AL101, a gamma-secretase inhibitor,has potent antitumor activity against adenoid cystic carcinoma with activated NOTCH signaling

Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy with limited treatment options for recurrent or metastatic disease. Due to chemotherapy resistance and lack of targeted therapeutic approaches, current treatment options for the localized disease are limited to surgery and radiation, which fails to prevent locoregional recurrences and distant metastases in over 50% of patients. Approximately 20% of patients with ACC carry NOTCH-activating mutations that are associated with a distinct phenotype, aggressive disease, and poor prognosis. Given the role of NOTCH signaling in regulating tumor cell behavior, NOTCH inhibitors represent an attractive potential therapeutic strategy for this subset of ACC. AL101 (osugacestat) is a potent γ-secretase inhibitor that prevents activation of all four NOTCH receptors. While this investigational new drug has demonstrated antineoplastic activity in several preclinical cancer models and in patients with advanced solid malignancies, we are the first to study the therapeutic benefit of AL101 in ACC. Here, we describe the antitumor activity of AL101 using ACC cell lines, organoids, and patient-derived xenograft models. Specifically, we find that AL101 has potent antitumor effects in in vitro and in vivo models of ACC with activating NOTCH1 mutations and constitutively upregulated NOTCH signaling pathway, providing a strong rationale for evaluation of AL101 in clinical trials for patients with NOTCH-driven relapsed/refractory ACC.Subject terms: Head and neck cancer, Targeted therapies     

A novel tubulin inhibitor, 6h,suppresses tumor-associated angiogenesis and shows potent antitumor activity against non–small cell lung cancers

Tumor angiogenesis is closely associated with the metastasis and progression of non–small cell lung cancer (NSCLC), a highly vascularized solid tumor. However, novel therapeutics are lacking for the treatment of this cancer. Here, we developed a series of 2-aryl-4-(3,4,5-trimethoxy-benzoyl)-5-substituted-1,2,3-triazol analogs (6a–6x) as tubulin colchicine-binding site inhibitors, aiming to find a novel promising drug candidate for NSCLC treatment. We first identified 2-(2-fluorophenyl)-3-(3,4,5-trimethoxybenzoyl)-5-(3-hydroxyazetidin-1-yl)-2H-1,2,3-triazole (6h) as a hit compound, which inhibited angiogenesis induced by NSCLC cells both in vivo and in vitro. In addition, our data showed that 6h could tightly bind to the colchicine-binding site of tubulin and inhibit tubulin polymerization. We also found that 6h could effectively induce G2/M cell cycle arrest of A549 and H460 cells, inhibit cell proliferation, and induce apoptosis. Furthermore, we showed 6h had the potential to inhibit the migration and invasion of NSCLC cells, two basic characteristics of tumor metastasis. Finally, we found 6h could effectively inhibit tumor progression in A549 xenograft mouse models with minimal toxicity. Taken together, these findings provide strong evidence for the development of 6h as a promising microtubule colchicine-binding site inhibitor for NSCLC treatment.     

Lorvotuzumab mertansine,a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.     

Lorvotuzumab mertansine,a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.     

BEBT-109, a pan-mutant-selective EGFR inhibitor with potent antitumor activity in EGFR-mutant non-small cell lung cancer

EGFR mutation-positive NSCLC tumors are highly heterogeneous, therefore, exploring an agent simultaneously targeting multiple EGFR mutations may be valuable for clinical practice. Compared with osimertinib, BEBT-109 shows more sensitive and extensive antitumor activity in EGFR mutant NSCLC, while sparing wild-type EGFR cell lines. Meanwhile, unlike the metabolite of osimertinib AZ5104, the main metabolites of BEBT-109 are found lacking in activity against wild-type EGFR cell lines. Preclinical and clinical studies demonstrate a unique pharmacokinetic profiles of BEBT-109 with rapid absorption and quick in vivo clearance without accumulation, which are conducive to minimizing the off-target toxicity of the covalent irreversible EGFR inhibitor. Oral administration of BEBT-109 induces tumor regression in EGFR exon 20 insertion xenografts, and even tumor disappearance in PC-9, HCC827 and H1975 xenograft models. Furthermore, in clinical trials, the objective responses were observed in NSCLC patients with EGFR T790M mutation in the first and second dosing cohorts. These findings demonstrate that BEBT-109, a potent pan-mutant-selective EGFR inhibitor with improved pharmacokinetic properties, might offer a promising new option for the treatment of multiple mutant-EGFR-driven NSCLC.     

