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PI3K plays a key role in cellular metabolism and cancer. Using a mass spectrometry–based metabolomics platform, we discovered that plasma concentrations of 26 metabolites, including amino acids, acylcarnitines, and phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors compared with non–tumor-bearing controls and in addition were increased following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These candidate metabolomics biomarkers were evaluated in a phase I dose-escalation clinical trial of pictilisib. Time- and dose-dependent effects were observed in patients for 22 plasma metabolites. The changes exceeded baseline variability, resolved after drug washout, and were recapitulated on continuous dosing. Our study provides a link between modulation of the PI3K pathway and changes in the plasma metabolome and demonstrates that plasma metabolomics is a feasible and promising strategy for biomarker evaluation. Also, our findings provide additional support for an association between insulin resistance, branched-chain amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther; 15(6); 1412–24. ©2016 AACR.

MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor.

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.

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Mol Cancer Ther; 9(1); 89–100

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]

CHK1 is a serine/threonine kinase that is activated in response to single strand breaks (SSBs) in DNA caused by either direct DNA damage (e.g. by genotoxic chemotherapeutic agents) or replication stress. Activation of CHK1 initiates a signaling cascade culminating in cell cycle arrest leading to DNA repair, senescence or death. Inhibition of CHK1 abrogates cell cycle arrest, inhibits DNA repair and enhances tumor cell death following DNA damage by a range of chemotherapeutic agents. Cells lacking intact G1 checkpoints through inactivation of p53 are particularly dependent on S and G2/M checkpoints and therefore expected to be more sensitive to genotoxic treatment in the presence of a CHK1 inhibitor, whereas normal cells with functional G1 checkpoints would be predicted to undergo less cell death. Thus CHK1 is a validated therapeutic target for cancer treatment. In a collaborative program with Sareum Ltd (Cambridge, UK) we used structure-based design to progress fragment hits to potent inhibitors of CHK1, including our recently published inhibitor SAR-020106. Guided by both biochemical and molecular pharmacological studies, we identified a lead series with pM to nM activity against the CHK1 enzyme in vitro and high selectivity vs CHK2 (200- to >500-fold) and CDK1. The lead series compounds have both good in vitro ADME and in vivo pharmacokinetic (PK) properties. From this lead series we identified CCT244747 as a potent and selective CHK1 inhibitor with oral efficacy. Specifically, CCT244747 is an 8nM inhibitor of CHK1 with >1000-fold selectivity versus CDK1 and CHK2 and excellent selectivity in a kinase panel screen of 120 kinases (only 8/120 with > 80% inhibition at 10μM). CCT244747 shows sub-micromolar activity in a cell-based checkpoint abrogation assay and potentiates the cytotoxicity of a selection of genotoxic chemotherapeutics including gemcitabine in colon, pancreatic and lung human tumor cell lines. CCT244747 has oral bioavailability of 61% in mice with a plasma half-life of approximately 1 hour. Further PK/PD evaluation demonstrated biomarker modulation in human tumor xenografts (pSer296 CHK1) at 6 and 24 hours post CCT244747 administration, in combination with gemcitabine. Efficacy studies of CCT244747 in combination with both irinotecan and gemcitabine, showed significant oral antitumor activity. For example, in the HT29 human colon tumor xenograft model the average percentage treated/control (%T/C) tumor weights were 15.4% for gemcitabine +CCT244747 combined, versus 62.5% and 88.4%, for gemcitabine and CCT244747 alone, respectively as measured on day 18 of therapy. There is now also evidence that CHK1 inhibitors may have single agent activity in certain cancers. In particular, CHK1 was recently identified in an RNAi screen as a therapeutic target in MYCN amplified neuroblastoma and we have shown significant efficacy of CCT244747 in a MYCN-driven transgenic mouse model of neuroblastoma (TH-MYCN) using MRI tumor volume measurements and explant tumor weights as measures of outcome. In summary, we have identified CCT244747 as a novel, potent and selective inhibitor of CHK1, which demonstrates biomarker modulation and efficacy both alone in neuroblastoma and in combination with genotoxic chemotherapeutic agents in common solid tumors, when delivered by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A228.

Internal tandem duplication or kinase domain point mutations of the fms-like tyrosine kinase 3 gene (FLT3-ITD, -TKD) result in constitutive FLT3 kinase activation and promote leukemic proliferation. FLT3-ITD occurs in 20–35% of adults and 15% of children with acute myeloid leukemia and confers a poor prognosis in both age groups. Activated FLT3 therefore represents a valid therapeutic target and several FLT3 inhibitors are currently in phase II-III clinical trials. The clinical impact of FLT3 inhibitors has thus far been limited by transient responses when used as single agents due to lack of sustained FLT3 inhibition and the emergence of secondary acquired resistance following treatment. Aurora kinases are a family of serine-threonine kinases that play a key role in several stages of mitosis. Although studies in leukemia have been limited to cell lines and small patient cohorts, over-expression of Aurora A has been consistently demonstrated and Aurora kinase inhibitors show potential in the treatment of AML. Our hypothesis is that simultaneous FLT3 / Aurora inhibition will improve single-agent efficacy and reduce susceptibility to FLT3-ligand-driven and FLT3 mutation-driven resistance in FLT3-ITD positive AML. In addition, we have generated a model of FLT3 inhibitor resistance by prolonged treatment of MOLM-13 cells with the selective FLT3 inhibitor MLN518. These resistant cells, termed MOLM-13-RES, are doubly-mutated with FLT3-ITD and a FLT3-TKD (D835Y) point mutation and over-express Survivin. Here we demonstrate that such doubly-mutated AML cells are sensitive to the dual FLT3-Aurora inhibitor CCT241736. CCT241736, an imidazo[4,5-b]pyridine derivative discovered at our Institution, is a novel, orally bioavailable, potent and highly selective FLT3 and pan-Aurora inhibitor [S(10) = 0.057; a fraction of 386 kinases inhibited >90% when screened at 1 μM of CCT241736]. In biochemical assays, CCT241736 has IC50 values against FLT3, Aurora A and Aurora B of 0.035, 0.015 and 0.1 μM respectively. Furthermore, CCT241736 inhibits a wide range of FLT3 mutants, including FLT3-ITD, -D835Y, -D835H, -K663Q and -N841I. In cellular assays, CCT241736 inhibits viability of the human FLT3-ITD positive AML cell lines MOLM-13 and MV-4;11 with EC50 values of 0.1 and 0.27 M respectively. In vivo, mouse tumor xenograft models of MOLM-13, MV-4;11 and the doubly-mutated cell line, MOLM-13-RES, are also sensitive to CCT241736, with biomarker modulation consistent with dual inhibition of FLT3 and Aurora kinases. In summary, dual inhibition of FLT3 and the critical mitotic kinase Aurora by CCT241736 may represent a novel treatment strategy for FLT3-mutated AML by overcoming the effects of high FL levels and limiting resistance caused by secondary mutations of the FLT3 gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B74.