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Purpose: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity.Experimental Design: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay.Results: CCT244747 inhibited cellular CHK1 activity (IC50 29–170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G2 arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma.Conclusion: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies. Clin Cancer Res; 18(20); 5650–61. ©2012 AACR.

PDF file, 79K, Supplementary Table 3 - Mouse body weights for data in Figure 4A Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with gemcitabine (100mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 4 - Mouse body weights for data in Figure 4C Body Weights expressed as a percentage of the weight on day 0 for mice bearing HT29 xenografts and treated with irinotecan (25mg/kg ip), CCT244747 (150mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 5 - Mouse body weights for data in Figure 4D Body Weights expressed as a percentage of the weight on day 0 for mice bearing Calu6 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6. Supplementary Table 6 - Mouse body weights for data in Supplementary Table 2 Body Weights expressed as a percentage of the weight on day 0 for mice bearing SW620 xenografts and treated with gemcitabine (100 mg/kg iv), CCT244747 (75mg/kg po) or the combination. Values are mean�SE, n= 3 to 6.

PDF file, 92K, Supplementary Figure 2. A constrained, scaffold docking model of CCT244747 in the ATP pocket of human CHK1 realized using the SAR-020106 X-ray crystal structure (PDB 2ym8).

PDF file - 81K, Supplementary data comprising the materials and methods for (i) crystallography (ii) gene expression studies (iii) siRNA studies and Supplementary Figure legends 1-7

PDF file, 118K, Supplementary Table 2 Summary of Effects of Irinotecan, Gemcitabine and CCT244747 alone or in combination on human tumor xenografts growth delay.

PDF file, 810K, Supplementary Figure 3 Characterisation of the effects of minimally toxic concentrations of CCT244747 alone or in combination with genotoxic agents (+) in HT29 and SW620 colon cancer cell lines. A, HT29 cells were treated with SN38 (100nM) or CCT244747 alone or in combination for 24h. B, SW620 cells were treated with gemcitabine (200nM) or CCT244747 alone or in combination for 24h. Cells were pre-treated with CCT244747 alone 1h prior to cytotoxic exposure. Protein expression was assessed by western blotting (40μg per lane) as described in Figure 2 and Materials and Methods.

PDF file, 76K, Supplementary Table 1 Kinome selectivity profile of CCT244747 CCT244747 was tested at 1 μM (140 enzymes) and 10 μM (121 enzymes) for inhibition of human kinases in a radiometric protein phosphorylation assay (MRC Protein Phosphorylation Unit, Dundee University, UK). Assays were conducted in the presence of ATP at the approximate Km,ATP for each kinase. Results are quoted as % control kinase activity remaining (100% = no inhibition, 0% = complete inhibition). Kinases inhibited by >50% at each concentration are highlighted. n.d. = not determined.

XLS file - 703K, Gene expression data for CCT128930: Genes that showed at least a 1.5 Fold Change in at least one treated sample compared to vehicle control and were differentially expressed between the treatment groups (Welch ANOVA FDR5%)

PDF file, 67K, Supplementary Figure 4. Effects of scheduling and exposure times on the ability of two different CHK1 inhibitors to potentiate gemcitabine cytotoxicity in SW620 cells in vitro. A, Effect of different contact times on the capacity of SAR-020106 to enhance gemcitabine cytotoxicity in SW620 cells. Gemcitabine was continuously present (96h) at an IC50 concentration and SAR- 020106 was added at the start of treatment over a range of concentrations for the intervals shown (i.e. 0-T1, 0-T2, 0-T3, etc). Growth delay and potentiation were measured using an SRB assay as described in Materials and Methods. Potentiation Index (PI) was expressed as a percentage of the maximum PI. Values are mean�SE, for n=3-5 independent experiments. B, Treatment of synchronized SW620 cells with gemcitabine and SAR-020106. Cells were synchronized with nocodazole (100ng/ml x 16h). Eight hours following release (at G1/S boundary) cells were treated with a fixed concentration of gemcitabine (GI50) for 96h combined with different concentrations of SAR-020106 for 0-96, 0-48 or 0-24h. Values are mean�SE for 3 independent determinations. C, Effects of contact time on the potentiation of gemcitabine cytotoxicity in synchronized compared to asynchronous SW620 cells. See B and Materials and Methods for further details. Values are mean�SE, n=3-5. D, Effects of different contact times and treatment schedules on the ability of CCT244747 to enhance gemcitabine cytotoxicity in SW620 cells. Values are mean�SE, for 3-7 independent experiments.

