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PDF file - 10K, SEN461 affects canonical WNT ligands mediated transcription.

PDF file - 20K, Comparative WNT transcriptional activity and growth inhibition with different compound AXIN stabilizers.

PDF file - 112K, Average body weight graph.

PDF file - 98K, In vitro response of T98G cells to WNT signaling inhibition.

PDF file - 94K, Consequences of inhibition of WNT signaling in glioblastoma cells: a less proliferative and less tumorigenic phenotype.

PDF file - 32K, Effects of different compound AXIN stabilizers on TNKS1 and TNKS2.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule–mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics. Mol Cancer Ther; 12(7); 1180–9. ©2013 AACR.

PDF file - 81K

Supplementary Figures and Materials and Metholds from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Table 2 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Table 1 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Supplementary Table 3 from SB939, a Novel Potent and Orally Active Histone Deacetylase Inhibitor with High Tumor Exposure and Efficacy in Mouse Models of Colorectal Cancer

Although clinical responses in liquid tumors and certain lymphomas have been reported, the clinical efficacy of histone deacetylase inhibitors in solid tumors has been limited. This may be in part due to the poor pharmacokinetic of these drugs, resulting in inadequate tumor concentrations of the drug. SB939 is a new hydroxamic acid based histone deacetylase inhibitor with improved physicochemical, pharmaceutical, and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines. It has very favorable pharmacokinetic properties after oral dosing in mice, with >4-fold increased bioavailability and 3.3-fold increased half-life over suberoylanilide hydroxamic acid (SAHA). In contrast to SAHA, SB939 accumulates in tumor tissue and induces a sustained inhibition of histone acetylation in tumor tissue. These excellent pharmacokinetic properties translated into a dose-dependent antitumor efficacy in a xenograft model of human colorectal cancer (HCT-116), with a tumor growth inhibition of 94% versus 48% for SAHA (both at maximum tolerated dose), and was also effective when given in different intermittent schedules. Furthermore, in APCmin mice, a genetic mouse model of early-stage colon cancer, SB939 inhibited adenoma formation, hemocult scores, and increased hematocrit values more effectively than 5-fluorouracil. Emerging clinical data from phase I trials in cancer patients indicate that the pharmacokinetic and pharmacologic advantages of SB939 are translated to the clinic. The efficacy of SB939 reported here in two very different models of colorectal cancer warrants further investigation in patients. Mol Cancer Ther; 9(3); 642–52

AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.

Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.

Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

[Image: see text] SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3′s enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure–activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents.

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti- M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti- M. tuberculosis activity.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, β-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased β-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule–mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics. Mol Cancer Ther; 12(7); 1180–9. ©2013 AACR.

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human pharmacokinetics (PK) was predicted using Simcyp and allometric scaling. SB939 showed high aqueous solubility with high Caco-2 permeability. Metabolic stability was relatively higher in dog and human liver microsomes than in mouse and rat. The major metabolites formed in human liver microsomes were also observed in preclinical species. Human cytochrome P450 (P450) phenotyping showed that SB939 was primarily metabolized by CYP3A4 and CYP1A2. SB939 did not significantly inhibit human CYP3A4, 1A2, 2D6, and 2C9 (>25 mu M) but inhibited 2C19 (IC50 = 5.8 mu M). No significant induction of human CYP3A4 and 1A2 was observed in hepatocytes. Plasma protein binding in mouse, rat, dog, and human ranged between similar to 84 and 94%. The blood-to-plasma ratio was similar to 1.0 in human blood. SB939 showed high systemic clearance (relative to liver blood flow) of 9.2, 4.5, and 1.5 l . h(-1) . kg(-1) and high volume of distribution at steady state (>0.6 l/kg) of 3.5, 1.7, and 4.2 l/kg in mouse, rat, and dog, respectively. The oral bioavailability was 34, 65, and similar to 3% in mice, dogs, and rats, respectively. The predicted oral PK profile and parameters of SB939, using Simcyp and allometric scaling, were in good agreement with observed data in humans. Simcyp predictions showed lack of CYP3A4 and 2C19 drug-drug interaction potential for SB939. In summary, the preclinical ADME of SB939 supported its preclinical and clinical development as an oral drug candidate.

A series of 3-(1,2-disubstituted-1H-benzimidazol-5-yl)-N-hydroxyacrylamides (1) were designed and synthesized as HDAC inhibitors. Extensive SARs have been established for in vitro potency (HDAC1 enzyme and COLO 205 cellular IC(50)), liver microsomal stability (t(1/2)), cytochrome P450 inhibitory (3A4 IC(50)), and clogP, among others. These parameters were fine-tuned by carefully adjusting the substituents at positions 1 and 2 of the benzimidazole ring. After comprehensive in vitro and in vivo profiling of the selected compounds, SB939 (3) was identified as a preclinical development candidate. 3 is a potent pan-HDAC inhibitor with excellent druglike properties, is highly efficacious in in vivo tumor models (HCT-116, PC-3, A2780, MV4-11, Ramos), and has high and dose-proportional oral exposures and very good ADME, safety, and pharmaceutical properties. When orally dosed to tumor-bearing mice, 3 is enriched in tumor tissue which may contribute to its potent antitumor activity and prolonged duration of action. 3 is currently being tested in phase I and phase II clinical trials.

