Development Of Anticancer Therapeutic Agents Research Articles

β-Lapachone induces ferroptosis of colorectal cancer cells via NCOA4-mediated ferritinophagy by activating JNK pathway

β-Lapachone is a natural product that can promote ROS generation and ultimately triggers tumor cells death by inducing DNA damage. Recent studies have indicated that the targeting of ferroptosis or iron metabolism is a feasible strategy for treating cancer. In this study, bulk RNA-seq analysis suggested that β-Lapachone might induce ferroptosis in CRC cells. We further tested this hypothesis using a xenograft model of human colorectal cancer as an animal model and in SW620 and DLD-1 of CRC cell lines. Western blot was used to determine the key proteins of ferroptosis (SLC7A11, GPX4), autophagy (LC3B, P62, ATG7), ferritinophagy (NCOA4, FTH1, TFRC), and JNK pathway (p-JNK, JNK, p-c-Jun, c-Jun). The levels of MDA, GSH/GSSG, lipid ROS, and intracellular ferrous iron were determined after β-Lapachone treatment, and inhibitors of various pathways, including NAC, Ferrostatin-1, DFO, 3-MA, and SP600125 were utilized to explore the molecular mechanism underlying β-Lapachone-mediated ferroptosis. As the result, we identified that β-Lapachone inhibited cell proliferation and induced apoptosis, autophagy, and ROS generation. In addition, β-Lapachone induced ferroptosis as demonstrated by intra-cellular iron overload, increased levels of lipid ROS and MDA. Mechanistically, JNK signaling pathway was involved in β-Lapachone-induced xCT/GPX4-mediated ferroptosis and NCOA4-mediated ferritinophagy in CRC cells. In vivo experiments in nude mice demonstrated that β-Lapachone significantly inhibited CRC growth and induced ferroptosis and NCOA4-mediated ferritinophagy. These findings not only identify a novel role for β-Lapachone in ferroptosis but also indicate that β-Lapachone may be a valuable candidate for the research and development of anti-cancer therapeutic agents.

Current status of organoid culture in urological malignancy.

Urological cancers are common malignancies worldwide. Several conventional models, for example, two-dimensional cell culture and animal models have been used for decades to study tumor genetics. Nonetheless, these methods have limitations in reflecting the real tumor microenvironment invivo, thereby hindering the development of anti-cancer therapeutic agents. Recently, three-dimensional culture models have gained attention because they can overcome the drawbacks of traditional methods. Above all, three-dimensional organoid models are able to mimic the tumor microenvironment in human bodies more closely as they are able to demonstrate the interactions between cells and extracellular matrix. This type of model has therefore extended our understanding of urological cancers. Tumor cells in organoid models can also be co-cultured with other cellular components, such as peripheral blood lymphocytes, and allow further understanding of the effect of tumor microenvironments on tumor growth. Furthermore, organoid models allow a prolonged culturing period, therefore, tumor evolution, progression and maintenance can also be assessed. Organoid models can be derived from each specific patient, and this facilitates investigation of individual cancer-specific mutations and their subtypes. As a result, the development of personalized medication targeting the signaling pathways or biomolecules of interest will be possible. In the present review, we summarize the development and applications of three-dimensional organoid cultures in urological cancers, mainly focusing on prostate, urinary bladder and kidney cancers, and assess the future prospects of this model.

Multiomic Analysis of Cereblon Expression and Its Prognostic Value in Kidney Renal Clear Cell Carcinoma, Lung Adenocarcinoma, and Skin Cutaneous Melanoma

Cereblon (CRBN) is a component of the E3 ubiquitin ligase complex that plays crucial roles in various cellular processes. However, no systematic studies on the expression and functions of CRBN in solid tumors have been conducted to date. Here, we analyzed CRBN expression and its clinical value using several bioinformatic databases. CRBN mRNA expression was downregulated in various cancer types compared to normal cells. Survival analysis demonstrated that overall survival was significantly positively correlated with CRBN expression in some cancer types including lung adenocarcinoma (LUAD), kidney renal clear cell carcinoma (KIRC), and skin cutaneous melanoma (SKCM). CRBN expression was downregulated regardless of clinicopathological characteristics in LUAD and KIRC. Analysis of genes that are commonly correlated with CRBN expression among KIRC, LUAD, and SKCM samples elucidated the potential CRBN-associated mechanisms of cancer progression. Overall, this study revealed the prognostic value of CRBN and its potential associated mechanisms, which may facilitate the development of anti-cancer therapeutic agents.

Abstract 1926: Ba/F3 NTRK cell panel, a powerful cell model for NTRK inhibitor discovery and development

