SKOV3 Xenograft Research Articles

Abstract 2368: Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth

Abstract Her-2 is a well-known driver of carcinogenesis that is overexpressed in breast, ovarian and other tumors. Immunotherapy with bispecific antibodies is a promising cancer therapy approach. Her-2 (Herceptin) antibody was used to test bispecific antibody against ovarian cancer cells. First, Her-2-CAR-T cells with Her-2 (4D5) ScFv and 4-1BB costimulatory domain were generated and tested using in vitro killing and IFN-gamma assays, and in vivo efficacy SKOV-3 xenograft NSG mouse model. Her-2-CAR-T cells killed SKBR-3 breast and SKOV-3 ovarian cancer cells and stopped SKOV-3 xenograft tumor growth. This Her-2-ScFv was used to generate bispecific Her-2 ScFv-CD3-ScFv human Fc antibody using in vitro transcribed mRNA. The 5’capped Her-2-CD3-hFc mRNA with 5’UTR, 3’UTR and poly A tail was generated by in vitro transcription using DNA template. The mRNA was embedded into lipid nanoparticles (LNP) using microfluidic PreciGenome Flex S NanoGenerator. Western blotting detected >160 kDa bispecific antibody secreted from A1847 and 293 cells transfected with mRNA-LNPs. The bispecific antibody was binding Her-2-positive A1847 ovarian cancer cell lines and T cells by FACS analysis. The Her-2-CD3-hFc bispecific antibody with T cells effectively killed Her-2-positive target cells in a dose-dependent manner in RTCA assay and produced high level of IFN-gamma. Moreover, intratumoral delivery of Her-2-CD3-hFc mRNA-LNP with intravenous injection of T cells completely blocked A1847 xenograft tumor growth. In conclusion. intratumoral delivery of Her-2-CD3 mRNA-LNP blocks ovarian xenograft tumor growth and provides a solid basis for future pre-clinical and clinical studies. Citation Format: Vita M. Golubovskaya, John Sienkiewicz, Jinying Sun, Shiming Zhang, Yanwei Huang, Liang Hu, Hua Zhou, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2368.

Discovery of 4-(N-dithiobenzyl piperazine)-1,8-naphthalimide as a potent multi-target antitumor agent with good efficacy, limited toxicity, and low resistance

A series of 4-(N-dithiobenzyl piperazine)-1,8-naphthalimide derivatives 4–6 were designed, synthesized, and evaluated as novel multi-target antitumor agents. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) results showed that compounds 5j, 5k, and 6j exhibited superior in vitro antiproliferative activity in MGC-803, HepG-2, SKOV-3, and T24 cancer cell lines and the cisplatin-resistant cell line A549/DDP. HepG-2, SKOV-3, and T24 xenograft assay results revealed that compounds 5j, 5k, and 6j exhibited good antitumor effects compared with amonafide. The pathology results indicated that compound 5j exhibited the least comprehensive toxicity among the three compounds, identifying compound 5j as a good candidate antitumor agent with good efficacy, limited toxicity, and low resistance. Compound 5j was thus chose for further antitumor mechanism investigation. Results from the omics research, confocal immunofluorescence, Western blot, transmission electron microscopy, and flow cytometry indicated that compound 5j exerted antitumor effects through multiple mechanisms, including ferroptosis, autophagy, apoptosis, and cell cycle arrest. These results suggest that screening novel 1,8-naphthalimide-based antitumor agents for good efficacy, limited toxicity, and low resistance based on a multi-target drug strategy is feasible.

Effects of Nicotine on the Progression and Metastasis of HumanOvarian Cancer cells by Exacerbating EMT

Does nicotine, a main constituent of toxicants in Cigarette Smoke, relate to EMT and contribute to the progression ofovarian cancer? Treat SK-OV3 xenograft mice with increasing nicotine and measure TGF-β in blood by ELISA assayand tumor cell metastasis by confocal microscopy, looking for cancer cells in the liver, bone, brain, stomach, ovary andthen measure xenograft tumor size by weight. A positive control is benzene treatment, and the negative control is PBS.The results will reveal the effects of nicotine on ovarian cancer cell proliferation, which will improve our understandingof the harm environmental toxicants can do to human health. At the same time, the physiological processes that lead tocarcinogenesis will also provide prevention awareness and treatment ideas for ovarian cancer caused by smoking.

