Panel Of Tumor Cell Lines Research Articles

Abstract 6318: Preclinical characterization of ARX305, a next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers

Abstract CD70 has emerged as an attractive tumor target with its over-expression in multiple solid and hematological cancers, yet tightly controlled and limited expression in normal cells. In the last decade, multiple anti-CD70 antibody drug conjugates (ADCs) have been developed, but most, if not all, of these ADC programs have been discontinued. Using an expanded genetic code to create Engineered Precision Biologics (EPBs), Ambrx has developed ARX305, a CD70-targeted next-generation antibody drug conjugate (ADC) to potentially overcome the challenges associated with earlier ADCs and treat patients with cancers over-expressing CD70. ARX305 is comprised of a proprietary, high-affinity, humanized anti-CD70 antibody stably and site-specifically conjugated to AS269, a potent microtubule inhibitor. After binding to CD70 expressed on target tumor cells, ARX305 internalizes and releases the cytotoxic payload pAF-AS269 which inhibits cellular proliferation. In vitro testing across a panel of solid and liquid tumor cell lines showed ARX305 elicited highly potent and specific cell killing across multiple tumor types with medium to high CD70 expression levels. In multiple in vivo CD70-expressing xenograft or disseminated models, ARX305 induced significant tumor growth inhibition or regression whereas unconjugated antibody exhibited poor activity. ARX305 utilizes stable oxime conjugation chemistry, a non-cleavable PEG linker, and a membrane-impermeable payload to minimize premature payload release in circulation and associated off-target toxicity. These properties of ARX305 result in high serum stability, with rodent pharmacokinetic studies confirming a similar exposure profile of ARX305 compared to unconjugated antibody and a long serum half-life. Repeat dose toxicokinetic studies in cynomolgus monkeys demonstrated ARX305 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, the highly potent anti-tumor activity in multiple tumor types and wide therapeutic index of ARX305 support clinical evaluation of this next generation anti-CD70 ADC. ARX305 is currently in a Phase 1 dose escalation study in China, and the IND in the United States is open. Citation Format: Lillian Skidmore, Jingjing Zhu, David Mills, Ting Jin, Suqin Li, Keith Tatsukawa, Nick A. Knudsen, Liying Gong, Jay Nelson, Kari Cox, Amha G. Hewet, Jianing Wang, Tim Buss, Feng Tian, Gang Xia, Shawn Zhang. Preclinical characterization of ARX305, a next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6318.

Abstract 6279: CTS2190, a next-generation epigenetic inhibitor for targeting type I PRMT deregulated human tumors

Abstract Methylation of arginine residues by protein arginine methyltransferases (PRMTs) is involved in the regulation of fundamental cellular processes. Type I PRMTs are frequently overexpressed in a variety of human cancers and associated with poor prognosis. Knockout (KO) of Prmt1 in mice results in decreased asymmetric dimethylarginine (ADMA) on cellular proteins, together with increased monomethylarginine (MMA) and symmetric dimethylarginine (SDMA) (Dhar et al., 2013). A structure-based drug discovery effort led to the identification of CTS2190 that showed single-digit nanomolar enzymatic inhibitory activity on type I PRMTs. Here, we report the development and preclinical characterization of CTS2190, a potent, orally bioavailable small molecule type I PRMT inhibitor. CTS2190 demonstrated strong cellular proliferation inhibitory activity in a large, diverse panel of human hematological and solid tumor cell lines across several lineages. In vivo, oral CTS2190 administration once daily resulted in tumor regression in a variety of xenograft (both CDX and PDX) models across different lineages with good tolerance. The anti-tumor activity was dose-dependent and correlated with the inhibition of type I PRMT activity as measured by decrease in ADMA and increase in MMA. In summary, CTS2190 is a promising type I PRMT inhibitor and clinical trial is underway to explore the proof-of-concept anti-tumor benefit for patients. Citation Format: Hui Shi, Xingnian Fu, Qiugeng Ouyang, Jiaxin Huang, Yiqin Wang, Youzhen Wang, Guoliang Xu, Yuan Mi, Haiping Wu. CTS2190, a next-generation epigenetic inhibitor for targeting type I PRMT deregulated human tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6279.