PDF file, 36K, Supplementary Figure 5. Effects of acute CCT244747 administration on non-tumor bearing GEMM mouse body weights. CCT244747 was administered at a dose of 150mg/kg p.o. x 7days and mouse body weights were measured daily. Results are mean�SD body weights for 5 mice. There was no apparent body weight loss, toxicity or morbidity following this treatment and hemizygous MYC-N tumor bearing GEMM mice were therefore treated with 100mg/kg CCT244747 p.o. x 7 days (see Figure 5).

Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component.Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148.Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed.Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

PDF file, 58K, Supplementary Figure 1. Comparison of assays for measuring CHK1 potentiation of cytotoxic agents in vitro. A, Determination of CCT244747 GI50 alone or in combination with a fixed concentration (GI50) of gemcitabine or SN38 in SW620 colon tumor cells in vitro. The potentiation index (PI, ratio of SRB GI50 of CCT244747 alone : Combination GI50) was 4.0 and 14.8 for SN38 and gemcitabine, respectively (see Table 1 for mean�SD values). Survival curves for combinations were corrected to 100%. B, Determination of SN38 or gemcitabine GI50 alone or in combination with a fixed concentration of CCT244747 (1μM) in SW620 colon tumor cells in vitro. The potentiation index (PI, ratio of SRB GI50 of genotoxic alone : genotoxic + 1μM CCT244747) was 1.4 and 3.2 for gemcitabine and SN38, respectively. PI values for several independent experiments as shown in B were 1.2�0.058 and 2.9�0.33 (mean�SD, n=4) for SN38 and gemcitabine, respectively.

XLS file - 66K, Genes that showed at least a 1.5 Fold Change in at least one treated sample compared to vehicle control and were differentially expressed between the treatment groups (Welch ANOVA FDR5%) for both CCT128930 and AT13148

PDF file - 553K, Supplementary Figure 1. Phosphorylation of AKT substrates remains suppressed, despite pSer473 AKT induction. Supplementary Figure 2. The pharmacokinetic-pharmacodynamic profile and antitumor activity of AT13148 in PTEN-deficient PC3 human prostate cancer xenografts. Supplementary Figure 3. Pharmacodynamic biomarker study of AT13148(40mg/kg) in MES-SA human tumor xenografts. Supplementary Figure 4. The antitumour activity of AT13148 in mutant KRAS A549 human lung cancer xenografts. Supplementary Figure 5. Mouse body weight data for the MES-SA (Figure 4B) and BT474 (Figure 4C) efficacy studies. Supplementary Figure 6. The effect of AT13148 versus CCT128930 on apoptosis and cell cycle distribution in U87MG human glioblastoma cells. Supplementary Figure 7. Differential effects of 24h treatment of 10M each of the AGC kinase inhibitor AT13148 and the selective AKT inhibitor CCT128930 on cell cycle effects in PTEN-deficient U87MG human glioblastoma cells. Supplementary Table 1. Summary of the inhibitory activity of 10��M AT13148 against in vitro. Supplementary Table 2. Summary of the inhibitory activity of AT13148 against a panel of protein kinases Supplementary Table 3. Summary of the in vitro cytotoxicity of AT13148 against a panel of human tumor cell lines harboring different defects in the PI3K-AKT signaling pathway. Supplementary Table 4. Summary of the pharmacokinetics of AT13148 in mouse plasma and tissues following iv or oral administration

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