Thirty-six novel acylurea connected straight chain hydroxamates were designed and synthesized. Structure–activity relationships (SAR) were established for the length of linear chain linker and substitutions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19 showed 10–20-fold enhanced HDAC1 potency compared to SAHA. In general, the cellular potency pIC50 (COLO205) correlates with enzymatic potency pIC50 (HDAC1). Compound 5b (SB207), a structurally simple and close analogue to SAHA, is more potent against HDAC1 and HDAC6 compared to the latter. As a representative example of this series, good in vitro enzymatic and cellular potency plus an excellent pharmacokinetic profile has translated into better efficacy than SAHA in both prostate cancer (PC3) and colon cancer (HCT116) xenograft models.

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.

Polo-like kinase 1 (PLK1) has been shown to be involved in cell cycle progression of rapidly proliferating, nontransformed cells as well as tumor cells. In cancer, overexpression of PLK1 contributes to the malignant state by aberrant cell cycle regulation at the G2/M phase. We attempted to validate PLK1 function in cell-based assays using antisense oligonucleotide knockout approaches. Our own results and published findings of other groups have led to the conclusion that PLK1 is a logical point for pharmacological intervention. HTS campaigns performed against the recombinant PLK1-protein led to the identification of two PLK1 inhibitory scaffolds, one containing a 7-azapteridine ring system, the other a fused tetracyclic ketone system. These compounds showed low micromolar antiproliferative activity towards SW620, a human colon cancer cell line, most likely through cell cycle arrest at the G2/M phase. Our data suggest that PLK1 may serve as a target for discovering small molecule anticancer agents. Drug Dev. Res. 62:349–361, 2004. © 2004 Wiley-Liss, Inc.

Porcupine (PORCN), a muti-pass integral membrane-bound-O-Acyl acyltransferase (MBOAT), resides in the in the endoplasmic reticulum (ER) and is required for biogenesis of Wnt ligands. The secreted mature Wnt ligands bind to their cognate receptors (Frizzled, LRP5/6 and transmembrane receptor ROR) to form the ligand - receptor complex which is capable to activate the Wnt-β-catenin signalling cascade and downstream signalling pathways such as mTOR, GSK3, Akt, and PKC. The deregulation of and aberrant activation of the various components of Wnt-β-catenin signalling pathway have been implicated in tumorigenesis, cell proliferation, survival and differentiation. Hyperactivity of PORCN is found to be associated with cancerous cell growth. Knockdown of Porcn mRNA significantly reduced the proliferation of breast cancer cells and resulted in the delay of MDA-MB-231 tumor formation in mouse xenograft models (Covey et al 2012). Loss of function mutations of RNF43, a negative regulation of Wnt-signalling via Frizzled receptor, is recently reported to be involved with pancreatic ductal adenocarcinoma. Inhibition of the PDAC cell lines bearing RNF43 mutations enhanced Wnt-β-catenin signalling and resulted in suppression of proliferation and differentiation of PDAC tumor cells (Jiang et al 2013). Taken together porcupine could be an attractive therapeutic approach for a particular Wnt-driven cancer population. We have identified the porcupine lead compounds (ETC-159, ETC-535, ETC-611 and ETC-539) from different novel chemical scaffolds. The aim of this study was to evaluate their pharmacokinetic properties and antitumor efficacy in different cancer mouse models, the murine MMTV-Wnt1 breast cancer and the human pancreatic HPAF-II cancer. All porcupine lead compounds had good oral pharmacokinetic properties with the absolute oral bioavailability greater than 42%. They had the maximum tolerated dose (MTD-7d) up to 200 mg/kg. They produced antitumor efficacy ranging from 24% to 79% at 1 mg/kg, 38% to 89% at 3 mg/kg, and 58 to 97% at 10 mg/kg in MMTV-Wnt1 tumor mouse model. In vivo inhibition of PORCN led to reduce the expression level of Axin2 in MMTV-Wnt tumors upto 8h. At 100 mg/kg, they produced antitumor efficacy ranging from 34% to 91% in human HPAF-II pancreatic xenograft mouse model. Of 4 porcupine lead compounds, ETC-159 demonstrated great oral pharmacokinetic properties and produced significantly antitumor efficacy (p value < 0.0001) in both cancer mouse models. ETC-1922159 was selected as the preclinical development candidate and currently is under investigation in Phase 1 clinical trial. Citation Format: Vishal Pendharkar, Yun Shan Chew, Vithya Manoharan, Choon Bing Low, Hongqian Esther Ong, Soo Yei Ho, Wei Ling Wang, Jeyaraj Duraiswamy Athisayamani, Babita Madan, David Virshup, Thomas Hugo Keller, May Ann Lee, Alex Matter, Jeffrey Hill, Kanda Sangthongpitag. In vivo pharmacokinetic properties and antitumor efficacy of porcupine lead inhibitors in the orthotopic murine MMTV-Wnt1 breast tumor model and the human HPAF-II pancreatic xenograft mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1172. doi:10.1158/1538-7445.AM2017-1172