Abstract 6298: A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors

Abstract Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some lymphoma, myeloma and some types of solid tumors including melanoma, non-small cell lung cancers and liver cancers. Previous attempt to target ICAM-1 with a monoclonal antibody bersanlimab (BI505, BioInvent) showed that it was well-tolerated but with limited clinical efficacy. To improve the effectiveness of targeting ICAM-1, we developed a novel ICAM-1 antibody drug conjugate (ADC). The ICAM-1 ADC is comprised of a humanized anti-ICAM-1 hIgG1 antibody conjugated to a novel topoisomerase I inhibitor through a cleavable linker at a drug-to-antibody ratio (DAR) of about 8. The antibody moiety of the ADC targets a unique epitope on ICAM-1 which is far from its ligand binding domain. Potent in vitro cytotoxicity of ICAM-1 ADC, with nanomolar to sub-nanomolar EC50, was observed on a panel of hematologic and solid tumor cell lines. In preclinical studies, one or two doses of ICAM-1 ADC at 5-6 mg/kg were generally sufficient to induce tumor regression in multiple cell line-derived xenograft models, including those of lung and liver cancers. In all models tested so far, the novel topoisomerase I inhibitor conjugated ICAM-1 ADC is at least as efficacious as the same ICAM-1 antibody conjugated to the benchmark topoisomerase I inhibitor linker payload deruxtecan (ICAM-1-DXd). Furthermore, in a repeat dose exploratory non-human primate safety study, the benchmark conjugate ICAM-1-DXd (DAR 8) was well-tolerated at 41 mg/kg, the highest dose tested. In conclusion, ICAM-1 is an attractive target for topoisomerase I inhibitor based ADC and warrants further investigation. Citation Format: Oi Kwan Wong, David Jackson, Lei Shi, Qi Fei, Xiaocheng Chen, Leonard Post. A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6298.

Abstract 694: TG6050 an oncolytic vaccinia virus armed with interleukin 12 and anti-CTLA4 antibody induces TME remodeling and strong anti-tumoral responses

Abstract Background: By replicating specifically into tumor cells, oncolytic vaccinia viruses (VACV) can turn “cold” tumors “hot” while delivering therapeutic payloads into tumor. Thanks to its large genome capacity, multiple cloning loci, and availability of different promoters, VACV is an optimal vector for the design of advanced immunotherapies. Payloads with an established clinical efficacy but limited in terms of tolerability require local delivery. We report here the preclinical characterization of TG6050, a VACV encoding single chain interleukin 12 (IL12) and anti-CTLA4, targeting multiple cancer indications. Methods: TG6050 was generated by insertion of both the single chain IL-12p70, and an anti-CTLA-4 full-length (heavy and light chains independently) into the thymidine kinase (TK) and ribonucleotide reductase (RR) loci, of a triple deleted (ΔJ2R,ΔI4L,ΔM2L) VACV, Copenhagen strain. TG6050 was fully characterized in vitro, i.e. replication in tumor and normal cells, transgenes expression, functionality, and genetic stability. Its preclinical surrogate mTG6050, expressing murine payloads, was used to investigate the in vivo anti-tumoral activities in a broad range of immunocompetent murine models, as well as its mode of action in the reprograming of the tumor microenvironment (TME). Results: TG6050 displayed the same replicative, oncolytic activity, and genetic stability features as benchmark recombinant vaccinia viruses. IL-12 and anti-CTLA4 were both expressed at high levels, and as functional molecules by a broad panel of reference tumor cell lines. Local administration of mTG6050 induced accumulation of transgenes into tumor, with low systemic exposure. Noteworthy, expression of IL-12 did not accelerate the clearance of the viral vector. The combined effects of viral replication, IL-12 and anti-CTLA4 expression translated into impressive antitumoral activities in several syngeneic tumor models including immune-resistant ones such as B16F10 or LLC1. Transcriptomic analyses of the TG6050-treated tumors demonstrated a strong dynamic of infiltration by innate and adaptive immune cells. IFNy-ELISpot analysis on splenocytes confirmed induction of a strong systemic and specific anti-tumoral immune response. Moreover, the combination of TG6050 with ICI improved tumor regression in several challenging tumor models. Conclusion: TG6050 is a novel oncolytic VACV designed for multiple routes of administration, and several tumor indications. Its strong impact on the TME, by massive infiltration of innate and adaptive immune cells, translated into very remarkable therapeutic activities in resistant tumors. This anti-tumor activity was further enhanced by combination with anti-PD-1. Citation Format: Jean-Baptiste Marchand, Fadi Azar, Christelle Demeusoit, Patricia Kleinpeter, Jules Deforges, Fend Laetitia, Chantal Hoffmann, Huguette Schultz, Nathalie Silvestre, Eric Quéméneur. TG6050 an oncolytic vaccinia virus armed with interleukin 12 and anti-CTLA4 antibody induces TME remodeling and strong anti-tumoral responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 694.