The preclinical study of 177Lu-DOTA-LTVSPWY as a potential therapeutic agent against HER2 overexpressed cancer.

Peptide receptor radionuclide therapy (PRRT) has evolved in cancer therapy and diagnosis. LTVSPWY, as a peptide, can target HER2 receptor; on the other hand, 177Lu emits β- which is helpful for cancer therapy. The radiolabeling of LTVSPWY with 177Lu results in a therapeutic agent (177Lu-DOTA-LTVSPWY) capable of cancer treatment. 177Lu-DOTA-LTVSPWY was prepared with high radiochemical purity (RCP). The stability was investigated in saline and human serum. The radiotracer affinity toward the SKOV-3 cell line with overexpression of the HER2 receptor was evaluated. Then the impact of the radiotracer on the colony formation of the SKOV-3 cell line was investigated with colony assay. Moreover, the biodistribution of this radiotracer in SKOV-3 xenograft tumor-bearing nude mice were also studied to determine the radiotracer accumulation in the tumor site. The mice were treated with 177Lu-DOTA-LTVSPWY and subjected to histopathological evaluation. The RCP of 177Lu-DOTA-LTVSPWY after radiolabeling and stability tests was more than 97.7%. The radiotracer displayed high affinity toward the SKOV-3 cell line (KD = 6.6 ± 3.2nM). Treatment of the SKOV-3 cell line with the radiotracer reduces the SKOV-3 colony survival to less than 3% for 5MBq of the radiotracer. Tumor-to-muscle (T/M) ratio is the highest at 48h and 1h post-injection (2.3 and 4.75, respectively). The histopathological study also confirms the cellular damage to the tumor tissue. 177Lu-DOTA-LTVSPWY can recognize HER2 receptors in vivo and in vitro; hence, it can serve as a therapeutic agent.

Siderocalin fusion proteins enable a new 86Y/90Y theranostic approach.

The mammalian protein siderocalin binds bacterial siderophores and their iron complexes through cation-π and electrostatic interactions, but also displays high affinity for hydroxypyridinone complexes of trivalent lanthanides and actinides. In order to circumvent synthetic challenges, the use of siderocalin-antibody fusion proteins is explored herein as an alternative targeting approach for precision delivery of trivalent radiometals. We demonstrate the viability of this approach in vivo, using the theranostic pair 90Y (β-, t1/2 = 64 h)/86Y (β+, t1/2 = 14.7 h) in a SKOV-3 xenograft mouse model. Ligand radiolabeling with octadentate hydroxypyridinonate 3,4,3-LI(1,2-HOPO) and subsequent protein binding were achieved at room temperature. The results reported here suggest that the rapid non-covalent binding interaction between siderocalin fusion proteins and the negatively charged Y(iii)-3,4,3-LI(1,2-HOPO) complexes could enable purification-free, cold-kit labeling strategies for the application of therapeutically relevant radiometals in the clinic.

Signal Amplification Pretargeted PET/Fluorescence Imaging Based on Human Serum Albumin-Encapsulated GdF3 Nanoparticles for Diagnosis of Ovarian Cancer.

Different modal imaging techniques could be complementary in tumor diagnosis. Human serum albumin (HSA)-encapsulated GdF3 nanoparticles were developed as T1 magnetic resonance imaging (MRI) contrast agents. However, no significant T1 enhancement in the tumor site of the SKOV3 human ovarian cancer xenograft tumor model was observed within 3 h after injection of tetrazine-modified GdF3@HSA NPs through small-animal MRI. After intravenous injection of 18F (or Cy7)-labeled Reppe anhydride, pretargeted positron emission tomography (PET) (near-infrared (NIR) fluorescence) imaging was used to reveal the pharmacokinetics of GdF3@HSA NPs in the SKOV3 xenograft mouse model to locate the tumor. The probe based on Reppe anhydride achieved rapid ligation with tetrazine-modified GdF3@HSA nanoparticles (NPs), which accumulated in tumor through Reppe anhydride/tetrazine bioorthogonal chemistry. This pretargeting strategy enabled excellent tumor visualization and quantification at an early period after nanoparticle injection (3 h p.i.), while the MRI images with significant T1 enhancement could be obtained until 24 h after injection of Gd-based contrast agents only. In vivo pretargeted multimodal imaging based on the tetrazine/Reppe anhydride system using HSA-encapsulated GdF3 nanoparticles would be beneficial for amplification of the imaging signal in the disease site and enhancing diagnostic efficiency.