Elevated levels of the phosphorylated m7G cap binding protein, eukaryotic initiation factor 4E (eIF4E) are associated with neoplasia. An observed oncogenic signaling effect upon activation of Erk1/2 or p38 MAPKs in cells is the phosphorylation of eIF4E specifically at Serine 209 by downstream kinase MAP kinase-interacting kinase 1 and 2 (Mnks). Therefore, inhibiting Mnks could be a potential therapeutic approach that selectively targets cancer cells without introducing toxicity to normal cells. To measure the target engagement and efficacy of Mnk inhibitors, we have established a sensitive method to quantitatively detect phospho-eIF4E and non-phospho-eIF4E. Here, we report using NanoPro 100 system, an automated capillary-based immunoassay platform that could separate the phosphorylated and non-phosphorylated eIF4E isoforms based on different isoelectric points. Primary antibodies against both non-phospho eIF4E and Serine 209 phosphorylated eIF4E were used for identification and verification of eIF4E isoforms in protein samples obtained from cell lysate, blood samples and tumor samples. Two major isoforms of eIF4E with isoelectric point at 5.38 for phospho-eIF4E and 5.88 for non-phospho-eIF4E were identified in eIF4E-overexpressing K562 cells. siRNA knockdown of eIF4E expression reduced both isoforms, while treatment with Mnk inhibitor only reduced phospho-eIF4E level. The measurement and quantification of the relative changes of phosphorylated eIF4E by this method can potentially be used in monitoring the efficacy of Mnk inhibitors in xenograft and future clinical trials. Citation Format: Zhiyuan Ke, Sifang Wang, Vithya Manoharan, Susmitha Vuddagiri, Esther Ong, Sharon Lim, Sin Tiong Ong, Kanda Sangthongpitag, Nacro Kassoum, Jeffrey Hill, May Ann Lee. Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2017-3819

Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3′-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.

Tankyrases constitute potential drug targets for cancer and myelin-degrading diseases. We have applied a structure- and biophysics-driven fragment-based ligand design strategy to discover a novel family of potent inhibitors for human tankyrases. Biophysical screening based on a thermal shift assay identified highly efficient fragments binding in the nicotinamide-binding site, a local hot spot for fragment binding. Evolution of the fragment hit 4-methyl-1,2-dihydroquinolin-2-one (2) along its 7-vector yields dramatic affinity improvements in the first cycle of expansion. A crystal structure of 7-(2-fluorophenyl)-4-methylquinolin-2(1H)-one (11) reveals that the nonplanar compound extends with its fluorine atom into a pocket, which coincides with a region of the active site where structural differences are seen between tankyrases and other poly(ADP-ribose) polymerase (PARP) family members. A further cycle of optimization yielded compounds with affinities and IC50 values in the low nanomolar range and with good solubility, PARP selectivity, and ligand efficiency.

Chronic Phase Chronic Myelogenous Leukemia (CP-CML) is well treated with tyrosine kinase inhibitors (TKI) through BCR-ABL kinase modulation. However, TKI are ineffective for late-stage or blast crisis (BC) CML in which phase, granulocyte macrophage progenitors (GMPs) have acquired the ability to function as leukemic stem cells (LSCs), and are thought to act as a reservoir for TKI resistance with poor prognosis. An effective BC-CML therapy will likely have to target the BC-LSC population to ensure long-term disease control. Recent studies have shown the importance of the MAP kinase interacting serine/threonine kinase (MNK1/2)-eukaryotic translation initiation factor 4E (eIF4E) axis in sustaining BC-LSC self-renewal. Thus, a single agent which inhibits both BCR-ABL and MNK1/2 kinase simultaneously represents a rational approach to target BC-LSCs. Such an agent should be able to distinguish between normal hematopoietic stem cells (HSC) and LSCs. Here, we report the discovery of a potential BC-CML therapy that relies on the inhibition of MNK1/2 and BCR-ABL kinases using a dual specific MNK/ABL inhibitor. We will also report the in vitro and in vivo biological data, pharmacokinetic properties, and biochemical characteristics of such compounds. These compounds are able to prevent eIF4E phosphorylation, thus selectively inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs as a single agent while leaving normal HSCs unscathed. Citation Format: Joseph Cherian, Kassoum Nacro, Zhi Ying Poh, Samantha Guo, Melvyn Wai Tuck Ho, Haiyan Yang, Sharon Lim, Meng Ling Choong, Joma Kanikadu Joy, Perlyn Z. Zekui, Boping Liu, Esther Hongqian Ong, Vishal Pendharkar, Lijun Ding, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Discovery of dual MNK 1 and 2 and BCR-ABL kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2137.

Various mutations in the Wnt pathway contribute to aberrant activation of Wnt signaling, which is implicated in multiple cancers. Besides the mutation of Adenomatous polyposis coli (APC) or beta-catenin which causes the continuous activation of Wnt signaling by stabilizing beta-catenin, there are mutations of other genes such as RNF43 that regulate Wnt signaling at the level of the ligand or cell surface receptor. Inhibition of the secretion of all human Wnts by blocking their palmitoleoylation by the O-acyltransferase, Porcupine (PORCN), could be an alternative therapeutic approach. Here we have developed a compound with a novel pharmacophore that can inhibit PORCN activity and hence Wnt signaling in nanomolar concentration. In vivo efficacy study demonstrated that the compound is highly efficacious in preventing tumor growth in genetic modification model MMTV-WNT1 mouse. We have identified a subset of pancreatic cancer, cholangiocarcinoma and mucinous ovarian cancer cell lines that are sensitive to the compound. Most of these cell lines harbor mutations in RNF43. Xenograft tumor models derived from the cancer cell lines were found to be sensitive to the compound. Our results demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors in a subset of cancers with mutation in RNF43 is a feasible and nontoxic strategy to overcome the problem of redundancy of Wnts, thereby, providing new option for therapy in diseases with up regulated Wnt expression. Citation Format: Zhiyuan Ke, Babita Madan, Shermaine Q.y. Lim, Sifang Wang, Jenefer Alam, Soo Yei Ho, Duraiswamy Athisayamani Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Li Jun Ding, Vithya Monoharan, Vishal Pendharkar, Esther Ong, Jeffrey Hill, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2015-4449