Abstract 4884: Targeted investigational oncology agents (IOA) in the NCI60: a phenotypic systems-based resource

Abstract The NCI60 human tumor cell line panel is a useful tool in the discovery and development of new anticancer agents. The publicly available cell-line characterization and compound screening data from the NCI60 screen have contributed to identifying cellular mechanisms of potential new anticancer agents. Mean-graph sensitivity/resistance patterns in the NCI60 screen serve as a fingerprint for molecular target identification and mechanism of action (MOA). A new NCI60 resource was developed based on screening of 175 FDA-approved oncology drugs (AOD) plus >825 investigational oncology agents (IOA), representing >250 therapeutic targets and MOAs. Compounds targeting different components in a biochemical pathway tend to show high correlations in their GI50 patterns through COMPARE analysis forming clusters with similar NCI60 mean-graph patterns. COMPARE evaluation of compounds that target components in the PI3K/AKT/mTOR pathway form correlation clusters linked by both target and pathway. Only 3 of 15 AKT inhibitors in the IOA set are not found in the connected cluster with a COMPARE correlation of 0.7 including the multi-kinase inhibitors perifosine and AT-13148. The AKT cluster includes both allosteric (MK-2206) and competitive inhibitors highlighting the NCI60 is a functional cell assay. More than 75% of PI3K inhibitors (32/42 agents), including the selective PI3K alpha and PI3K beta inhibitors, form a highly connected cluster at a 0.7 COMPARE correlation. The heat map view showed that >50% of the non-connected PI3K outliers were inactive in the NCI60 assay. The mTOR inhibitors (12/20) form a connected cluster at 0.7 COMPARE correlation, with half of the non-connected mTOR singletons representative of the rapamycin class of inhibitors. The BRAF/MEK/ERK pathway compounds (37) form a highly connected correlation cluster (13/15 BRAF, 14/15 MEK, 8/9 ERK inhibitors) at a 0.75 COMPARE correlation. The NCI60 heat maps for BRAF inhibitors show a consistent pattern of the melanoma cell lines and 2 of the colon cancer lines (COLO 205, HT29). The ERK inhibitors display sensitivity patterns similar to the BRAF agents with additional activity observed against a leukemia (HL-60), ovarian (OVCAR-5) and renal (A498) cell line and 3 other colon cell lines (HCC-298, HCT-116, SW-620). This data analysis resource will be available to the public (https://ioa.cancer.gov). NCI60 compound suppliers can incorporate their test compound(s) data to use the interactive visualization tools with AOD and IOA agents. This project was funded in part with federal funds from the NCI, NIH, under Contract # 75N91019D00024/75N91020F00003. Citation Format: Joel Morris, Mark W. Kunkel, Stephen L. White, Donn G. Wishka, Omar D. Lopez, Lori Bowles, Penny Sellers, Patricia Ramsey, Julie Grams, Tiffany Rohrer, Karen Martin, Thomas Dexheimer, Dmitriy Sonkin, John D. Williams, Jerry M. Collins, James H. Doroshow, Beverly A. Teicher. Targeted investigational oncology agents (IOA) in the NCI60: a phenotypic systems-based resource. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4884.

Therapeutic impact of purified Trichoderma virideL-asparaginase in murine model of liver cancer and in vitro Hep-G2 cell line

