D) Cell lysates from four KRAS-mutant and four KRAS wild-type cells growing at steady-state conditions were probed with the indicated antibodies. (E) IGF1 and IGF2 concentrations in supernatant of NSCLC cell lines growing in steady-state conditions.

  • Fig. 5 Combination of mTOR, IGF1R, and MEK inhibitors promotes tumor regression in KRAS-driven lung tumors.

    (A) KrasLSL-G12D;Trp53Flox/Flox mice were treated with linsitinib (25 mg/kg), everolimus (2.5 mg/kg), and/or trametinib (2 mg/kg) (four to five mice per group). A waterfall representation of the response of each tumor after 4 weeks of treatment is shown. The inset box shows an enlargement of the combination treatments. (B and C) Mice with urethane-induced lung tumors were treated with vehicle or linsitinib (17 mg/kg), everolimus (1.7 mg/kg), and trametinib (1.3 mg/kg) (six to seven mice per group). Waterfall representation of the response of each tumor after 4 weeks of treatment (B) or after a second cycle of 4 weeks of treatment (C). (D) KrasLSL-G12D;Stk11Flox/Flox mice were treated with vehicle, linsitinib (25 mg/kg), everolimus (2.5 mg/kg), and/or trametinib (2 mg/kg) (four mice per group). A waterfall representation of the response of each tumor after 3 weeks of treatment is shown. The inset box shows an enlargement of the combination treatments. Statistics were done using Mann-Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Drug combinations with a direct KRAS inhibitor inhibit the viability in KRAS-mutant cells.

    (A) KRAS-G12C mutant cells were treated with the KRAS-G12C inhibitor ARS-1620 (1 μM; Ars) as single treatment or in combination with the indicated drugs. Cell confluence was followed over time using Incucyte. (B) KRAS-mutant cells were treated with different drug combinations and apoptosis (caspase-3 cleavage) was monitored over time using Incucyte. (C) KRAS-mutant cells were treated with several drug combinations and stained with crystal violet at different time points. (D) KRAS-mutant cells were treated for 24 hours with several drug combinations, and cell lysates were probed with the indicated antibodies. (E) KRAS-mutant cells were treated with either ARS-1620 or the combination of ARS-1620 + linsitinib + everolimus. Cell lysates were probed with the indicated antibodies or were used to measure RAS-GTP by RAF-RAS binding domain (RBD) pull-down. (F) KRAS wild-type cells were treated with several drug combinations and stained with crystal violet at different time points. (G and H) NSCLC cells were grown in 2D-adherent monolayers (G) or 3D-spheroid suspension (H) and treated with 1 μM ARS-1620, 500 nM linsitinib and 40 nM everolimus (Lins + Ever), or the combination of the three-drugs (Ars + Lins + Ever). Cell viability was measured after 5 days. Mean ± SD of two to three independent experiments, unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. When not otherwise indicated, treatments were done using 1 μM linsitinib, 40 nM everolimus, 1 μM ARS-1620, and/or 5 nM trametinib.

  • Fig. 7 Combination of KRAS-G12C inhibitor with IGF1R and mTOR inhibitors increases in vivo efficacy.

    (A) Tumor volume of HCC44, H358, and H1373 xenografts treated with ARS-1620, linsitinib and everolimus, or the three-drug combination (five to seven mice per group, mean ± SEM). Growth curves were compared using two-way ANOVA. (B) Waterfall representation of the response of each individual tumor after 21 days of treatment. In H1373, # indicates values after 17 days of treatment. Mice were euthanized at day 17 because they had reached the maximum tumor volume. (C) 3LL cells (parental and two NRAS knockout clones) were treated with DMSO or 1 μM ARS-1620 for 24 hours. Cell lysates were probed with the indicated antibodies. (D) 3LL cells were treated with serial dilutions of ARS-1620 in the presence or absence of 1 μM linsitinib and 40 nM everolimus. Cell viability was measured after 5 days. Mean ± SEM of three independent experiments. (E) 3LL cells were treated for 24 hours with different combinations containing 1 μM ARS-1620, 1 μM linsitinib, 40 nM everolimus, and/or 5 nM trametinib. Cell lysates were probed with the indicated antibodies. (F) Relative apoptosis induction (caspase-3 cleavage) in 3LL cells after 72 hours of treatment with the same treatments as in (E). Mean ± SD of two independent experiments. (G) Tumor volume of 3LL ΔNRAS-63 cells grown subcutaneously in C57BL/6 mice. Mice were treated with different drug combinations containing ARS-1620, linsitinib, everolimus, and/or trametinib (five to seven mice per group, mean ± SEM). Growth curves were compared using two-way ANOVA. (H) Survival analysis of C57BL/6 mice bearing subcutaneous 3LL ΔNRAS-86 tumors treated with ARS-1620, linsitinib and everolimus, or the three-drug combination (four to five mice per group). All mice were treated with ARS-1620 (100 mg/kg intraperitoneally or 200 mg/kg oral gavage), linsitinib (17 mg/kg), everolimus (1.7 mg/kg), and/or trametinib (1.3 mg/kg). *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/510/eaaw7999/DC1

    Materials and Methods

    Fig. S1. A whole-genome shRNA screen identifies combinatorial drug targets.

    Fig. S2. Combination of mTOR inhibitors with IGF1R and MEK inhibitors reduces the viability of KRAS-mutant NSCLC cells.

    Fig. S3. KRAS-mutant cells show increased sensitivity to the combination of mTOR, IGF1R, and MEK inhibitors.

    Fig. S4. Combination of IGF1R with mTOR inhibitors blocks PI3K/AKT and mTOR pathways.

    Fig. S5. mTOR inhibition activates the IGF1R pathway in KRAS-mutant cells.

    Fig. S6. Combination of mTOR, IGF1R, and MEK inhibitors results in regression of KRAS-driven lung tumors.

    Fig. S7. Drug combinations with a KRAS-G12C inhibitor cause inhibition of viability in KRAS-mutant cells.

    Fig. S8. Combination of KRAS-G12C inhibitor, IGF1R, and mTOR inhibitors is effective in vivo.

    Data file S1. shRNA screen hit lists.

    Date file S2. Primary data.

    Reference (55)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. A whole-genome shRNA screen identifies combinatorial drug targets.
    • Fig. S2. Combination of mTOR inhibitors with IGF1R and MEK inhibitors reduces the viability of KRAS-mutant NSCLC cells.
    • Fig. S3. KRAS-mutant cells show increased sensitivity to the combination of mTOR, IGF1R, and MEK inhibitors.
    • Fig. S4. Combination of IGF1R with mTOR inhibitors blocks PI3K/AKT and mTOR pathways.
    • Fig. S5. mTOR inhibition activates the IGF1R pathway in KRAS-mutant cells.
    • Fig. S6. Combination of mTOR, IGF1R, and MEK inhibitors results in regression of KRAS-driven lung tumors.
    • Fig. S7. Drug combinations with a KRAS-G12C inhibitor cause inhibition of viability in KRAS-mutant cells.
    • Fig. S8. Combination of KRAS-G12C inhibitor, IGF1R, and mTOR inhibitors is effective in vivo.
    • Reference (55)

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    Other Supplementary Material for this manuscript includes the following:

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