A novel microsatellite marker was found within 48.5 kb of the Fas gene. The observed heterozygosity in 160 healthy unrelated controls was 0.78. There was no evidence of linkage to type I diabetes mellitus in 120 diabetic children using the transmission disequilibrium test.

Although targeting the BCR-ABL kinase with tyrosine kinase inhibitors (TKI) is effective in chronic phase (CP) CML, targeting BCR-ABL alone is not sufficient to treat late-stage or blast crisis (BC) CML. In BC CML, granulocyte macrophage progenitors (GMPs) acquire the ability to function as leukemic stem cells (LSCs), and are thought to act as a reservoir for TKI resistance. While long-term remission in BC CML is possible with allogeneic stem cell transplantation, only a minority of patients are eligible for transplantation, and may experience high morbidity and mortality. Safe and effective BC CML therapy will likely have to target the BC LSC population to ensure long-term disease control. Recent studies have shown the importance of the MAP kinase interacting serine/threonine kinase (MNK1/2)-eukaryotic translation initiation factor 4E (eIF4E) axis in sustaining BC LSC self renewal. Thus, a single agent which inhibits both BCR-ABL and MNK1/2 kinase simultaneously represents a rational approach to target BC LSCs. Such an agent should be able to distinguish between normal hematopoietic stem cells (HSC) and LSCs. Here, we report the discovery, structure activity relationships, pharmacokinetic properties, and biochemical characteristics of dual MNK1/2 and BCR-ABL inhibitors, ETC-027 and ETC-219. These compounds are able to prevent eIF4E phosphorylation, BCR-ABL-driven growth and proliferation of BC CML cells, as well as inhibit the MNK-eIF4E-dependent self-renewal function of BC LSCs, while leaving normal HSCs untouched. Citation Format: Joseph Cherian, Kassoum Nacro, Zhi Ying Poh, Samantha Guo, Melvyn Ho, Haiyan Yang, Sharon Lim, Meng Ling Choong, Jun Li Ding, Joma Kanikadu Joy, Zekui Perlyn Kwek, Boping Liu, Hongqian Esther Ong, Vishal Pendharkar, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Discovery of dual MNK 1 and 2 and BCR-ABL kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 948. doi:10.1158/1538-7445.AM2014-948