BackgroundHepatocellular carcinoma (HCC) is among the common cancers, but difficult to diagnose and treat. l-asparaginase has been introduced in the treatment protocol of pediatric acute lymphoblastic leukemia (ALL) since the 1960s with a good outcome and increased survival rates to nearly 90%. Moreover, it has been found to have therapeutic potential in solid tumors. Production of glutaminase-free-l-asparaginase is of interest to avoid glutaminase-related toxicity and hypersensitivity. In the current study, an extracellular l-asparaginase that is free of l-glutaminase was purified from the culture filtrate of an endophytic fungus Trichoderma viride. The cytotoxic effect of the purified enzyme was evaluated in vitro against a panel of human tumor cell lines and in vivo against male Wister albino mice intraperitoneally injected with diethyl nitrosamine (200 mg/kg bw), followed by (after 2 weeks) oral administration of carbon tetrachloride (2 mL/kg bw). This dose was repeated for 2 months, and after that, the blood samples were collected to estimate hepatic and renal injury markers, lipid profiles, and oxidative stress parameters. Resultsl-asparaginase was purified from T. viride culture filtrate with 36 purification folds, 688.1 U/mg specific activity, and 38.9% yield. The highest antiproliferative activity of the purified enzyme was observed against the hepatocellular carcinoma (Hep-G2) cell line, with an IC50 of 21.2 g/mL, which was higher than that observed for MCF-7 (IC50 34.2 g/mL). Comparing the DENA-intoxicated group to the negative control group, it can be demonstrated that l-asparaginase adjusted the levels of the liver function enzymes and the hepatic injury markers that had previously changed with DENA intoxication. DENA causes kidney dysfunction and altered serum albumin and creatinine levels as well. Administration of l-asparaginase was found to improve the levels of the tested biomarkers including kidney and liver function tests. l-asparaginase treatment of the DENA-intoxicated group resulted in a significant improvement in the liver and kidney tissues to near normal similar to the healthy control group. ConclusionThe results suggest that this purified T. viridel-asparaginase may be able to delay the development of liver cancer and may be used as a potential candidate for future application in medicine as an anticancer medication.

Study on the Phototoxicity and Antitumor Activity of Plant Extracts from Tanacetum vulgare L., Epilobium parviflorum Schreb., and Geranium sanguineum L.

Our country is rich in medicinal plants with a thousand-year tradition of use for therapeutic and cosmetic purposes. World Health Organization estimates that around 70% of population is using traditional (folk) medicine to cure various ailments. In many cases, herbal preparations prove to be useful, but some of them can also exert toxic effects. Therefore, herbal extracts should be tested both as curatives and for safety application. The aim of the present work was to investigate extracts from Tanacetum vulgare (TVA), Epilobium parviflorum (EPE) and Geranium sanguineum (GSA), obtained by different organic solvents, for their phototoxicity and anticancer activity. The standard BALB/c 3T3 Neutral Red Uptake phototoxicity/cytotoxicity assay was used to evaluate the possible phototoxic properties of the extracts. The antitumor activity of the extracts was studied in vitro on a panel of human tumor cell lines in comparison to the non-tumorigenic MCF-10A cells. The selectivity indices and the photo-irritancy factors were determined. The results show that the studied extracts are not phototoxic at concentration range from 4 to 1000 µg/ml. Determined photo-irritancy factors (PIF) for the extracts was < 2 (PIF for EPE = 1.72, GSA = 1.04, TVA = 1.43), which assigns them to the category of non-phototoxic substances. In addition, at concentrations lower than < 60 µg/ml, no statistically significant cytotoxicity was observed. The selectivity index was calculated relative to the IC50 value for non-tumorigenic MCF-10A cells. The highest selective index obtained with respect to the HT-29 cells was shown by all testing extracts (selective index (SI) for EPE = 2.6, GSA = 2.4 and TVA = 2.5), to the HeLa cells by extract GSA (SI = 2.0) and to the PC3 cells by extracts GSA (SI = 2.6) and TVA (SI = 2.0). Presented data shows that the studied extracts have a high level of photosafety with a promising antitumor activity in certain cell lines.

In silico and In vitro Determination of Antiproliferative Activity of Series N-Pyrrolyl Hydrazide-Hydrazones and Evaluation of their Effects on Isolated Rat Mycrosomes and Hepatocytes.

