Article Figures & Data
Figures
- Figure 1.
AZD0364: structure, binding mode, potency, and selectivity profile. A, Chemical structure of AZD0364. B, Crystal structure of AZD0364 bound to the ATP binding site of ERK2. C, Summary table of biochemical and cellular potency of AZD0364 and reported ERK1/2 inhibitors. D, Kinases in the Thermo Fisher Scientific 122-kinase panel that showed greater than 80% inhibition/binding after 1 μmol/L treatment with AZD0364 are highlighted in red and listed in Supplementary Fig. S1C.
- Figure 2.
AZD0364 reduces RAS/MAPK pathway output in a time- and dose-dependent manner in both BRAF- and KRAS-mutant cancer cell lines. Immunoblots of whole-cell lysates prepared from A375 (A), Calu-6 (B), and A549 (C) cell lines. Cells were treated with AZD0364 at the indicated concentrations for 2 and 24 hours. Western blots are representative of at least two experiments.
- Figure 3.
BRAF and NRAS mutations confer sensitivity to AZD0364. A, Ranked GI50 values for AZD0364 across 747 cancer cell lines; dotted line indicates GI50 sensitivity cutoff of <1 μmol/L. Sensitive cell lines are highlighted in green. B, Volcano plot of P value versus effect size for each cell line; dashed lines indicate cutoffs for log10 (P) and effect size that are considered significant. Sensitive cell lines have a negative Cohen D value. C, AZD0364 growth inhibition GI50 values in BRAF-, NRAS-, and KRAS-mutant cell lines versus WT (non-BRAF-, NRAS-, KRAS-mutant) cell lines.
- Figure 4.
In vivo antitumor efficacy of AZD0364 in RAS/MAPK-driven tumor models. A, Pharmacokinetic/pharmacodynamic relationship between AZD0364 blood-free plasma concentrations and downstream protein targets of ERK1/2, pFRA1, and p-p90RSK. B, Pharmacokinetic/pharmacodynamic relationship between AZD0364 blood-free plasma concentrations and downstream transcriptional targets of ERK1/2, DUSP6, and ETV4. Antitumor efficacy of AZD0364 at 50 mg/kg once daily (QD) treatment in A375 melanoma xenograft (mouse/group, n = 10), 70% regression from baseline on day 21 (C), Calu-6 NSCLC xenograft (mouse/group, n = 11), 10% regression reached from baseline on day 21 (D), and A549 NSCLC xenograft (mouse/group, n = 12), 68% TGI from baseline on day 21 (E). F, Efficacy of AZD0364 at various dosing schedules in the Calu-6 NSCLC xenograft (mouse/group, n = 10). G, Efficacy of AZD0364 in A375 xenograft model with acquired resistance to BRAF and MEK inhibition (mouse/group, n = 10), 89% TGI from baseline on day 21. All data are presented as mean ± SEM. BiD, twice daily.
- Figure 5.
Combined inhibition of ERK and MEK resulted in greater cell growth inhibition and greater downstream target modulation in KRAS-mutant NSCLC cell lines. Chart of growth inhibition (GI50) values in a panel of KRAS-mutant NSCLC cell lines for AZD0364 (A) and selumetinib (B). C, Loewe synergy scores for combined treatment of AZD0364 and selumetinib in a panel of KRAS-mutant NSCLC cell lines. Synergistic combinations are defined to have a Loewe score of ≥5; dashed line indicates synergy score of 10. D, Representative dose matrices for A549 and NCI-H358 cell lines showing the percent growth inhibition on a scale of 0%–200%, with 0%–100% representing inhibition of cell growth and 100%–200% representing cell death, relative to the day 0 values. E, Immunoblot from A549 NSCLC cells treated with 0.03 μmol/L AZD0364 and 0.03 μmol/L selumetinib as a single agents and in combination. F, Expression of selected RAS/MAPK-related transcripts was quantified by qRT-PCR in A549 NSCLC cells treated with AZD0364 and/or selumetinib, both at 500 nmol/L for 6, 24, 48, and 72 hours. Blue/red line indicates a 2-fold change in gene expression. These changes were significantly altered from the control for at least two timepoints, P < 0.05, pairwise Student t test.
- Figure 6.
Combined treatment of AZD0364 and selumetinib in vivo. A, Combined treatment of AZD0364 and selumetinib is more efficacious than single-agent treatment in vivo in NCI-H358 (mouse/group, n = 6–10) NSCLC and HCT-116 (mouse/group, n = 12) colorectal cancer tumor models. B, phospho-p90RSK expression was quantified by Western blotting from tumor samples taken after 21 days of dosing. C, phospho-p90RSK expression was quantified by Western blotting from A549 tumor samples taken after 6 days of dosing, AZD0364 and selumetinib dosed simultaneously. D, Ki67 was quantified by IHC from A549 tumor samples taken after 6 days of dosing; AZD0364 and selumetinib dosed simultaneously. E, Expression of selected RAS/MAPK-related transcripts over time, as quantified by qRT-PCR from A549 tumor samples taken after 6 days of dosing; AZD0364 and selumetinib dosed simultaneously. An experiment in A549 was run comparing different AZD0364 and selumetinib dosing schedules. F, Combined treatment of AZD0364 (once daily) and selumetinib (twice daily) is more efficacious than selumetinib (administered three times daily) treatment in vivo in A549 (mouse/group, n = 12) tumors. Combination dosing group reached 21% regression from baseline at day 21 and 38% regression from baseline at day 35. G, Intermittent dosing of the AZD0364 and selumetinib combination (dosed for 3.5 days every 4 days) resulted in tumor stasis (93% TGI at day 21), but not regression (mouse/group, n = 12). Tumors in continuous dosing group reach 21% regression from baseline at day 21. BiD, twice daily; QD, once daily. P value vs. vehicle: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001.
Additional Files
Supplementary Data
- Supplementary materials and methods - Supplementary materials and methods
- Supplementary Figure Legends - Supplementary Figure Legends
- Supplementary Table 1 - Primers used for targeted gene profiling on selected RAS/MAPK pathway genes either on Fluidigm or by qRT-PCR on Roche Lightcycler 480
- Supplementary Table 2 - Response of 747 cell lines to treatment with AZD0364 for 72 hours.
- Supplementary Table 3 - Cell lines defined as sensitive to AZD0364 from 747 cell panel screen
- Supplementary Table 4 - KRAS mutation status of cell lines included in in vitro combination screen
- Supplementary Figure 1 - Supplementary Figure 1
- Supplementary Figure 2 - Supplementary Figure 2
- Supplementary Figure 3 - Supplementary Figure 3
- Supplementary Figure 4 - Supplementary Figure 4
- Supplementary Figure 5 - Supplementary Figure 5
- Supplementary Figure 6 - Supplementary Figure 6
Volume 20, Issue 2