Pharmacologic inhibition of BCR-ABL by tyrosine kinase inhibitors (TKI) provides effective therapy for chronic phase but not blast crisis (BC) CML. The inability of TKIs to control BC CML is due to the reactivation of BCR-ABL through TKI resistance-conferring mutations, as well as the activation of alternative oncogenic pathways that mediate acquired leukemia stem cell (LSC) function and differentiation block in leukemic progenitors. Accordingly, the development of effective BC therapeutics will require reversing multiple contributors to the BC phenotype, and indicates that targeting BCR-ABL alone will not be sufficient to control BC CML. Indeed, despite the ability of third-generation TKIs to effectively inhibit the majority of clinically-relevant BCR-ABL kinase domain mutations, most patients with BC treated with these agents continue to relapse despite initial responses. Because our recent work has highlighted the importance of the MNK kinase-eIF4E axis in b-catenin-associated BC self-renewal (Lim et al., PNAS18;110(25):E2298-307, 2013), we set out to develop compounds capable of targeting BCR-ABL, TKI-resistant BCR-ABL mutants, as well as the MNK kinases simultaneously. Here, we report the discovery and characterization of two lead compounds ETC-219 and ETC-027 that inhibit clinically relevant BCR-ABL isoforms (E255K, T315I) as well as the MNK1/2 kinases at nanomolar potency (Table 1).Table 1In vitro kinase assays for ETC-219 and ETC-027.Compound IDIC50 MNK1 (µM)IC50 MNK2 (µM)IC50 ABL (µM)IC50 ABL (T315I) (µM)IC50 ABL (E255K) (µM)ETC-2190.2540.0150.0140.0130.148ETC-0275.0150.0240.2140.0913.19 Using a panel of 104 kinases, we showed that ETC-219 and ETC-027 inhibited 90% of the activity of 15 and 12 kinases respectively when tested at 1uM. ETC-219 and ETC-027 interact with only 27 and 20 of off-target kinases respectively, and offer significant selectivity over kinases outside of the MNK and BCR-ABL families. ETC-219 and ETC-027 have favorable pharmacokinetic properties, and include the following ADME characteristics (Table 2).Table 2ADME characteristics of ETC-219 and ETC-027.ETC-219ETC-027ParametersUnitIVPOIVPODosemg/kg1050550C0ng/ml21241714Cmaxng/ml12122490Tmaxh12T1/2h22.21.82.1CLL/h/kg39.72.83.5VzL/kg8.831.37.410.8VssL/kg7.25.8AUC0-t(last)ng.h/ml32905128179514337AUC0-infng.h/ml33025133179814349Extrapolated area for AUC0-inf%0.37000.11000.16000.0800F%3180 Regarding targeting of BCR-ABL-dependent cellular proliferation and survival, we found that ETC-219 and ETC-027 exhibited potent anti-proliferative activity against a panel of BC CML cell lines (K562, BV-173, EM-2, KCL-22, JURL-MK1), as well as Ba/F3-BCR-ABL cells containing the T315I mutation. In an in vivo xenograft model, employing K562-eIF4E cell lines in NOD-SCID mice, we found that both compounds inhibited tumor growth in a dose-dependent manner, and a dose-dependent decrease in eIF4E phosphorylation in tumor explants were also observed. Tumor regression was observed at 100 mg/kg for ETC-219. Importantly, mice given ETC-219 and ETC-027 at 100 mg/kg and 200 mg/kg doses did not exhibit significant toxicity. With respect to MNK-eIF4E-b-catenin axis inhibition, ETC-219 and ETC-027 inhibited eIF4E phosphorylation at IC50s of 0.186 nM and 0.28 nM respectively in HeLa cells. In parallel, both compounds inhibited Wnt/b-catenin reporter activity in BC CML cell lines, as well as the expression of a panel of Wnt-regulated genes. Using the serial replating assay as a readout for LSC function and self-renewal capacity, we found that ETC-219 and ETC-027 were effective at 1nM at preventing serial replating using primary CD34+ cells from patients in BC. Furthermore, ETC-219 and ETC-027 inhibited the serial replating efficacy of BC cells from patients with clinical resistance to the third generation TKI, ponatinib. Conclusion Our results demonstrate that: i) small molecule compounds with favorable ADME properties can be developed to potently and specifically inhibit the BCR-ABL and MNK kinases simultaneously; ii) Dual specific MNK and BCR-ABL inhibitors are effective at abrogating BCR-ABL-driven growth and proliferation, as well as the MNK-eIF4E-dependent self-renewal function of BC LSCs. We conclude that dual specific BCR-ABL and MNK inhibitors may warrant testing in patients with BC CML. Disclosures: Chuah: Novartis: Honoraria; BMS: Honoraria.

Dysregulation of the Wnt signaling cascades is implicated in multiple disorders. There are 19 human Wnts that mediate signaling through diverse downstream pathways. To achieve maximum benefit from inhibition of Wnt signaling, targeting all of these pathways may be useful. The secretion and biological activity of all human Wnts requires palmitoylation mediated by Porcupine (PORCN), an endoplasmic reticulum-localized membrane bound O-acyltransferase. Several small molecule inhibitors of PORCN have been developed. Here we report a novel pharmacophore with derivatives that are nanomolar inhibitors of Wnt signaling. By a number of criteria, these compounds potently inhibit PORCN catalytic activity and hence suppress downstream Wnt-activated signaling pathways. The compounds effectively reduce autocrine Wnt signaling activity in selected cancer cell lines. The inhibitory activity is stereospecific, as an (R) enantiomer is inactive. Compounds with good oral bioavailability were tested for their in vivo activity and found to be highly efficacious in reversing tumor growth in both MMTV-WNT1 mice and of tumor xenografts. Treated tumors showed marked nuclear exclusion and decreased cytoplasmic staining of beta-catenin compared to vehicle controls. Importantly the treatment modulated downstream markers of Wnt signaling. No signs of toxicity were observed in mice at therapeutically effective doses. These results and our published results on C59 demonstrate that inhibiting the Wnt/beta-catenin pathway by targeting PORCN with small-molecule inhibitors is a feasible and nontoxic strategy. Use of porcupine inhibitors overcomes the problem of redundancy of Wnts, thereby, providing new options for therapy in diseases with high Wnt activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C248. Citation Format: Babita Madan, Zhiyuan Ke, Shermaine Q.y. Lim, Jenefer Alam, Soo Yei Ho, Duraiswamy A. Jeyaraj, Kakaly Ghosh, Yun Shan Chew, Jamal Aliyev, Li Jun Ding, Vishal Pendharkar, Sifang Wang, Kanda Sangthongpitag, Thomas Keller, May Ann Lee, David M. Virshup. Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C248.