The significant increase in patients suffering from different types of cancer, guides scientists to take prompt measures in the development of novel and effective antiproliferative agents, where the intercalation of heterocyclic fragments in the designed molecules has proven to be a useful practice. The newly synthesized compounds were obtained from the corresponding 1,4-dicarbonyl derivative through multicomponent reactions to produce biologically active target molecules and assessed by in silico and in vitro assays for their possible antitumor activity. The pharmacological bioassay was conducted in the panel of human tumor cell lines (i) SKW-3 (ACC 53) - human T-cell leukemia and (ii) HL-60 (ACC 3) - human acute myeloid leukemia (AML). The statistical processing of MTT data included the paired Student's t-test with p ≤ 0.05 set as significance level. All evaluated structures displayed a higher cytotoxic effect against the acute myeloid leukemia HL-60 with 11o and 11p as the most active compared to the activity against SKW-3 cell line. Throughout the cytotoxicity screening two molecules, 11l and 12o, displayed comparable chemosensitivity on both cell lines. The corresponding hepatotoxicity on isolated rat hepatocytes and microsomes was also established, identifying 11, 12 and 12a as the least toxic and 11x, 11d, 12x and 12d as the most toxic derivatives. As the most promising compound is underlined ethyl 1-(2-(2-((1-acetyl-1H-indol-3-yl)methylene)hydrazinyl)-2- oxoethyl)-5-(4-bromophenyl)-2-methyl-1H-pyrrole-3-carboxylate (11l) demonstrating highest activity on both evaluated tumor cell lines, decreased hepatotoxicity on all evaluated parameters and docking score of -7.517 kcal/mol.

Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells.

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties-minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

Cisplatin and zoledronic acid: two drugs combined in a Pt(II) complex with potential antitumor activity towards bone tumors and metastases.

Treatment of primary bone malignancies comprises surgery, radiotherapy, chemotherapy, and analgesics. Platinum-based chemotherapeutics, such as cisplatin, are commonly used for the treatment of bone cancer but, despite their success, outcomes are limited by toxicity and resistance. Recently, dinuclear Pt complexes with a bridging geminal bisphosphonate ligand proved to be endowed with selective accumulation in bone tumors or metastases leading to improved efficacy and reduced systemic toxicity. Further improvement could be expected by the use of a bisphosphonate ligand with intrinsic pharmacological activity such as zoledronic acid (ZL). In the present work is reported the synthesis and full characterization of the dinuclear Pt(II) complex [{cis-Pt(NH3)2}2(ZL)]HSO4 which combines two drugs with antitumor activity, cisplatin and zoledronic acid. Both drugs, individually, are already approved by the U.S. Food and Drug Administration and the European Medicinal Agency for clinical use. The in vitro cytotoxicity of the new Pt(II)-ZL complex has been tested against a panel of human tumor cell lines.

Novel pyrazolo[3,4-d]pyrimidine derivatives: design, synthesis, anticancer evaluation, VEGFR-2 inhibition, and antiangiogenic activity

A novel series of 12 pyrazolo[3,4-d]pyrimidine derivatives were created and evaluated in vitro for their antiproliferative activity against the NCI 60 human tumor cell line panel.

Synthesis and Antiproliferative Activity against Cancer Cells of Indole-Aryl-Amide Derivatives

Indoles constitute a large family of heterocyclic compounds widely occurring in nature which are present in a number of bioactive natural and synthetic compounds, including anticancer agents or atypical opioid agonists. As a result, exponential increases in the development of novel methods for the synthesis of indole-containing compounds have been reported in the literature. A series of indole-aryl amide derivatives 1-7 containing tryptamine or an indolylacetic acid nucleus were designed, synthesized, and evaluated as opioid ligands. These new indole derivatives showed negligible to very low affinity for μ- and δ-opioid receptor (OR). On the other hand, compounds 2, 5 and 7 showed Ki values in the low μM range for κ-OR. Since indoles are well known for their anticancer potential, their effect against a panel of tumor cell lines was tested. The target compounds were evaluated for their in vitro cytotoxicity in HT29, HeLa, IGROV-1, MCF7, PC-3, and Jurkat J6 cells. Some of the synthesized compounds showed good activity against the selected tumor cell lines, with the exception of IGROV1. In particular, compound 5 showed a noteworthy selectivity towards HT29 cells, a malignant colonic cell line, without affecting healthy human intestinal cells. Further studies revealed that 5 caused the cell cycle arrest in the G1 phase and promoted apoptosis in HT29 cells.