Shikonin Mediates Apoptosis through G Protein-Coupled Estrogen Receptor of Ovarian Cancer Cells.

This study was intended to establish the predictive target of Shikonin (SK) against ovarian cancer using network pharmacology and to clarify the potential mechanism of SK in promoting apoptosis in ovarian cancer. Cell Counting Kit-8 assay, plate clone assays, LDH assay, flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide staining, and western blotting were used to assess the effect of SK on apoptosis of ovarian cancer cell lines (SKOV3 and A2780). Pharmacodynamic targets were used to predict the targets of SK and ovarian cancer. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses were used to analyze the biological functions and signal pathways of these targets. SK promoted apoptosis in ovarian epithelioid adenocarcinoma cells. SK-ovarian cancer pharmacodynamic target analysis screened 17 related genes. GO and KEGG analyses showed that SK affected the estrogen signaling pathway. SK inhibited the expression of GPER in SKOV3 and A2780 cells and downregulated the expression of EGFR, p-EGFR, PI3K, and p-AKT in a concentration-dependent manner. The apoptosis-promoting effect of SK was enhanced by GPER-specific agonist G1 and inhibited by the specific inhibitor G15. The expression of EGFR, p-EGFR, PI3K, and p-AKT was decreased by G1 and reversed by G15. SK also inhibited tumor growth in the SKOV3 xenograft model, and it acted synergistically with G1. However, the effect can be attenuated by G15 in vivo. In summary, SK may affect the apoptosis of ovarian cancer cells through GPER/EGFR/PI3K/AKT, and GPER may be a key target of SK in ovarian cancer cell apoptosis.

Targeting CD44-positive ovarian cancers via engineered paclitaxel prodrug nanoparticles for enhanced chemotherapeutic efficacy

Ovarian cancer (OvCa) is currently the fifth most lethal malignancy affecting women health owing to the lack of early diagnosis and treatment choices available before the disease has progressed to a later stage. Paclitaxel (PTX) has shown substantial antineoplastic action against a variety of human cancers, including OvCa, for multiple decades. Despite this, the therapeutic use of this drug is not yet adequate owing to surfactant-related toxicities and off-target effects. In response to these constraints, nanoparticle carriers have evolved as delivery tools for the biocompatible and target delivery of PTX. In this work, a novel polymeric PTX formulation was developed for targeted therapy of OvCa cells, which was achieved by prodrug engineering and HA decoration strategies. Further studies indicated that HA-coated nanodrugs (HA-PLA-PTX NPs) could preferentially accumulate in the CD44-expressing SKOV3 cells, which induced elevated cytotoxicity, reduced cell proliferation, and increased cell apoptosis. In vivo study also demonstrated that equivalent doses of HA-PLA-PTX NPs surpassed the clinical PTX formulation Taxol in a SKOV3 xenograft tumor model. In conclusion, HA-PLA-PTX NPs might be a potentially feasible delivery system for effective OvCa treatment.

A novel form of docetaxel polymeric micelles demonstrates anti-tumor and ascites-inhibitory activities in animal models as monotherapy or in combination with anti-angiogenic agents.

Malignant ascites (MA) is caused by intraperitoneal spread of solid tumor cells and results in a poor quality of life. Chemotherapy is a common first-line treatment for patients with MA. Taxotere ® (DTX) is widely used in solid tumor therapies. However, the low water solubility and side effects caused by additives in the formulation restrict the clinical application of docetaxel. HT001 is a clinical stage docetaxel micelle developed to overcome the solubility issue with improved safety profiles. To support clinical development and expand clinical application of HT001, this study used in vitro and in vivo approaches to investigate the anti-tumor effects of HT001 when applied as monotherapy or in combination with anti-angiogenic agents. HT001 demonstrated comparable anti-proliferative activities as docetaxel in a broad range of cancer cell lines in vitro. Furthermore, HT001 suppressed tumor growth in a dose-dependent manner in A549, MCF-7, and SKOV-3 xenograft tumor mouse models in vivo. In a hepatocellular carcinoma H22 malignant ascites-bearing mouse model, HT001 presented a dose-dependent inhibition of ascites production, prolonged animal survival, and reduced VEGF levels. When dosed at 20 mg/kg, the HT001-treated group exhibited curative results, with no ascites formation in 80% of mice at the end of the study while all the mice in the vehicle control group succumbed. Similar results were obtained in HT001 treatment of mice bearing malignant ascites produced by human ovarian cancer ES-2 cells. Notably, the combination of HT001 with Endostar not only significantly reduced ascites production but also prolonged survival of H22 ascites-bearing mice. HT001 showed similar PK and tissue distribution profiles as DTX in non-rodent hosts. Collectively, these results demonstrate potent anti-tumor activity of HT001 in multiple solid tumor models or malignant ascites models, and reveal synergistic effects with anti-angiogenic agents, supporting the clinical development and clinical expansion plans for HT001.

