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Cellular activity of BOS172722

Metabolite changes that constitute the Venn diagram in Figure 2. Significant changes listed are reductions (labelled "-1") or increases (labelled "1") in concentrations relative to the relevant controls.

Pediatric glioblastoma (pGBM), although rare, is one of the leading causes of cancer-related deaths in children, with tumors essentially refractory to existing treatments. We have identified IGF1R to be a potential therapeutic target in pGBM due to gene amplification and high levels of IGF2 expression in some tumor samples, as well as constitutive receptor activation in pGBM cell lines. To evaluate the therapeutic potential of strategies targeting the receptor, we have carried out in vitro and in vivo preclinical studies using the specific IGF1R inhibitor NVP-AEW541. A modest inhibitory effect was seen in vitro, with GI50 values of 5 to 6 μmol/L, and concurrent inhibition of receptor phosphorylation. Specific targeting of IGF1R with short interfering RNA decreased cell viability, diminished downstream signaling through phosphoinositide 3-kinase (PI3K), and induced G1 arrest, effects mimicked by NVP-AEW541, both in the absence and presence of IGF2. Hallmarks of PI3K inhibition were observed after treatment with NVP-AEW541 by expression profiling and Western blot analysis. Phospho–receptor tyrosine kinase (RTK) arrays showed phosphorylation of platelet-derived growth factor receptor (PDGFR) α/β in pGBM cells, suggesting coactivation of an alternative RTK pathway. Treatment of KNS42 with the PDGFR inhibitor imatinib showed additional effects targeting the mitogen-activated protein kinase pathway, and cotreatment of the PDGFR inhibitor imatinib with NVP-AEW541 resulted in a highly synergistic interaction in vitro and increased efficacy after 14 days therapy in vivo compared with either agent alone. These data provide evidence that inhibition of IGF1R, in combination with other targeted agents, may be a useful and novel therapeutic strategy in pGBM. Mol Cancer Ther; 10(8); 1407–18. ©2011 AACR.

Supplementary Figure 3 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

PDF file - 101K

Supplementary Figure 1 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

Summary of baseline reproducibility of candidate plasma metabolite biomarkers (n=17 patients).

Characteristics of dose-dependent changes in candidate plasma metabolite pharmacodynamic biomarker concentrations in 41 patients, as determined on day 1 of pictilisib clinical trial. Cells with p-values

Supplementary Figures 1-4 from Preclinical Pharmacology, Antitumor Activity, and Development of Pharmacodynamic Markers for the Novel, Potent AKT Inhibitor CCT128930

Supplementary Figure Legends and Supplementary Table 1 from Preclinical Pharmacology, Antitumor Activity, and Development of Pharmacodynamic Markers for the Novel, Potent AKT Inhibitor CCT128930

Horizon PTEN +/- cell line panel and Enrichment of mutated genes in cell lines sensitive to BOS172722

Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora A and B kinase overexpression has also been associated with various human cancers, and as such, they have been extensively studied as novel antimitotic drug targets. Here, we characterize the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC50 values in both biochemical and cellular assays and exhibits antiproliferative activity against a wide range of human solid tumor cell lines. CCT137690 efficiently inhibits histone H3 and transforming acidic coiled-coil 3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumor cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy, and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 downregulation, and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumor growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN-amplified neuroblastoma. Mol Cancer Ther; 10(11); 2115–23. ©2011 AACR.

Changes in candidate plasma metabolite pharmacodynamic biomarker concentrations over first 2 weeks in 17 patients treated with {greater than or equal to}330 mg once-daily of pictilisib. Cells with p-values

Heat map of changes (relative to vehicle control) in candidate plasma and tumor metabolite biomarkers at 8 hours post-dosing of mice bearing U87MG xenografts with pictilisib 75mg/kg or buparlisib 60mg/kg (a, acyl; aa, acyl-acyl; ae, acyl-alkyl; Cx:y, where x is the number of carbons in the fatty acid side chain; y is the number of double bonds in the fatty acid side chain; AC, acylcarnitine; DC, decarboxyl; M, methyl; OH, hydroxyl; PC, phosphatidylcholine)

Changes in plasma metabolites across timepoints by patient/sample.

