Apparent Toxicity Research Articles

Design, synthesis, and biological evalution of bifunctional inhibitors against Hsp90-HDAC6 interplay

HDAC6 and Hsp90, existing as a cytosolic complex play an important role in maintaining the protein homeostasis. The interplay of HDAC6 and Hsp90 has attracted wide attention due to their important role and promise as therapeutic targets in malignant cancers. Therefore, the discovery of dual inhibitors targeting HDAC6 and Hsp90 is of high importance. In the present study, we describe the design, synthesis, and biological evaluation of bifunctional inhibitors against HDAC6 and Hsp90 interplay. In particular, compound 6e shows a significant inhibitory activity against both HDAC6 and Hsp90 with IC50 values of 106 nM and 61 nM, respectively. Compound 6e promotes the acetylation of HDAC6 substrate proteins such as α-tubulin and Hsp90 via HDAC6 inhibition, and also induces the degradation of Hsp90 clients such as Her2, EGFR, Met, Akt, and HDAC6 via Hsp90 inhibition. Compound 6e consequently furnishes potent antiproliferative effect on gefitinib-resistant H1975 non-small cell lung cancer (NSCLC) with a GI50 value of 1.7 μM. In addition, compound 6e successfully achieved significant tumor growth inhibition in H1975 NSCLC xenograft model without noticeable abnormal behavior, body weight changes, and apparent ocular toxicity. We conclude that compound 6e constitutes an excellent tool as well as a valuable lead for assessment of Hsp90 and HDAC6 dual inhibition with a single molecule.

Nano-Iron and Nano-Zinc Induced Growth and Metabolic Changes in Vigna radiata

The widespread industrial use and consequent release of nanosized iron (nFe3O4) and zinc oxide (nZnO) particles into the environment have raised concerns over their effects on living organisms, including plants. These nanoparticles are the source of their respective metal ions and although plants require both Fe and Zn ions for proper growth, excessive levels of these metals are toxic to them. A better understanding of the effects of these nanoparticles on plants also offers an opportunity for their useful applications in agriculture. The present work evaluates the changes in seed germination, plant growth, photosynthetic capacity, levels of biomolecules and antioxidant enzymes in Vigna radiata (L.) Wilczek when grown in the presence of nFe3O4 (size 1–4 nm) and nZnO (size 10–20 nm) and compared to the control plants. The plants were raised hydroponically for up to 14 days at two different concentrations of nanoparticles, viz. 10 and 100 mg/L. Inductively coupled plasma mass spectrometry (ICP-MS) results established that V. radiata can accumulate Fe and Zn in shoots with high efficiency. The results indicated that nFe3O4 had a favourable effect on V. radiata, whereas no apparent benefit or toxicity of nZnO was observed at the tested concentrations.

Epimedin C Alleviates Glucocorticoid-Induced Suppression of Osteogenic Differentiation by Modulating PI3K/AKT/RUNX2 Signaling Pathway.

Secondary osteoporosis is triggered mostly by glucocorticoid (GC) therapy. Dexamethasone (DEX) was reported to inhibit osteogenic differentiation in zebrafish larvae and MC3T3-E1 cells in prior research. In this research, we primarily examined the protective impacts of epimedin C on the osteogenic inhibition impact of MC3T3-E1 cells and zebrafish larvae mediated by DEX. The findings illustrated no apparent toxicity for MC3T3-E1 cells after administering epimedin C at increasing dosages from 1 to 60 μM and no remarkable proliferation in MC3T3-E1 cells treated using DEX. In MC3T3-E1 cells that had been treated using DEX, we discovered that epimedin C enhanced alkaline phosphatase activities and mineralization. Epimedin C could substantially enhance the protein expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL) in MC3T3-E1 cells subjected to DEX treatment. Additionally, epimedin C stimulated PI3K and AKT signaling pathways in MC3T3-E1 cells that had been treated using DEX. Furthermore, in a zebrafish larvae model, epimedin C was shown to enhance bone mineralization in DEX-mediated bone impairment. We also found that epimedin C enhanced ALPL activity and mineralization in MC3T3-E1 cells treated using DEX, which may be reversed by PI3K inhibitor (LY294002). LY294002 can also reverse the protective impact of epimedin C on DEX-mediated bone impairment in zebrafish larval. These findings suggested that epimedin C alleviated the suppressive impact of DEX on the osteogenesis of zebrafish larval and MC3T3-E1 cells via triggering the PI3K and AKT signaling pathways. Epimedin C has significant potential in the development of innovative drugs for the treatment of glucocorticoid-mediated osteoporosis.

