Cytotoxic activity and cell specificity of a novel LHRH peptide drug conjugate, D-Cys6-LHRH vedotin, against ovarian cancer cell lines.

Ovarian cancer is the most deadly female gynaecological malignancy in developed countries and new treatments are urgently needed. The luteinising hormone releasing hormone (LHRH) peptide drug conjugate Zoptarelin doxorubicin is one such potential new drug modality that entered clinical trials for treating LHRH receptor-positive gynaecological cancers. However, development stopped after disappointing Phase 3 results in 2017. We believe the lack of efficacy was due to linker instability and payload potency. In this work, we replaced its linker-toxin with vedotin (MC-VC-PABC-MMAE), yielding the novel peptide drug conjugate D-Cys6-LHRH vedotin. A GI50 and cell specificity comparison against cancerous and non-cancerous ovarian cell lines showed significantly superior bioactivity and selectivity over Zoptarelin doxorubicin (GI50 4 vs. 453 nM) and other chemotherapeutic drugs used for treating ovarian cancers. Our results suggest D-Cys6-LHRH vedotin can potentially be used as a treatment for ovarian cancer.

Fragment-based lead discovery of indazole-based compounds as AXL kinase inhibitors.

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.

WNT inhibition creates a BRCA-like state in Wnt-addicted cancer.

Wnt signaling maintains diverse adult stem cell compartments and is implicated in chemotherapy resistance in cancer. PORCN inhibitors that block Wnt secretion have proven effective in Wnt-addicted preclinical cancer models and are in clinical trials. In a survey for potential combination therapies, we found that Wnt inhibition synergizes with the PARP inhibitor olaparib in Wnt-addicted cancers. Mechanistically, we find that multiple genes in the homologous recombination and Fanconi anemia repair pathways, including BRCA1, FANCD2, and RAD51, are dependent on Wnt/ß-catenin signaling in Wnt-high cancers, and treatment with a PORCN inhibitor creates a BRCA-like state. This coherent regulation of DNA repair genes occurs in part via a Wnt/ß-catenin/MYBL2 axis. Importantly, this pathway also functions in intestinal crypts, where high expression of BRCA and Fanconi anemia genes is seen in intestinal stem cells, with further upregulation in Wnt-high APCmin mutant polyps. Our findings suggest a general paradigm that Wnt/ß-catenin signaling enhances DNA repair in stem cells and cancers to maintain genomic integrity. Conversely, interventions that block Wnt signaling may sensitize cancers to radiation and other DNA damaging agents.

PORCN inhibition synergizes with PI3K/mTOR inhibition in Wnt-addicted cancers.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is aggressive and lethal. Although there is an urgent need for effective therapeutics in treating pancreatic cancer, none of the targeted therapies tested in clinical trials to date significantly improve its outcome. PORCN inhibitors show efficacy in preclinical models of Wnt-addicted cancers, including RNF43-mutant pancreatic cancers and have advanced to clinical trials. In this study, we aimed to develop drug combination strategies to further enhance the therapeutic efficacy of the PORCN inhibitor ETC-159. To identify additional druggable vulnerabilities in Wnt-driven pancreatic cancers, we performed an in vivo CRISPR loss-of-function screen. CTNNB1, KRAS, and MYC were reidentified as key oncogenic drivers. Notably, glucose metabolism pathway genes were important in vivo but less so in vitro. Knockout of multiple genes regulating PI3K/mTOR signaling impacted the growth of Wnt-driven pancreatic cancer cells in vivo. Importantly, multiple PI3K/mTOR pathway inhibitors in combination with ETC-159 synergistically suppressed the growth of multiple Wnt-addicted cancer cell lines in soft agar. Furthermore, the combination of the PORCN inhibitor ETC-159 and the pan-PI3K inhibitor GDC-0941 potently suppressed the in vivo growth of RNF43-mutant pancreatic cancer xenografts. This was largely due to enhanced suppressive effects on both cell proliferation and glucose metabolism. These findings demonstrate that dual PORCN and PI3K/mTOR inhibition is a potential strategy for treating Wnt-driven pancreatic cancers.

Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3.

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.

Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

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.

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors.

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.

A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.

BACKGROUND: Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. OBJECTIVE: A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. RESULTS: Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. CONCLUSION: This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.

Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

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.

NOTUM is a potential pharmacodynamic biomarker of Wnt pathway inhibition.

Activation of Wnt signaling due to Wnt overexpression or mutations of Wnt pathway components is associated with various cancers. Blocking Wnt secretion by inhibiting PORCN enzymatic activity has shown efficacy in a subset of cancers with elevated Wnt signaling. Predicting response to upstream Wnt inhibitors and monitoring response to therapeutics is challenging due to the paucity of well-defined biomarkers. In this study we identify Notum as a potential biomarker for Wnt driven cancers and show that coordinate regulation of NOTUM and AXIN2 expression may be a useful predictor of response to PORCN inhibitors. Most importantly, as NOTUM is a secreted protein and its levels in blood correlate with tumor growth, it has potential as a pharmacodynamic biomarker for PORCN and other Wnt pathway inhibitors.

Discovery and Optimization of a Porcupine Inhibitor.

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.

Pharmacophore Model for Wnt/Porcupine Inhibitors and Its Use in Drug Design.