Agrimonolide inhibits cancer progression and induces ferroptosis and apoptosis by targeting SCD1 in ovarian cancer cells

BackgroundOvarian cancer is a gynaecological tumour has high incidence and mortality rates. Agrimonolide, isolated from Agrimonia pilosa Ledeb, has multiple biomedical activities, including anticancer activity. PurposeHere, we aimed to reveal the function of agrimonolide on ovarian cancer progression. MethodsMTT assay, colony-formation assay, flow cytometry, transwell assay, scratch test, western immunoblotting, reactive oxygen species (ROS) detection, and ferroptosis analysis were performed to reveal the role and underlying mechanisms of agrimonolide in ovarian cancer cell lines (A2780 and SKOV-3). The effects of agrimonolide on the SKOV-3 xenograft model were also studied. ResultsAgrimonolide dose-dependently inhibited proliferation, migration, and invasion and promoted apoptosis in A2780 and SKOV-3 cells. Agrimonolide induced ferroptosis in tumour cells, evidenced by the increased levels of ROS, total iron, and Fe2+ and downregulation of ferroptosis indicators (SLC7A11 and GPX4). The SwissTargetPrediction and Comparative Toxicogenomics Database predicted SCD1 as a target protein for agrimonolide. Molecular Operating Environment software docked agrimonolide in the SCD1 protein, and the binding energy of interaction was -8.21 kcal/mol. The effects of agrimonolide on proliferation, invasion, and induction of apoptosis and ferroptosis were attenuated by SCD1 overexpression in A2780 and SKOV-3 cells. Additionally, agrimonolide attenuated the tumour growth of ovarian cancer in the SKOV-3 xenograft model and significantly downregulated SCD1 in tumour tissues. ConclusionOur study is the first to suggest that agrimonolide acts as a novel apoptosis- and ferroptosis-inducing agent in ovarian cancer cells by targeting SCD1. Agrimonolide may be a novel therapeutic agent for treating ovarian cancer.

Fuzheng Jiedu Decoction Induces Apoptosis and Enhances Cisplatin Efficacy in Ovarian Cancer Cells In Vitro and In Vivo through Inhibiting the PI3K/AKT/mTOR/NF-κB Signaling Pathway

Objectives This study is aimed at investigating the anticancer activity of Fuzheng Jiedu decoction (FJD) alone or in combination with cisplatin in ovarian cancer (OC) models, as well as its underlying mechanisms of action. Methods The anticancer activities of FJD, cisplatin, and the combination of the PI3K inhibitor (LY294002, LY) or activator (IGF-1) were evaluated in OC cell lines in vitro and in a SKOV3 xenograft mouse model in vivo. The cell proliferation and invasion ability were measured using MTT, EdU, and transwell assays, respectively. The cell apoptosis was examined by flow cytometry and JC-1 assays. The expression levels of the Bcl-2 family and the PI3K/AKT/mTOR/NF-κB pathway-related proteins were analyzed by Western blot. Results The in vivo and in vitro studies showed that FJD administration could significantly inhibit cell proliferation and promote cell apoptosis in two OC cell lines SKOV3 and 3AO and partially decreased the tumor volumes and weights. In addition, FJD could significantly downregulate the protein levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, NF-κB, p38, and Bcl-2 and upregulate the Bax, Cyt-C, and cleaved caspase-3 in OC tumor tissues and cells. FJD cotreatment increased the efficacy of cisplatin, including inhibiting OC cell proliferation and invasion, promoting cell apoptosis, and inhibiting the PI3K/AKT/mTOR signaling pathway, while this enhancement was suppressed by IGF-1. Similarly, LY also enhanced the anticancer efficacy of cisplatin. Conclusions This study indicated that FJD could improve the efficacy of cisplatin by inhibiting the PI3K/AKT/mTOR/NF-κB signaling pathway. It is suggested that FJD may be a valuable adjuvant drug for the treatment of OC.