COBRA-1, a rationally-designed epoxy-THF containing compound with potent tubulin depolymerizing activity as a novel anticancer agent

A novel mono-THF containing synthetic anticancer drug, COBRA-1, was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of alpha-tubulin. COBRA-1 inhibited GTP-induced tubulin polymerization in cell-free tubulin turbidity assays. Treatment of human breast cancer and brain tumor (glioblastoma) cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Like other microtubule-interfering agents, COBRA-1 activated the proapoptotic c-Jun N-terminal kinase (JNK) signal transduction pathway, as evidenced by rapid induction of c-jun expression.     

Bromodomain and extraterminal domain inhibitor enhances the antitumor effect of imatinib in gastrointestinal stromal tumours

In gastrointestinal stromal tumours (GISTs), the function of bromodomain‐containing 4 (BRD4) remains underexplored. BRD4 mRNA abundance was quantified in GISTs. In the current study, we investigated the role of BRD4 in GISTs. Our results show a significant enhancement in BRD4 mRNA and a shift from very low‐risk/low‐risk to high‐risk levels as per NCCN specifications. Overexpression of BRD4 correlated with unfavourable genotype, nongastric location, enhanced risk and decreased disease‐free survival, which were predicted independently. Knockout of BRD4 in vitro suppressed KIT expression, which led to inactivation of the KIT/PI3K/AKT/mTOR pathway, impeded migration and cell growth and made the resistant GIST cells sensitive to imatinib. The expression of KIT was repressed by a BRD4 inhibitor JQ1, which also induced myristoylated‐AKT‐suppressible caspases 3 and 9 activities, induced LC3‐II, exhibited dose‐dependent therapeutic synergy with imatinib and attenuated the activation of the PI3K/AKT/mTOR pathway. In comparison with their single therapy, the combination of JQ1/imatinib more efficiently suppressed the growth of xenografts and exhibited a reduction in KIT phosphorylation, a decrease in Ki‐67 and in the levels of phosphorylated PI3K/AKT/mTOR and enhanced TUNEL staining. Thus, we characterized the biological, prognostic and therapeutic implications of overexpressed BRD4 in GIST and observed that JQ1 suppresses KIT transactivation and nullifies the activation of PI3K/AKT/mTOR, providing a potential strategy for treating imatinib‐resistant GIST through dual blockade of KIT and BRD4.     

Discovery of a novel chimeric ubenimex–gemcitabine with potent oral antitumor activity

Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13 activity of ubenimex and the cytotoxic activity of gemcitabine in vitro. Further characterization also demonstrated that compound BC-A1 exhibited significant anti-invasion and anti-angiogenesis effects in vitro. The preliminary stability test of BC-A1 revealed that it could release gemcitabine in vitro. The in vivo anti-tumor results in liver cancer showed that at the same dosage, oral administration of BC-A1 was as potent as intraperitoneal administration of gemcitabine. This warranted the further research and development of the orally active prodrug BC-A1 because gemcitabine can not be orally administrated in clinic.     