Abstract 180 Background and Aims. The main pathogenic molecular events associated with myeloproliferative neoplasms (Polycythemia Vera, Essential Thrombocytosis, and Primary Myelofibrosis) are mutations in Janus kinase 2 (JAK2) or in the thrombopoietin receptor that arise in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. The approved JAK2 inhibitor (Ruxolitinib) is not expected to be selective for the mutant JAK2/receptor signaling or to completely suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. We postulate that myeloproliferative neoplasms can be treated more effectively if we target the constitutive JAK2 signaling by a JAK2 inhibitor together with another kinase inhibitor targeting a specific pathway that is co-activated by the aberrant JAK2 signaling. This should increase targeting specificity, reduce JAK2 inhibitor dosages, and minimize potential side effects of these drugs. To this end, we constructed cell line models of myeloproliferative neoplasms and tested the models using a JAK2 inhibitor in combination with a panel of kinase inhibitors to identify combination pairs that give the best synergism. The synergistic pair was further confirmed in mouse models of myeloproliferative neoplasms. Methods. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-Abl. The effect of two JAK2 inhibitors (Ruxolitinib and TG101348) in combination with a panel of 15 various kinase inhibitors (one JNK, one B-Raf, one ROCK-1, one TIE-2, one PI3K, two CDK, two MAPK, three p38, and three mTOR inhibitors). An 8×8 constant ratio Latin square design were used for testing inhibition of cell proliferation/survival in these cell line models. Calculations were carried out using the Chou-Talalay method to determine which drug-pair demonstrated synergism in inhibiting cell growth. Further eight PI3K inhibitors were acquired and tested when we found strong synergism between the JAK2 inhibitors and the PI3K inhibitor ZSTK474 in the first panel. The engineered Ba/F3 cells were also inoculated into female BALB/c nude mice to generate the JAK2 mutant mouse model. These mice were treated intravenously with Ruxolitinib and the PI3K inhibitor GDC0941. Blood profile and physical parameters of the mice were measured for 14 days post treatment. Bone marrow cells from mice reconstituted with bone marrow from JAK2 V617F knock-in mice were plated for colony formation in the presence or absence of Ruxolitinib and the PI3K inhibitor GDC0941. Primary Epo-independent colonies from CD34+ cells of one PV patient were assessed in two independent experiments in the presence or absence of combination drugs. Results. Out of 15 kinase inhibitors tested, three PI3K inhibitors (ZSTK474, GDC0941 and BEZ235), synergized with JAK2 inhibitors (Ruxolitinib and TG101348) in inhibiting cell growth. The combination index was less than 0.5 in all 8×8 dose combination ratios. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as growth of Ba/F3 cells expressing Bcr-Abl (at equivalent STAT5 activation levels) was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight due to proliferation of autonomous cells. Our combination treatment using Ruxolitinib and GDC0941 could drastically reduce spleen weight compared to treatment with either compound alone. Endogenous erythroid colony forming unit (CFU-E) and burst forming unit (BFU-E) formation from JAK2 V617F knock-in bone marrow cells was reduced significantly by the combined use of Ruxolitinib and GDC0941 compared to individual drugs. Similarly, Epo-independent BFU-E colony formation from peripheral CD34+ cells of one JAK2 V617F-positive PV patient was reduced significantly by the drug combination. Conclusions. Our findings of strong synergy between the JAK2 inhibitors and PI3K inhibitors suggested that we may be able to administer these drugs at lower concentrations than when the drugs are used individually. It provides a framework for combination trials using compounds in these two classes in patients with myeloproliferative neoplasms. Disclosures: No relevant conflicts of interest to declare.

This study focuses on three main myeloproliferative neoplasms, namely, polycythaemia vera, essential thrombocytosis, and primary myelofibrosis. The main pathological causes of these three diseases are a mutation in the JAK2 protein or a mutation in the thrombopoietin receptor that arises in the hematopoietic stem/progenitor cells. Both type of mutations lead to constitutive activation of the JAK2 signaling pathways. Current JAK2 inhibitors in clinical trials are not expected to be selective and specific enough to suppress the multiple signaling pathways activated by the aberrant JAK2 signaling. Hence, we are seeking a combination treatment using a specific JAK2 inhibitor with another specific kinase inhibitor to inhibit the multiple signaling pathways activated by the constitutively active JAK2. Mouse Ba/F3 cells were engineered to express either JAK2 WT, or JAK2 V617F, or TpoR W515L, or TpoR JAK2 WT, or TpoR JAK2 V617F, or Bcr-abl. The effect of JAK2 inhibitors in combination with a panel of kinase inhibitors in an 8×8 constant ratio drug combination design were used to test for the inhibition of cell viability in the cell models. Calculation of drug synergism was carried out using the Chou-Talalay method. We found that JAK2 inhibitors synergized with PI3K inhibitors. The JAK2-PI3K inhibitors combination was specific for JAK2 signaling as survival of Ba/F3 cells expressing Bcr-abl was unaffected by this combination treatment. Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F were found to have increased spleen weight. Our combination treatment could drastically reduce spleen weight compared to treatment with individual compound alone. The combination treatment also resulted in significant survival benefit in Balb/c mice inoculated with Ba/F3 cells expressing TpoR JAK2 V617F compared to untreated mice or mice given either compound alone. 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 B239.