Evaluation of an 131I-labeled HER2-specific single domain antibody fragment for the radiopharmaceutical therapy of HER2-expressing cancers

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH_1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH_1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[131I]iodobenzoate (iso-[131I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [131I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[131I]SGMIB-VHH_1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[131I]SGMIB-VHH_1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc2-iso-[131I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[131I]SGMIB-VHH_1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[131I]SGMIB-VHH_1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted.

Half-Sandwich Cyclometalated RhIII Complexes Bearing Thiolate Ligands: Biomolecular Interactions and In Vitro and In Vivo Evaluations.

A class of cyclometalated RhIII complexes [Cp*Rh(ppy)(SR)] bearing thiolate ligands, Cp* = pentamethylcyclopentadienyl, ppy = 2-phenylpyridinate, and R = pyridyl (Spy, 2), pyrimidyl (SpyN, 3), benzimidazolyl (Sbi, 4), and benzothiazolyl (Sbt, 5), were produced and identified by means of spectroscopic methods. The in vitro cytotoxicity of the RhIII compounds in three different human mortal cancerous cell lines (ovarian, SKOV3; breast, MCF-7; lung, A549) and a normal lung (MRC-5) cell line were evaluated, indicating the selectivity of these cyclometalated RhIII complexes to cancer cells. Complex 5, selected for in vivo experiment, has shown an effective inhibition of tumor growth in SKOV3 xenograft mouse model relative to control (p-values < 0.05 and < 0.01). Importantly, the outcomes of H&E (hematoxylin and eosin) staining and hematological analysis revealed negligible toxicity of 5 compared to cisplatin on a functioning of the main organs of mouse. Molecular docking, UV-vis, and emission spectroscopies (fluorescence, 3D fluorescence, synchronous) techniques were carried out on 1-5 to peruse the mechanism of the anticancer activities of these complexes. The obtained data help to manifest the binding affinity between the rhodium compounds and calf thymus DNA (CT-DNA) through the interaction by DNA minor groove and moderate binding affinity with bovine serum albumin (BSA), particularly with the cavity in the subdomain IIA. It can be concluded that the Rh-thiolate complexes are highly promising leads for the development of novel effective DNA-targeted anticancer drugs.

Disruption of adenosine 2A receptor improves the anti-tumor function of anti-mesothelin CAR T cells both in vitro and in vivo

Chimeric antigen receptor (CAR) T cells have been successfully used for the treatment of hematological malignancies including acute and chronic lymphoblastic leukemia. However, results of CAR T cell projects in solid tumors have been less impressive to date, partly because of immunosuppressive tumor microenvironment (TME). It is widely known that high adenosine production is an important factor causing tumor-induced immunosuppression in TME, and adenosine mediates the suppression of anti-tumor T cell responses via binding and signaling through adenosine 2a receptor (A2aR). Previous studies have shown that adenosine generated by cancer cells significantly inhibits T cell anti-tumor activity through binding and then activating adenosine 2A receptors (A2aRs) of T cells. Based on the previous work, in our study, we evaluated whether A2aR disruption by shRNA could enhance the anti-tumor function of anti-mesothelin (MSLN) CAR T cells both in vitro and in vivo. For this goal above, we used MSLN-positive human ovarian serous carcinoma cells (SKOV3) and human colon cancer cells (HCT116) as target cancer cells while MSLN-negative human ovarian cancer cells (ES2) as non-target cancer cells. We observed that targeting cell-intrinsic A2aR through shRNA overexpression caused significant A2aR disruption in CAR T cells and profoundly increased CAR T cell efficacy in both CAR T cell cytokine production and cytotoxicity towards MSLN-positive cancer cells in vitro. More importantly, in SKOV3 xenograft mouse models, anti-MSLN CAR-T cells significantly reduced the tumor burden compared with non-transduced T cells, and the anti-tumor activity of A2aR-disrupted anti-MSLN CAR-T cells was stronger than that of wild-type anti-MSLN CAR-T cells.Altogether, our study showed enhanced anti-tumor efficacy caused by shRNA-mediated A2aR disruption in anti-MSLN CAR T cells both in vitro and in vivo, which proved that shRNA-mediated modification of gene expression might be an excellent strategy for improving CAR T cell function in immunosuppressive tumor microenvironment (TME) and could potentially improve the outcome of treatment in clinical trials.

Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer

ObjectivePoly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. MethodsWe assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. ResultsEntinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. ConclusionEntinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

A Novel Salt Inducible Kinase 2 Inhibitor, ARN-3261, Sensitizes Ovarian Cancer Cell Lines and Xenografts to Carboplatin.

Simple SummaryCarboplatin and paclitaxel constitute first-line treatment for ovarian cancer, producing tumor shrinkage in 70% of patients, but curing less than 20% with advanced stage disease. Previous studies have shown that treatment with ARN-3261, a small molecule inhibitor of the enzyme salt-induced kinase 2, can improve the response to paclitaxel in human ovarian cancer cells grown in culture and in immunocompromised mice. Here we have found that ARN-3261 also increases carboplatin’s ability to kill ovarian cancer cells grown in culture and in immunocompromised mice, causing additional DNA damage and decreasing levels of survivin, a protein that protects cancer cells from programmed cell death. Our studies encourage clinical evaluation of ARN-3261 and a Phase I clinical trial has been initiated to test the drug in ovarian cancer patients.Salt-induced kinase 2 (SIK2) is a serine-threonine kinase that regulates centrosome splitting, activation of PI3 kinase and phosphorylation of class IIa HDACs, affecting gene expression. Previously, we found that inhibition of SIK2 enhanced sensitivity of ovarian cancer cells to paclitaxel. Carboplatin and paclitaxel constitute first-line therapy for most patients with ovarian carcinoma, producing a 70% clinical response rate, but curing <20% of patients with advanced disease. We have asked whether inhibition of SIK2 with ARN-3261 enhances sensitivity to carboplatin in ovarian cancer cell lines and xenograft models. ARN-3261-induced DNA damage and apoptosis were measured with γ-H2AX accumulation, comet assays, and annexin V. ARN-3261 inhibited growth of eight ovarian cancer cell lines at an IC50 of 0.8 to 3.5 µM. ARN-3261 significantly enhanced sensitivity to carboplatin in seven of eight ovarian cancer cell lines and a carboplatin-resistant cell line tested. Furthermore, ARN-3261 in combination with carboplatin produced greater inhibition of tumor growth than carboplatin alone in SKOv3 and OVCAR8 ovarian cancer xenograft models. ARN-3261 enhanced DNA damage and apoptosis by downregulating expression of survivin. Thus, a SIK2 kinase inhibitor enhanced carboplatin-induced therapy in preclinical models of ovarian cancer and deserves further evaluation in clinical trials.

Identification of methyl (5-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-morpholinopyrrolo[2,1-f][1,2,4]triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)carbamate (CYH33) as an orally bioavailable, highly potent, PI3K alpha inhibitor for the treatment of advanced solid tumors.

In various human cancers, PI3Ks pathway is ubiquitously dysregulated and thus become a promising anti-cancer target. To discover new potent and selective PI3K inhibitors as potential anticancer drugs, new pyrrolo[2,1-f][1,2,4]triazines were designed, leading to the discovery of compound 37 (CYH33), a selective PI3Kα inhibitor (IC50=5.9nM, β/α, δ/α,γ/α=101-, 13-, 38-fold). Western blot analysis confirmed that compound 37 could inhibit phosphorylation of AKT in human cancer cells to modulate the cellular PI3K/AKT/mTOR pathway. And further evaluation invivo against SKOV-3 xenograft models demonstrated that a dose-dependent antitumor efficacy was achieved.