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.Subject terms: Breast cancer, Cancer models, Cancer therapeutic resistance, Drug development, Mitosis     

Phosphofructokinase from Dictyostelium discoideum is a potent inhibitor of tubulin polymerization

We identified the nonallosteric phosphofructokinase from the slime mold Dictyostelium discoideum as a potent protein factor that inhibits the rate of polymerization of tubulin at a molar ratio of 1 molecule to about 300 tubulin dimers for half-maximal action (IC50 = 32 nM). This effect was (i) assessed by turbidity measurements, pelleting of microtubules, and electron microscopy, (ii) observed when tubulin assembly was induced by taxol as well as by GTP in the presence of microtubule-associated proteins or glutamate, and (iii) specific as it was not produced by the phosphofructokinase from rabbit muscle. Also in contrast to the latter, neither tubulin nor microtubules modified the catalytic activity of the slime mold isozyme. Immunoelectron microscopy provided further evidence that D. discoideumphosphofructokinase physically interacts with tubulin, leading to the formation of aggregates. The process seems to be reversible since microtubules eventually formed in the presence of the inhibitor with concomitant reduction of tubulin aggregates. Limited proteolysis by subtilisin showed that the hypervariable C-termini of tubulin is not involved in the interaction with the enzyme. The possible physiological relevance of this novel function of D. discoideum phosphofructokinase different from its glycolytic action is discussed.     

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.     

Elaiophylin,a novel autophagy inhibitor,exerts antitumor activity as a single agent in ovarian cancer cells

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporation''s pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.     

Assessment of a novel VEGF targeted agent using patient-derived tumor tissue xenograft models of colon carcinoma with lymphatic and hepatic metastases

The lack of appropriate tumor models of primary tumors and corresponding metastases that can reliably predict for response to anticancer agents remains a major deficiency in the clinical practice of cancer therapy. It was the aim of our study to establish patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases useful for testing of novel molecularly targeted agents. PDTT of primary colon carcinoma, lymphatic and hepatic metastases were used to create xenograft models. Hematoxylin and eosin staining, immunohistochemical staining, genome-wide gene expression analysis, pyrosequencing, qRT-PCR, and western blotting were used to determine the biological stability of the xenografts during serial transplantation compared with the original tumor tissues. Early passages of the PDTT xenograft models of primary colon carcinoma, lymphatic and hepatic metastases revealed a high degree of similarity with the original clinical tumor samples with regard to histology, immunohistochemistry, genes expression, and mutation status as well as mRNA expression. After we have ascertained that these xenografts models retained similar histopathological features and molecular signatures as the original tumors, drug sensitivities of the xenografts to a novel VEGF targeted agent, FP3 was evaluated. In this study, PDTT xenograft models of colon carcinoma with lymphatic and hepatic metastasis have been successfully established. They provide appropriate models for testing of novel molecularly targeted agents.     

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.     

From fighting depression to conquering tumors: a novel tricyclic thiazepine compound as a tubulin polymerization inhibitor