Introduction: SB939 is an hydroxamic acid based class I, II and IV HDAC inhibitor, with very favourable physiochemical, pharmaceutical and pharmacokinetic properties, resulting in an excellent bioavailability and marked accumulation in tumor tissue. In clinical phase I studies with SB939 in solid tumors, drug exposure was demonstrated to be higher and side effects milder/fewer compared to first generation HDAC inhibitors such as SAHA. Only 6.7% Grade3/4 fatigue (2/30 patients) or 6.9% (2/29 patients) were observed in two independent Phase I clinical studies making SB939 ideally suited for combination therapies. The aim of the present studies was to explore the potential of SB939 for the treatment of hepatocellular carcinoma (HCC). Methods: The effects of SB939 as single agent and combination therapy on the proliferation of HCC cell lines were explored in vitro studies. Single agent and combination therapy together with Sorafenib (Nexavar), a multitargeted VEGFR-b-Raf kinase inhibitor, which is now standard therapy for HCC, was explored in an orthotopic xenograft model of HCC, using Hep3B cells in female SCID mice. Mice were dosed daily with either 10 or 45 mg/kg Sorafenib per oral (p.o.) or with 125 mg/kg SB939 p.o. every other day, alone or in combination with Sorafenib. Sorafenib was dosed first and SB939 dosed 6 h thereafter. Plasma and tumor concentrations of SB939 and Sorafenib were measured using HPLC/MS, analyzed with the MassLynx software and PK parameters were calculated by a non-compartmental method using WinNonlin 4.0 software. Pharmacodynamic (PD) parameters such as HDAC inhibition, inhibition of the Raf-ras MAPK pathway, as well as anti-angiogenic effects of the treatment were analyzed by Western blotting and immunohistochemistry of tumor tissue. Results: HCC cell lines did not show high sensitivity to the anti-proliferative effects of either Sorafanib or SB939 (IC50 values for SB939 ranging from 0.9 to 1.23 μM). In contrast, in the in vivo model of HCC, Sorafenib at a dose of 10 mg/kg had no significant effect on tumor growth (tumor growth inhibition [TGI] was −12.6%), but a dose of 45 mg/kg was very effective (82% TGI). SB939 at a maximum tolerated dose and schedule was well tolerated and moderately effective in arresting tumor grow (40% TGI). When combined with the ineffective lower dose of Sorafenib, the anti-tumor efficacy of SB939 was significantly enhanced (58% TGI), with an in vivo combinatorial index of 0.4, indicating synergy. Conclusion: Although the Hep3B cell line is quite resistant to SB939 in vitro, SB939 is moderately effective as single agent therapy. Anti-tumor efficacy of SB939 was synergistically enhanced in combination with a low and ineffective dose of the VEGFR-Raf tyrosine kinase inhibitor Sorafenib, and the combination was well tolerated, providing a rationale for a phase II trial in combination with Sorafenib in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5436.

Histone deacetylases (HDACs) are emerging new molecular targets for cancer therapy. Small-molecule HDAC inhibitors have been developed and shown to induce tumor cell cytostasis, differentiation and apoptosis in experimental models and efficacy in clinical trials in various hematological malignancies following intravenous and/or oral administration. SB939 is a novel HDAC inhibitor with improved metabolic, pharmacokinetic and pharmacological properties compared to other HDAC inhibitors currently in clinical trials1. The objective of this study was to characterize the anti-tumor efficacy of SB939 in preclinical models of hematological malignancies. SB939 selectively inhibits HDAC class I and II isozymes, with Ki values ranging from 16 to 247 nM. It inhibited the proliferation of cell lines from various haematological malignancies, including leukemia, lymphoma and multiple myeloma with IC50 values ranging from 80 nM to 200 nM. It induced cell cycle arrest leading to apoptotic cell death in tumor cell lines as well as primary cells isolated from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). SB939 has excellent pharmacokinetic properties and tolerability after oral administration in mice1. The oral anti-tumor efficacy of SB939 was evaluated in models of AML (MV4-11) and lymphoma (Ramos) with the tumors grown subcutaneously in nude mice. After daily oral treatment at 50mg/kg (21 days for MV4-11; 14 days for Ramos), SB939 significantly reduced tumor growth in both models (%TGI values were 116% and 100% respectively in MV4-11 and Ramos). In the MV4-11 model, SB939 induced complete tumor regression, in 6/10 mice. In conclusion, our data demonstrate that SB939 is a potent, orally active anti-tumor drug with potential for the treatment of various types of hematological malignancies. 1Kanda Sangthongpitag, Haishang Wang, Pauline Yeo, Liu Xin, Evelyn Goh, Lee Sun New, Peizi Zeng, Xiaofeng Wu, Changyong Hu, Tony Ng and Kantharaj Ethirajulu. ADME attributes of SB939, a best-in-class HDAC Inhibitor, and its PK/PD correlation in the Pharmacological Species. EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics Prague Congress Centre, 2006, Nov 7–10; Prague, Czech Republic, Abstract number 166

JAK2 is the most common mutated gene in bcr-abl-negative chronic myeloproliferative disorders (MPDs) making it an attractive target for drug discovery. Pre-clinical studies have shown that inhibition of JAK2 signaling pathways confer therapeutic benefits in animal models of MPD. We describe here SB1518, a potent, selective and orally active inhibitor of JAK2 with therapeutic potential for the treatment of MPDs. SB1518 is a potent ATP-competitive inhibitor of both JAK2 kinase (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) without significant activity against 40 other representative kinases across the human kinome. SB1518 selectively inhibits the proliferation of cell lines driven by JAK2 or its mutants (e.g. IC50 = 81 nM for a murine BaF3 cell line stably transfected to express erythropoietin receptor and GFP-labeled JAK2V617F). It reduces basal phosphorylation of JAK2 and STAT5 in BaF3-JAK2V617F cells in a concentration-dependent manner. The excellent physicochemical, metabolic and pharmacokinetic properties of SB1518 render it amenable to oral dosing. SB1518 was investigated in a model of advanced MPD established by intravenous injection of BaF3-JAK2V617F cells in nude mice. In several studies using different initial tumor burden and/or drug regimen, we observed dose-dependent and statistically significant therapeutic effects including normalization of elevated white blood cell count and reduction of GFP-labeled BaF3 cells in the peripheral blood, resolution of hepatosplenomegaly, reduction of phospho-STAT5 in diseased organs, prolonged survival and alleviation of terminal-stage anemia and thrombocytopenia. These therapeutic effects were observed at doses well tolerated by the animals. In conclusion, our data demonstrate the therapeutic potential of SB1518 for the treatment of myeloproliferative disorders caused by aberrant JAK2 signaling.

