Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

PURPOSE: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. EXPERIMENTAL DESIGN: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. RESULTS: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. CONCLUSIONS: Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.

FGF7-FGFR2 autocrine signaling increases growth and chemoresistance of fusion-positive rhabdomyosarcomas.

Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas.

Orally bioavailable CDK9/2 inhibitor shows mechanism-based therapeutic potential in MYCN-driven neuroblastoma.

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma.

Identification of ovarian high-grade serous carcinoma cell lines that show estrogen-sensitive growth as xenografts in immunocompromised mice.

Ovarian cancer remains a significant challenge in women worldwide. Tumors of the high-grade serous carcinoma (HGSC) type represent the most common form of the disease. Development of new therapies for HGSC has been hampered by a paucity of preclinical models in which new drugs could be tested for target engagement and anti-tumor efficacy. Here, we systematically assessed in vivo growth of ovarian cancer cells, including six validated HGSC cell lines, in highly immunocompromised NSG mice by varying the injection site. We found that, with the exception of OVCAR3, HGSC cell lines COV318, COV362, KURAMOCHI, OVCAR4, and OVSAHO, generally demonstrate poor growth as either subcutaneous or intraperitoneal xenografts. Intrabursal injections performed with KURAMOCHI and COV362 cells did not improve tumor growth in vivo. Additional analysis revealed that OVSAHO and COV362 express moderate levels of estrogen receptor (ERα), which translated into improved growth of xenografts in the presence of 17ß-Estradiol. Surprisingly, we also found that the growth of the widely used non-HGSC ovarian cell line SKOV3 could be significantly improved by estrogen supplementation. By describing successful establishment of estrogen-sensitive HGSC xenograft models, OVSAHO and COV362, this work will enable testing of novel therapies for this aggressive form of ovarian cancer.

Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736.

Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

Correction: Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

ALK2 inhibitors display beneficial effects in preclinical models of ACVR1 mutant diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal childhood brainstem tumour, with a quarter of patients harbouring somatic mutations in ACVR1, encoding the serine/threonine kinase ALK2. Despite being an amenable drug target, little has been done to-date to systematically evaluate the role of ACVR1 in DIPG, nor to screen currently available inhibitors in patient-derived tumour models. Here we show the dependence of DIPG cells on the mutant receptor, and the preclinical efficacy of two distinct chemotypes of ALK2 inhibitor in vitro and in vivo. We demonstrate the pyrazolo[1,5-a]pyrimidine LDN-193189 and the pyridine LDN-214117 to be orally bioavailable and well-tolerated, with good brain penetration. Treatment of immunodeprived mice bearing orthotopic xenografts of H3.3K27M, ACVR1R206H mutant HSJD-DIPG-007 cells with 25 mg/kg LDN-193189 or LDN-214117 for 28 days extended survival compared with vehicle controls. Development of ALK2 inhibitors with improved potency, selectivity and advantageous pharmacokinetic properties may play an important role in therapy for DIPG patients.

Suppression of interferon gene expression overcomes resistance to MEK inhibition in KRAS-mutant colorectal cancer.

Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.

Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N2-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N8-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722).

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.

Correction to: An in vitro and in vivo study of the combination of the heat shock protein inhibitor 17-allylamino-17-demethoxygeldanamycin and carboplatin in human ovarian cancer models.

The corresponding author of this article has informed us of concerns about the immunoblots in Fig. 2 which were carried out in the collaborating laboratory of Professor Ann Jackman.

Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

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. Mol Cancer Ther; 16(10); 2315-23. ©2017 AACR.

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737).

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach.

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.

Plasma Metabolomic Changes following PI3K Inhibition as Pharmacodynamic Biomarkers: Preclinical Discovery to Phase I Trial Evaluation.

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.

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19.

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.

The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma.

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4 nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220 nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.