A novel tricyclic thiazepine derivative, 6-(p-tolyl)benzo[f] pyrido[2,3-b][1,4] thiazepine 11,11-dioxide (TBPT), exhibits potent inhibitory effects in two non-small-cell lung cancer cell lines, H460 and its drug-resistant variant, H460_TaxR, while exhibiting much less toxic effects on normal human fibroblasts. After five injections of TBPT at a dose of 60 mg/kg, it inhibits H460_TaxR tumor growth in xenografted mouse models by 66.7% without causing observable toxicity to normal tissues. Based on gene perturbation data and a series of investigations, we reveal that TBPT is not a P-glycoprotein substrate and it inhibits microtubule formation by targeting tubulin, thereby causing cell cycle arrest at the G2/M stage and eventually inducing apoptosis. This redeployment of anti-depressant compound scaffold for anticancer applications provides a promising future for conquering drug-resistant tumors with fewer side effects.Drug resistance and nonselective toxicity have been two major clinical obstacles for cancer chemotherapy.^{1, 2, 3, 4, 5} Drug resistance is responsible for treatment failure in >90% of patients with metastatic cancers.⁶ A major cause of drug resistance is the overexpression of a 170-kDa transmembrane protein, P-glycoprotein (P-gp), on the cancer cell surface in response to drug treatment. P-gp acts as an ATP-dependent drug efflux pump.^{7, 8, 9} Many drugs, including paclitaxel (PTX), taxanes, doxorubicin and vinca alkaloids are substrates of P-gp and are readily pumped out of cells, thereby leading to reduced drug accumulation inside cells.^{1, 10} Furthermore, anticancer drugs often elicit serious nonselective toxicity to normal organs. Patients who have undergone chemotherapy often suffer from treatment-related morbidity or mortality, such as leukopenia, hepatic toxicity and even death.^{11, 12, 13, 14} Therefore, potent and selective agents against multidrug-resistant cancers are urgently needed.Tricyclic azepine derivatives, such as tianeptine, clomipramine and quetiapine, have been used as anti-psychotic drugs. Other reports have demonstrated that tricyclic azepine derivatives such as nevirapine work as anti-human immunodeficiency virus agents.^{15, 16} A recent report has also demonstrated that tricyclic anti-depressants such as clomipramine exhibit anticancer activity.¹⁷ In this work, we report the good activity and superb selectivity of a tricyclic thiazepine compound, 6-(p-tolyl)benzo[f] pyrido[2,3-b][1,4] thiazepine 11,11-dioxide (TBPT) against drug-resistant non-small-cell lung cancer (NSCLC). TBPT inhibited the growth of both drug-sensitive and drug-resistant human lung cancer cells in vitro and drug-resistant tumors in vivo without obvious side effects. Further examination indicated that TBPT evaded the P-gp-mediated drug efflux and acted as a novel microtubule depolymerizing agent, thereby inducing cancer cell G2/M phase arrest and apoptosis.     

Diaza- and triazachrysenes: potent topoisomerase-targeting agents with exceptional antitumor activity against the human tumor xenograft,MDA-MB-435

Several 5,12-diazachrysen-6-ones and 5,6,11-triazachrysen-12-ones were synthesized with varied substituents at the 5- or 11-position, respectively. Each compound was evaluated for its potential to stabilize the cleavable complex formed with TOP1 and DNA. Two analogues with very potent TOP1-targeting activity, 3a and 4a, exhibited cytotoxic activity with IC(50) values at or below 2nM against RPMI8402. Compound 3a was active in vivo by either ip or po administration in the human tumor xenograft athymic nude mice model.     

HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin

HURP is a newly discovered microtubule-associated protein (MAP) required for correct spindle formation both in vitro and in vivo. HURP protein is highly charged with few predicted secondary and tertiary folding domains. Here we explore the effect of HURP on pure tubulin, and describe its ability to induce a new conformation of tubulin sheets that wrap around the ends of intact microtubules, thereby forming two concentric tubes. The inner tube is a normal microtubule, while the outer one is a sheet composed of tubulin protofilaments that wind around the inner tube with a 42.5° inclination. We used cryo-electron microscopy and unidirectional surface shadowing to elucidate the structure and conformation of HURP-induced tubulin sheets and their interaction with the inner microtubule. These studies clarified that HURP-induced sheets are composed of anti-parallel protofilaments exhibiting P2 symmetry. HURP is a unique MAP that not only stabilizes and bundles microtubules, but also polymerizes free tubulin into a new configuration.     

Structural modifications of indolinones bearing a pyrrole moiety and discovery of a multi-kinase inhibitor with potent antitumor activity

Structural modifications of compound 2, an angiokinase inhibitor reported by our group were performed, which led to the discovery of methyl (Z)-3-(((4-(2-methyl-5-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (7h). Compound 7h exhibited excellent inhibitory activity against angiokinases including VEGFR-1/2/3, PDGFRα/β, and FGFR-1, as well as LYN and c-KIT kinases. At the cellular level, compound 7h significantly attenuated phosphorylation of AKT and ERK proteins, potently inhibited colony formation of HT-29, MKN74, and HepG2 cancer cells, and induced cell apoptosis. Upon incubation with human liver microsome, 7h exhibited comparable metabolic stability to nintedanib. Compound 7h has emerged as a promising lead compound for future drug design.