Studies on 1,4-Quinone Derivatives Exhibiting Anti-Leukemic Activity along with Anti-Colorectal and Anti-Breast Cancer Effects

Colorectal cancer (CRC), breast cancer, and chronic myeloid leukemia (CML) are life-threatening malignancies worldwide. Although potent therapeutic and screening strategies have been developed so far, these cancer types are still major public health problems. Therefore, the exploration of more potent and selective new agents is urgently required for the treatment of these cancers. Quinones represent one of the most important structures in anticancer drug discovery. We have previously identified a series of quinone-based compounds (ABQ-1-17) as anti-CML agents. In the current work, ABQ-3 was taken to the National Cancer Institute (NCI) for screening to determine its in vitro antiproliferative effects against a large panel of human tumor cell lines at five doses. ABQ-3 revealed significant growth inhibition against HCT-116 CRC and MCF-7 breast cancer cells with 2.00 µM and 2.35 µM GI50 values, respectively. The MTT test also showed that ABQ-3 possessed anticancer effects towards HCT-116 and MCF-7 cells with IC50 values of 5.22 ± 2.41 μM and 7.46 ± 2.76 μM, respectively. Further experiments indicated that ABQ-3 induced apoptosis in both cell lines, and molecular docking studies explicitly suggested that ABQ-3 exhibited DNA binding in a similar fashion to previously reported compounds. Based on in silico pharmacokinetic prediction, ABQ-3 might display drug-like features enabling this compound to become a lead molecule for future studies.

Synthesis and Antiproliferative Effect of Halogenated Coumarin Derivatives.

A series of 6- and 6,8-halocoumarin derivatives have been investigated as potential antiproliferative compounds against a panel of tumor and normal cell lines. Cytotoxic effects were determined by the MTT method. To investigate the potential molecular mechanism involved in the cytotoxic effect, apoptosis assay, cell cycle analysis, reactive oxygen species (ROS), and reduced glutathione analysis were performed. Among the screened compounds, coumarins 6,8-dibromo-2-oxo-2H-chromene-3-carbonitrile 2h and 6,8-diiodo-2-oxo-2H-chromene-3-carbonitrile 2k exhibited the most antiproliferative effect in thyroid cancer-derived cells TPC-1. The apoptosis assay showed that both 2h and 2k induced apoptosis in TPC-1 thyroid cancer cells. According to these experiments, both coumarins induced a slight increase in TPC-1 cells in the G2/M phase and a decrease in the S phase. A significant increase in ROS levels was observed in TPC-1 treated with diiodocoumarin 2k, while the dibromocoumarin 2h induced a decrease in ROS in a dose and time-dependent manner.

On the influence of the rhodamine substituents onto the cytotoxicity of mitocanic maslinic acid rhodamine conjugates

Maslinic acid was converted via a di-acetylated piperazinyl amide into rhodamine conjugates differing in their alkyl moieties. These conjugates were submitted to cytotoxicity assays employing a panel of human tumor cell lines. These conjugates held high cytotoxicity but also some selectivity especially for A2780 cells. Thereby, a propyl substituted rhodamine conjugate showed EC50 values as low as EC50 = 0.01 μM and was approx. 15 times more cytotoxic for the cancer cells than for non-malignant fibroblasts (NIH 3 T3). Cytotoxicity obviously parallels the lipophilicity of the residue and suggests - since the compounds act as mitocanes - an interaction of the conjugates with the inner mitochondrial membrane.

Synthesis and structures of polynuclear organotin(IV) complexes of a polyaromatic carboxylate ligand and cytotoxic evaluation in tumor cell lines

Three polynuclear organotin(IV) derivatives of composition [n-Bu3Sn(HL)]n1, [Ph3Sn(HL)]n2 and [n-Bu2Sn(HL)2]23 were synthesized by reacting 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H'HL) with (n-Bu3Sn)2O, Ph3SnOH and n-Bu2SnO, respectively. The structures 1–3 were fully characterized by elemental analysis, IR and NMR (1H, 13C, and 119Sn), 119Sn Mössbauer spectroscopy, and additionally, the molecular and crystal structures of 1–3 and its pro-ligand (H'HL) were established by single-crystal X-ray diffraction analysis. The tributyltin(IV) complex 1 is a one-dimensional coordination polymer, in which the azo ligand bridges adjacent Sn(IV) centres solely via the two carboxylate O-atoms. The hydroxy H atom forms an intramolecular O–H···N hydrogen bond with the imine N-atom, as observed in the crystal structure of H'HL. The triphenyltin(IV) complex 2 is also a one-dimensional coordination polymer, but in this case the azo ligand bridges adjacent Sn(IV) centres via its carboxylate group and the deprotonated phenol O-atom. Unlike in 1, the phenol H-atom has migrated to the imine N-atom, to give a zwitterionic form of the azo ligand. The tin centers in 1 and 2 are pentacoordinated and reveal a distorted trans-R3SnO2 trigonal-bipyramidal environment. The dibutyltin(IV) complex 3 crystallizes as discrete centrosymmetric dinuclear entities where the unique Sn(IV) center is heptacoordinated in a distorted pentagonal bipyramid coordination geometry. In vitro cytotoxicity studies of compound 1 were performed and compared with 2 across a panel of human tumor cell lines and the results were compared with the data of six clinically used anticancer drugs.