Key physicochemical properties such as solubility, lipophilicity (logD7.4, logP) and pKa (the negative log of the acid dissociation constant) can be used to predict protein binding, tissue distribution, and gastrointestinal (GI) absorption. More recent application of computational methods allows even better prediction of compound oral bioavailability from in vitro and/or in silico properties. During the lead optimization process in our multi-kinase inhibitor program a good correlation between physicochemical properties like logD and solubility (determined experimentally at pH 7) versus AUC(0-inf) (Area Under the Curve) was observed for a series of kinase inhibitors. A calculated logD*solubility value between 200 to 400 predicted a reasonably high oral AUC(0-inf) in mouse PharmacoKinetic (PK) studies for this series of compounds, with correlation coefficient of >0.9. The correlation of logD*solubility vs. AUC(0-inf) enabled us to prioritize compounds for PK studies. SB1317, a novel pyrimidine derivative, is an orally active multi-kinase inhibitor that evolved as a lead drug candidate from in vitro and in vivo pharmacokinetic studies. SB1317 is a highly permeable compound, does not undergo active P-glycoprotein transport, and has a solubility of 75 μg/ml. It is metabolically stable in dog and human liver microsomes and unstable in rodent liver microsomes. No P450 inhibition was observed up to 10 μM towards CYP3A4, CYP1A2, CYP2C9 and CYP2C19. In vivo pharmacokinetics in nude mice and Beagle dogs resulted in %F of 12 and 37% respectively. Tumor pharmacokinetics of SB1317 shows increased exposure in tumor than in plasma with a AUC(0-t) tumor/plasma ratio of 3. Excellent oral dose proportionality (10, 20 and 40mg/kg) was observed in both plasma and tumor. SB1317 has exhibited uniformly high plasma protein binding (>99%) across the preclinical species and human. It is the free or unbound portion of the compound in the plasma or at the tissue level that would yield effective anti-tumor activity. (AUC0-inf)unbound/GI50 and Cmax-unbound/GI50 were used as PK/PD surrogates for the measure of SB1317 efficacy. A factor of SB1317 unbound AUC (0-inf)/GI50 above 0.5 results in significant pharmacodynamic (PD) effects in hematological tumor models (MV4-11 and HL-60 engraft model). Linear pharmacokinetic extrapolation from preclinical species to human was determined by allometric scaling (predicted human % F = 36). PK/PD relationships established for SB1317 in preclinical species could form the basis of a PK/PD-driven clinical development program.

FLT3 is the most common mutated gene in acute myeloid leukemia (AML), and FLT3 mutations are strongly correlated with poor prognosis. Small-molecule inhibitors of FLT3 kinase have been evaluated in clinical trials but with limited success due to the emergence of activating mutants other than FLT3. CDK1, 2, 4 and 6 are well-established anti-cancer targets due to their direct role in cell cycle control. CDK7 and 9 are transcriptional CDKs that are emerging anti-cancer targets as a result of recent advances in the understanding of their effects on apoptotic regulators. Recent evidence suggests that the combined targeting of CDK1, 2 and 9 enhances apoptotic killing of tumor cells. Most CDK active compounds currently in clinical development do not target all of these CDKs and have poor pharmacokinetic/pharmacodynamic properties. Compounds targeting these CDKs, in addition to FLT3, may be more effective in AML and also be effective against other hematological as well as solid tumors. SB1317 is a novel potent inhibitor of FLT3 kinase (IC50 = 45 nM) and CDK2 (IC50 = 11 nM). The CDK spectrum also includes potent inhibition of CDK1 and 9 (IC50 = 19 and 10 nM respectively). SB1317 inhibits proliferation of a broad panel of tumor cell lines, and is particularly potent against the mutant FLT3-dependent AML cell line, MV4-11 (proliferation IC50 = 18 nM). SB1317 reduced phospho-Rb and phospho-FLT3 levels in MV4-11 cells in a dose-dependent manner. It induced apoptosis in MV4-11 cells as well as in primary AML cells from patients. The pharmaceutical, metabolic and pharmacokinetic properties of SB1317 render it amenable to oral dosing. In a nude mouse subcutaneous model of AML (MV4-11), SB1317 induced complete tumor regression after oral daily dosing (40 mg/kg for 21 days). In an orthotopic model of AML (HL60), SB1317 significantly prolonged median survival time (59 days versus 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. It also showed significant anti-tumor activity in a nude mouse model of B-cell lymphoma (Ramos) with a tumor growth inhibition of 63% (15 mg/kg i.p. 5d on/5d off). These results demonstrate the therapeutic potential of this novel kinase inhibitor for the treatment of hematological malignancies.