20(S)-Rg3 upregulates FDFT1 via reducing miR-4425 to inhibit ovarian cancer progression

We previously found that ginsenoside 20(S)-Rg3 diminishes the proliferative and invasive capacities of ovarian cancer cells by decreasing miR-4425 level. Yet the mechanism of action of miR-4425 in ovarian cancer remains unclear. Here we report that miR-4425 is upregulated in ovarian cancer tissues relative to normal ovarian tissues, and transfection of miR-4425 inhibitor impairs the proliferation, migration and invasion of SKOV3 and 3AO ovarian cancer cells. Further, miR-4425 antagomiR reduces cell proliferation in a subcutaneous SKOV3 xenograft model using BALB/c nude mice. We identifies farnesyl-diphosphate farnesyltransferase 1 (FDFT1) as a direct target of miR-4425 by Western blotting and a luciferase reporter assay. Forced expression of FDFT1 via transfection of an FDFT1-expressing plasmid into ovarian cancer cells not only retards cell proliferation, motility and invasiveness, but also negates the tumorigenic properties of a miR-4425 mimic. By contrast, silencing of FDFT1 by siRNAs abrogates suppression of the proliferation, migration and invasion of ovarian cancer cells treated with a miR-4425 inhibitor. Finally, transfection of either a miR-4425 mimic or FDFT1 siRNAs into 20(S)-Rg3-treated ovarian cancer cells counteracts the tumor-inhibitory activity of the ginsenoside. In conclusion, 20(S)-Rg3 exerts anti-ovarian cancer activity by downregulating oncogenic miR-4425 that inhibits the expression of the tumor suppressor gene FDFT1. These results expand our current understanding of the molecular pathways leading to ovarian cancer progression, and unveil the mechanism of action of 20(S)-Rg3 in ovarian cancer inhibition.

Suppression of c-Met-Overexpressing Tumors by a Novel c-Met/CD3 Bispecific Antibody.

IntroductionOverexpression of c-Met, or hepatocyte growth factor (HGF) receptor, is commonly observed in tumor biopsies and often associated with poor patient survival, which makes HGF/c-Met pathway an attractive molecular target for cancer therapy. A number of antibody-based therapeutic strategies have been explored to block c-Met or HGF in cancers; however, clinical efficacy has been very limited, indicating that blockade of c-Met signal alone is not sufficient. Thus, an alternative approach is to develop an immunotherapy strategy for c-Met-overexpressing cancers. c-Met/CD3 bispecific antibody (BsAb) could bridge CD3-positive T lymphocytes and tumor cells to result in potent tumor cell killing.Materials and MethodsA bispecific antibody, BS001, which binds both c-Met and CD3, was generated using a novel BsAb platform. Western blotting and T cells-mediated killing assays were utilized to evaluate the BsAb’s effects on cell proliferation, survival and signal transduction in tumor cells. Subcutaneous tumor mouse models were used to analyze the in vivo anti-tumor effects of the bispecific antibody and its combination therapy with PD-L1 antibody.ResultsBS001 showed potent T-cell mediated tumor cells killing in vitro. Furthermore, BS001 inhibited phosphorylation of c-Met and downstream signal transduction in tumor cells. In A549 lung cancer xenograft model, BS001 inhibited tumor growth and increased the proportion of activated CD56+ tumor infiltrating lymphocytes. In vivo combination therapy of BS001 with Atezolizumab (an anti-programmed cell death protein1-ligand (PD-L1) antibody) showed more potent tumor inhibition than monotherapies. Similarly, in SKOV3 xenograft model, BS001 showed a significant efficacy in tumor growth inhibition and tumor recurrence was not observed in more than half of mice treated with a combination of BS001 and Pembrolizumab.Conclusionc-Met/CD3 bispecific antibody BS001 exhibited potent anti-tumor activities in vitro and in vivo, which was achieved through two distinguished mechanisms: through antibody-mediated tumor cell killing by T cells and through inhibition of c-Met signal transduction.

Design and optimization of orally spleen tyrosine kinase (SYK) inhibitors for treatment of solid tumor

As the aim to discover orally SYK inhibitors for solid tumor treatment, a series of novel derivatives based on imidazo[1,2-a]pyrazine scaffold were designed, synthesized and evaluated. Structure-activity relationship study of both enzymatic and cellular assays led to the identification of compound 12f. The novel SYK inhibitor 12f showed potent antitumor activity against solid tumors with favorable drug-like properties of lipophilicity and solubility. 12f could induce cell apoptosis of ovarian and lung cancer cell lines. In SKOV3 xenograft mouse model, oral administration of 12f led to significant tumour regression without obvious toxicity. 12f improved the limited response of traditional SYK inhibitors in solid tumors in vitro and in vivo. Taken together, this compound may act as a promising lead compound for further development of new SYK inhibitors for solid tumor therapy.