Comprehensive Exploration of NK Cell Education in ADCC Triggered By a Monoclonal Antibody or a Bispecific Killer Engager Molecule (BiKE)

The responsiveness of NK cells to malignant cells that lack self-HLA class I (HLA-I) expression is controlled by NK cell education governed by the interaction between selected inhibitory NK cell receptors and their cognate HLA-I ligands. However, it remains unclear to what extent education impacts the ability of NK cells to perform antibody-dependent cellular cytotoxicity (ADCC) as previous studies show inconsistent results and are limited in resolution. Moreover, no study has yet addressed if NK cell education impacts ADCC triggered by novel CD16-binding therapeutics such as bispecific killer engagers (BiKEs). In this study we systematically characterized NK cell degranulation against a panel of tumor cell lines with defined HLA-I expression in the presence of either the monoclonal antibody rituximab or a recently developed CD34xCD16 BiKE. Using high-resolution flow cytometry, our data revealed that NK cells educated by one or two KIRs did not have significantly enhanced ADCC capacity over uneducated KIR+ or KIR- NK cells, especially when increasing the antibody concentration. Contrasting co-education by KIRs, NKG2A was consistently associated with improved responsiveness and superior ADCC. Surprisingly, these results were despite that KIR-education correlated with higher CD16 expression that was markedly lower on NKG2A+ NK cells compared to both KIR-educated and uneducated subsets. An extended analysis uncovered that the inhibitory KIR2DL1-C245 receptor, reported to mediate weak NK cell education compared to the KIR2DL1-R245 variant, further repressed the ADCC capacity of NKG2A+ NK cells from donors expressing its ligand HLA C2. This highlights that also inhibitory KIRs can dampen the ADCC potential of NK cells similar to that of activating KIRs which also were discovered to restrict ADCC even when stimulated by its ligand. Finally, our study unveiled that BiKE-triggered ADCC has a similar relationship to NK cell education and its hierarchies as that triggered by a monoclonal antibody. In conclusion, the ADCC capacity of an NK cell is more regulated by restrictive mechanisms of education conferred by activating or selected inhibitory KIRs in individuals carrying the cognate KIR ligand than that of inhibitory KIRs conferring higher education potential. Hence, this study establishes that NK cell education does not enhance the ADCC capacity of an NK cell per se while providing new insights that also inhibitory KIRs can dampen the ADCC potential even when the target cell lacks its ligand. Our results add to the fundamental understanding of NK cell biology as well as to avenues of therapeutic applications for NK cells.

Symmetric lipophilic polyamines exhibiting antitumor activity

Unsymmetric lipophilic polyamine derivatives are considered as potential antitumor agents. Here, a series of novel symmetric lipophilic polyamines (LPAs) based on norspermine and triethylenetetramine (TETA) backbones bearing alkyl substituents with different lengths (from decyl to octadecyl) at C(1) atom of glycerol were synthesized. Performed screening of the cytotoxicity of novel compounds on the panel of tumor cell lines (MCF-7, KB-3-1, B16) and non-malignant fibroblasts hFF3 in vitro revealed a correlation between the length of the aliphatic moieties in LPAs and their toxic effects – LPAs with the shortest decyl substituent were found to exhibit the highest cytotoxicity. Furthermore, norspermine-based LPAs displayed somewhat more pronounced cytotoxicity compared with their TETA-based counterparts. Further mechanistic studies demonstrated that hit LPAs containing the norspermine backbone and tetradecyl or decyl substituents efficiently induced apoptosis in KB-3-1 cells. Moreover, decyl-bearing LPA inhibited motility and enhanced adhesiveness of murine B16 melanoma cells in vitro, showing promising antimetastatic potential. Thus, developed novel symmetric norspermine-based LPAs can be considered as promising anticancer chemotherapeutic candidates.