Abstract NTRK (Neurotrophic tyrosine kinase receptor)family, also named as TRK (Tropomyosin receptor kinase),is composedof three transmembrane protein receptors, TrkA, TrkB and TrkC,which play important rolesin neuronal differentiation and survival. Over-activation of NTRK1, NTRK2 and NTRK3 due to chromosomal rearrangement and mutations, was found in congenital fibrosarcoma, secretory breast carcinoma cancer and other types ofcancer. The identification of NTRK fusions as diver cancer genes has led to rapid development of anticancer therapeutic agents, including LOXO101, LOXO195 and TPX0005.Ba/F3 line is a popular system for studying both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects. Our group used Ba/F3 cells togenerate a celllinepanel with 14 different NTRK fusion genes or mutations for NTRK inhibitor screening and development. Inaddition to use the cell lines for in vitro assays, thetransformed Ba/F3 linescan grow well in immune-deficient miceas in vivo models. LOXO101—a NTRK inhibitor—was used to test tumor growth inhibition, the results showedthat threewild-type fusion cell lineswere sensitive to LOXO101, but not other tumor models containing NTRK gatekeeper mutations. All the tumor models were sensitive to TPX0005, another NTRK inhibitor. Ourdata indicatethatthe celllinepanel of Ba/F3is apowerful cell model for new NTRK inhibitor discovery and development. Citation Format: Feng Hao, Zhaoshuai Bai, Guoqian Wang, Feng He, Changpeng Liu, Jinying Ning. Ba/F3 NTRK cell panel, a powerful cell model for NTRK inhibitor discovery and development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1926.

Global reactivity indexes and nonparametric statistics in the study of the proapoptotic activity of coumarins

AbstractCoumarins are widely distributed as secondary metabolites and are popular structural motifs among known anticancer agents; their planarity and electronic environment are very important building blocks for bioactive compounds. Due to the broad scope of their biological activity, anticancer properties of these molecules remain an important goal for medicinal chemistry. In this work, the knowledge and understanding of the correlation between electronic environment and global reactivity of a series of hydroxycoumarins lead to develop a statistical model to study the proapoptotic activity of this type of compounds. Thus, the analysis of the charge distribution within the molecule and experimental data of proapoptotic activity of known coumarins are analyzed through a regression model. This model offers the possibility of predicting whether a structural related coumarin will show the desired activity or not, which increases the ability to a target‐directed approach in the development of anticancer therapeutic agents.

Abstract 4203: Kinase inhibitor demonstrates efficacy in patient derived xenograft model of fibrolamellar hepatocellular carcinoma featuring DNAJB1-PRKACA gene fusion

Abstract Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare and distinct primary hepatic malignancy that has conventionally been considered to be a histologic variant of hepatocellular carcinoma. However, it has more recently been recognized as a distinct clinical entity with respect to its epidemiology and prognosis. The etiology of FL-HCC is unclear and there is no clinical or histological evidence for association with chronic liver disease. Recent genetic studies have identified the fusion gene DNAJB1-PRKACA as a recurrent genetic lesion in the disease. Molecular pathogenesis of FL-HCC; however, is not well understood and has been reported to occur in association with focal nodular hyperplasia. Overexpression of genes in PIK3, MAPK and RAS pathways is observed in FL-HCC and in contrast to viral-associated HCC, epigenetic instability is rare in FL-HCC. Here we describe development of a novel FL-HCC Patient Derived Xenograft (PDX) model LI5132. RNASeq analysis revealed presence of DNAJB1-PRKACA gene fusion on chromosome 19, a molecular signature of FL-HCC. Clinical diagnosis as HCC of fibrolamellar type was confirmed by H&E staining in the PDX tumor. The PDX tumor had a distinctive intratumoral fibrosis with a lamellar pattern. PDX model was established by inoculating patient tumor cells subcutaneously in NOD-SCID mice. Robust tumor growth was observed with a 100% take rate in NOD-SCID mice. To evaluate the translational relevance of this model toward therapeutic development, the efficacy of a novel multi-target Aurora kinase A and angiokinase inhibitor, ENMD-2076, was evaluated in a therapeutic mode in this model. ENMD-2076 showed significant efficacy in suppressing tumor growth in NOD-SCID mice, at the 200 mg/kg dose evaluated in this study. Weight loss observed during the treatment period could not be attributed to ENMD-2076 as it was similar to weight loss observed with vehicle treatment. ENMD-2076 has now progressed in to phase II trials in the clinic for treatment of FL-HCC. This demonstrates the translational utility of this FL-HCC model in development of anti-cancer therapeutic agents. Citation Format: Jayant Thatte, Elvira C. Talaoc, Colleen Scott, Ken Ren, Thomas Broudy. Kinase inhibitor demonstrates efficacy in patient derived xenograft model of fibrolamellar hepatocellular carcinoma featuring DNAJB1-PRKACA gene fusion [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 4203. doi:10.1158/1538-7445.AM2017-4203

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

Regulation of p53 Stability and Apoptosis by a ROR Agonist

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anti-cancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORα. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer.

Identifying G-quadruplex-binding ligands using DNA-functionalized gold nanoparticles

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.

A convenient method for synthesis of tetraoxazole peptide macrocycles

G-quadruplex DNA plays important regulatory roles in the maintenance of telomere length and transcriptions inhibition of such oncogenes as c-myc, and thus has become an attractive target for the development of anti-cancer therapeutic agents. Poly-oxazole macrocycles are a promising new class of G-quadruplex binding ligands. Herein is described the synthesis of a tetraoxazole peptide macrocycle and an epimerization product. The synthetic unit was prepared by cyclization-oxidation of a diserine with one hydroxyl protected. Two-cycle coupling of the unit gave a linear tetraoxazole amide. The targeted molecule and the epimerization product were obtained after macrocyclization of the linear precursor and following removal of the protecting benzyl groups. With structural similarities to the most potent G-quadruplex stabilizer telomestatin, these two molecules might potentially be used to probe the biological significance of G-quadruplex' in vivo.