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. Mol Cancer Ther; 10(2); 360–71. ©2010 AACR.

Supplementary Figure 2 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

Supplementary Figure 4 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

Time of day variation in metabolites in healthy volunteers and in patients treated with the MEK inhibitor.

Supplementary Figure Legends 1-4 from Enhanced Efficacy of IGF1R Inhibition in Pediatric Glioblastoma by Combinatorial Targeting of PDGFRα/β

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.

CCR Translation for This Article from Dual Blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) Pathways Synergistically Inhibits Rhabdomyosarcoma Cell Growth In Vitro and In Vivo

T2*-weighted image (TE=4.9 ms) for a stage IV HNSCC patient (Pt No: 5). Marker lines represent image positions used in the assessment of motion correction. B: Time traces of nodal (LN) and primary (P) projections without (S(t)) and with motion correction (S(t)MOCO). Pronounced global motion (i.e. swallowing) is marked with an asterisk. Persistent motion artifacts present in the primary tumor are indicated with an arrow.

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.

Supplementary Data from Mechanistic Evaluation of the Novel HSP90 Inhibitor NVP-AUY922 in Adult and Pediatric Glioblastoma

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, 63KB, Kinase inhibitory profile of SAR-020106 at 10 μMa.

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.

Scatter plots comparing individual MRI measurements (tumor volume, baseline R2* and percentage of fluctuating volume) against pathology (Hoechst 33342 uptake for perfusion, pimonidazole adduct formation for hypoxia) determined from the cohort of CALR xenografts.

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.

Fluctuating volume percentages detected for HNSCC metastatic lymph node ROIs between two MR sessions 48 hours apart (solid lines - responders, dashed lines - non responders).

Examples of Ktrans and R2* distributions for fluctuating and non-fluctuating parts of human HNSCC (Patient No 5, N2). The non-enhancing part of the tumor (Ktrans = 0) is indicated with an asterisk.

Supplementary Figure Captions

Examples of R2* time series (left column, R2* range 40 s-1) and corresponding power frequency spectra (R2* power range displayed: 0 - 30) for voxels within human HNSCC classified as significantly fluctuating (tumor - top two rows) and without significant fluctuations (tumor - middle two rows, adjacent muscle - bottom row).

Supplementary Table & Figure Legends - PDF file 41K, Detailed captions for: Suppl Table S1. Immunohistochemical analysis indicates correlation between pAKT(Ser473),pERK1/2(Thr202/Tyr204) and pS6(Ser235/236) in primary tumours Fig S1. High level expression of p110alpha in two of four ARMS cell lines. Fig S2. PIK3CA KD does not induce cell cycle arrest or apoptosis in the growth inhibited RMS cells. Fig S3. Downregulation of PTEN expression appears to be associated with delayed inhibition of AKT phosphorylation in RH30 cells following p110alpha KD. Fig S4 Increased sensitivity of the RMS-1 PIK3CA KD line to TGX221, and of the RMS-1, RD and RMS-YM KD lines to ZSTK474. Fig S5 Equivalent changes in the biomarkers of MAPK and PI3K activity at 6h in vivo, to those seen in vitro following acute dosing with AZD6244 and AZD8055 alone, and in combination, with recovery to control levels correlating with plasma and tumor clearance of both drugs. Fig S6 Tolerability study of twice daily dosing of 10mk/kg po AZD6244 and AZD8055 indicates reversible weight loss with combination treatment, while PK analysis of tail vein blood spots confirms drug-drug interactions. Fig S7 Dose-finding therapeutic study defines a once daily treatment schedule of AZD6244, 10mg/kg p.o., and AZD8055, 20mg/kg p.o., as well tolerated and active when given in combination

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).

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials. [Mol Cancer Ther 2009;8(7):1725–38] [Mol Cancer Ther 2009;8(7):1725–38]

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.

Supplementary Figure 1 from Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Supplementary Methods - PDF file 44K, Method for the preparation and analysis of AZD8055, AZD6244 and BEZ235 by LC-MS/MS Drug-drug interaction assay