Shikonin from Chinese herbal medicine induces GSDME-controlled pyroptosis in tumor cells

To investigate the potential anti-tumor mechanisms of naphthoquinone compound shikonin (SKN) extracted from the root of Chinese herbal medicine plant lithospermum ( Lithospermum erythrorhizon Sieb. & Zucc.). We first observed that SKN treatment led to swelling and bubbles in HeLa cells that were similar to the phenotype of cell pyroptosis. Subsequently, the HeLa cells experienced a pyroptotic process with SKN, and this was then assessed using lactate dehydrogenase (LDH) release and propidium iodide (PI)/Hoechst double staining experiments. Pyroptosis is defined as gasdermin-mediated programmed necroptosis. To identify the potential pyroptosis machinery, two strategies were utilized that included a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 screening experiment and a pyroptosis reconstitution assay executed by each of the five known gasdermins (GSDMA-E). Moreover, endogenous cleavage was also detected in a panel of tumor cell lines. Compared with the control, both the LDH release and PI/Hoechst double-staining experiments suggested that SKN induced perforation and enhancement of the permeability of the cell membranes that resulted in pyroptosis in HeLa cells ( P = .028 and P = .032, respectively). In addition, the reconstitution assays in human embryonic kidney 293T (HEK-293T) cells and endogenous cleavage assays in HeLa cells indicated that the pyroptosis was controlled by GSDME. In addition, we also found SKN could trigger pyroptosis in a panel of tumor cell lines in which the cellular morphologies were proportional to the GSDME expression levels. Additionally, the cleavage of GSDME was also detected, and this was indicative of a similar GSDME-mediated mechanism. Our study not only explained the molecular mechanism of cytotoxicity of SKN to various tumor cells, but also provided additional information for the potential clinical application of natural naphthoquinone compounds against cancer.

225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

Background: Targeted protein degradation (TPD) using the endogenous ubiquitin (Ub) proteasome system (UPS) is a rapidly growing drug discovery approach to eliminate pathogenic proteins. Strategies for TPD have focused on designing heterobifunctional PROTACs that utilize ligand binding to select E3 ligases (e.g., cereblon and VHL), often resulting in compounds with poor drug-like properties. Monovalent degraders represent an alternative approach, in which small molecules are designed to bind the target protein and induce its degradation through the recruitment of an E3 ligase complex. Monovalent degraders generally have better physico-chemical properties than PROTACs; however, designing compounds to induce degradation is not well precedented and relies on serendipitous discovery. Methods: An E3 ligase agnostic chemical library was screened using our ultra-high throughput cell-based screening (uHTS) platform. Hits were identified by measuring protein degradation of BRD4 using immunofluorescence. A Ub pathway focused CRISPR screen was used to identify the E3 ligase responsible for compound-induced degradation of BRD4. The degradation mechanism was validated using western blot, luciferase-based protein-protein interaction (PPI) and mutational analyses. In vitro antitumor activity was assessed by proliferation and apoptosis assays. NOD/ SCID mice were implanted sub-Q with acute myeloid leukemia (AML) xenograft model MV-4-11 and treated with compounds administered QD. Results: uHTS resulted in identification of selective monovalent degraders that target the bromodomain extra-terminal (BET) protein, BRD4. Degrader series optimization produced PLX-3618, which demonstrated potent degradation of BRD4, without depleting BET family members BRD2 and BRD3. Proteasome and neddylation inhibitors confirmed degradation was mediated via the UPS, and a Ub-focused CRISPR screen identified CUL4DCAF11 as the E3 complex responsible for PLX-3618 induced degradation of BRD4. PPI studies verified a BRD4/PLX-3618/DCAF11 ternary complex; BRD4 point mutational analyses provided further insights into the DCAF11-mediated degradation mechanism. Degradation of BRD4 by PLX-3618 resulted in downregulation of the MYC oncogene and potent anti-proliferative activity against a panel of tumor cell lines, with high sensitivity observed in AML. in vivo, PLX-3618 resulted in complete tumor regression, whereas a pan-BET inhibitor only resulted in tumor growth inhibition without regression. PK/PD analyses provided insight into the unique exposure/response profile of targeted protein degraders. Conclusion: These results demonstrate the efficient discovery of novel monovalent degraders using our proprietary platform. Characterization of the degradation mechanism highlights the discovery of DCAF11 as a novel E3 ligase substrate receptor amenable to recruitment and degradation of BRD4. No conflict of interest.