Porcupine is a component of the Wnt pathway which regulates cell proliferation, migration, stem cell self-renewal, and differentiation. The Wnt pathway has been shown to be dysregulated in a variety of cancers. Porcupine is a membrane bound O-acyltransferase that palmitoylates Wnt. Inhibiting porcupine blocks the secretion of Wnt and effectively inhibits the Wnt pathway. Using high throughput screening, we have identified a number of novel porcupine inhibitors with diverse scaffolds. The pharmacophore requirements for our porcupine inhibitors were elucidated, and a pharmacophore model is proposed. Our compounds as well as all currently published porcupine inhibitors may be fitted to this model in low energy conformations with good superimposition of the pharmacophore elements. The model also explains the stereochemical requirements of our chiral porcupine inhibitors. The pharmacophore model was successfully used for designing 3 new series of porcupine inhibitors having a tricyclic xantine, a phtalimide, or a piperidine-maleimide scaffold.

Molecular integrative clustering of Asian gastric cell lines revealed two distinct chemosensitivity clusters.

Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy.

Characterization of ductal carcinoma in situ cell lines established from breast tumor of a Singapore Chinese patient.

BACKGROUND: Five cell lines were established from a Singaporean patient of Chinese origin with breast ductal carcinoma in situ (DCIS). These five cell lines express exogenous human telomerase reverse transcriptase (hTERT) which confers the ability to proliferate indefinitely. METHODS: Cells were isolated from the DCIS excision and transfected with a plasmid expressing hTERT, a catalytic subunit of telomerase. Five immortalized colonies were propagated and characterized by karyotyping, array comparative genomic hybridization (CGH), immunostaining and Western blots for biomarkers, in vitro anchorage independent growth, in vivo mouse tumorigenicity, drug sensitivity, species authentication and virology safety testing. RESULTS: Array CGH analysis showed that the cell lines harbored different specific genetic aberrations. Common mutations observed in most breast cancer cell lines such as the general loss of heterozygosity (LOH) throughout chromosome X and chromosome 17 are also observed in our cell lines. The cell lines were further characterized as human breast cells that are estrogen- and progesterone-receptor positive, and sensitive to tamoxifen. The cell lines showed anchorage-independent growth in the soft agar assay and can grow in common culture medium without supplementation with growth factor, therefore demonstrating transformed characteristics. Four of the cell lines can engraft and form measureable tumors after 50 days when injected subcutaneously into immune-deficient (SCID) mice. The weak tumorigenicity of these cell lines corresponded well with their non-malignant growth origin. The cell lines were authenticated to be of human origin based on DNA fingerprint of the cells. The cell lines were free from contamination of 20 viruses and mycoplasma in the virological safety test panel. CONCLUSIONS: Unlike most available breast cell lines, our cell lines are derived from primary breast cancer tissues that represent earlier grades or tumor progression stages. They would be useful as study models for basic and clinical research programs directed at early diagnosis and intervention.

Establishment and characterization of a singaporean chinese lung adenocarcinoma cell line with four copies of the epidermal growth factor receptor gene.

We have established a lung adenocarcinoma cell line, ETCC016, from lung pleural effusion of a male Singaporean Chinese with advanced lung adenocarcinoma. The subject smoked 20 cigarettes per day for more than 30 years. The cell line arose from spontaneous transformation of cells grown in a collagen-coated culture dish. Transformed characteristics of the cell line include the ability to reach high confluency in a culture dish, low cell doubling time, ability to form colonies in soft agar, and ability to form solid tumor in immune-compromised SCID mice. Immunostaining showed that the cells originated from lung epithelial cells. Genomic analysis revealed a large amount of chromosomal aberrations (gain and loss of genetic materials, and loss of heterozygosity [LOH]), indicative of a long history of smoking. The cells have four copies of epidermal growth factor receptor (EGFR) and three copies of MYC, but have lost one copy of the RB1 gene. LOH was detected in TP53 and BRAF genes. There is no anaplastic lymphoma kinase (ALK) gene rearrangement. The ETCC016 lung adenocarcinoma cell line has demonstrated susceptibility towards inhibitors specific for EGFR/HER2 and ALK targets, but resistance to MYC-specific inhibitor. This cell line will be a useful model for further understanding of lung adenocarcinoma.

Combination treatment for myeloproliferative neoplasms using JAK and pan-class I PI3K inhibitors.

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.

Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.

Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of ß-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC(50). C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/ß-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo.

Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X(L) function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X(L)/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X(L), whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X(L). Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X(L), which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.

Specific activation of the p53 pathway by low dose actinomycin D: a new route to p53 based cyclotherapy.

The activation of p53 has been proposed as a novel anti-cancer treatment in two distinct contexts. In the first activation of p53 in tumor cells can promote apoptosis and senescence and enhance the anti-tumor activity of cytotoxic chemotherapeutic drugs. In the second application activation of p53 in normal tissues can cause a reversible cell cycle arrest that can be used to protect normal cells from the action of anti-mitotics. In this cyclotherapy role p53 mutant tumor cells are not arrested and remain sensitive to anti-mitotics. The advent of specific p53 activating molecules such as nutlin-3 has encouraged both approaches. We have sought for a clinically approved drug that can mimic nutlin-3. We show here that low doses of actinomycin D mimic nutlin-3 in the highly specific activation of p53 dependant transcription, in the induction of a reversible protective growth arrest in normal cells and in the enhancement of the activity of chemotherapeutic drug induced killing of p53 positive human tumor cells. While high doses of actinomycin D reveal its more non-specific activities, low doses of the drug will allow exploration of the value of p53 activation in preclinical and clinical models before nutlin-3 like drugs are approved.