PEGylated Gold Nanoparticle Toxicity in Cardiomyocytes: Assessment of Size, Concentration, and Time Dependency.

Gold Nanoparticles (GNPs) have shown promising capabilities for use in many in-vivo applications such as gene and drug delivery, photothermal ablation of tumors, and tracking in many imaging modalities. Yet GNPs have thus far had limited use in cardiovascular medicine. Polyethylene glycol functionalized (PEGylated) GNPs have been extensively studied in a wide array of in vitro and in vivo models with results showing no apparent toxicity, but to our knowledge an investigation has never been performed to determine direct cardiomyocyte toxicity. In this study, we assessed if PEGylated GNPs exhibited direct toxicity to a primary culture of neonatal rat cardiomyocytes in order to establish PEGylated GNPs for potential future use in cardiovascular medicine applications. We present novel results that demonstrate both a particle size and concentration dependent relationship on cell viability. Cell viability was found to be significantly enhanced for many concentrations and sizes as compared to the control and increased linearly as a function of particle diameter. Additionally, viability increased in a parabolically dependent manner as a function of decreasing particle concentration. These new results could advance understanding of nanoparticle-cell interactions and lead to the development of new applications involving the use of gold nanoparticles in cardiovascular medicine.

Design and synthesis of NAD(P)H: Quinone oxidoreductase (NQO1)-activated prodrugs of 23-hydroxybetulinic acid with enhanced antitumor properties

A series of NQO1 selectively activated prodrugs were designed and synthesized by introducing indolequinone moiety to the C-3, C-23 or C-28 position of 23-hydroxybetulinic acid (23-HBA) and its analogues. Among them, the representative compound 32j exhibited significant antiproliferative activities against NQO1-overexpressing HT-29 cells and A549 cells, with IC50 values of 1.87 and 2.36 μM, respectively, which were 20–30-fold more potent than those of parent compound 23-HBA. More importantly, it was demonstrated in the in vivo antitumor experiment that 32j effectively suppressed the tumor volume and largely reduced tumor weight by 72.69% with no apparent toxicity, which was more potent than the positive control 5-fluorouracil. This is the first breakthrough in the improvement of in vivo antitumor activities of 23-HBA derivatives. The further molecular mechanism study revealed that 32j blocked cell cycle arrest at G2/M phase, induced cell apoptosis, depolarized mitochondria and elevated the intracellular ROS levels in a dose-dependent manner. Western blot analysis indicated that 32j induced cell apoptosis by interfering with the expression of apoptosis-related proteins. These findings suggest that compound 32j could be considered as a potent antitumor prodrug candidate which deserves to be further investigated for personalized cancer therapy.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

Simple SummaryCancer cells largely use glycolysis to obtain both chemical energy (ATP) and metabolic intermediates for anabolic reactions, and several studies proved that the blockage of the glycolytic pathway is an efficient anticancer strategy. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key tetrameric glycolytic enzyme, has raised considerable attention in recent years as a potential drug target in pathological conditions in which glycolytic flux has a crucial role. In cancers, a recognized involvement of the Warburg effect, as a key mechanism for cancer-cell energetic metabolism, favouring tumour progression and invasion, has contributed to highlight human GAPDH (hGAPDH) as an effective drug target to specifically hit cancer cells exhibiting metabolic dependence on glycolysis, without significantly affecting normal cells. In this study, we tested the effects of 3-bromo-isoxazoline derivatives specifically designed to bind and inhibit GAPDH activity, in pancreatic ductal-adenocarcinoma cells.A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of Plasmodium falciparum GAPDH (PfGAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH. They showed an antiproliferative effect on pancreatic ductal-adenocarcinoma cells (PDAC) and pancreatic-cancer stem cells (CSCs), and among them two promising compounds were selected to be tested in vivo. Interestingly, these compounds were not effective in fibroblasts. The AXP-3019 derivative was able to block PDAC-cell growth in mice xenograft without apparent toxicity. The overall results support the assumption that selective inhibition of the glycolytic pathway, by targeting GAPDH, is an effective therapy for pancreatic cancer and that 3-bromo-isoxazoline derivatives represent a new class of anti-cancer compounds targeting glycolysis.

Carfentanil Blood Concentrations in Impaired Driving Investigations in Ontario, Canada.

Carfentanil is a potent opioid with no medical use in humans; it presents a serious threat to public health and road safety due to its presence in the illicit drug supply, the potency of the drug and instances of use prior to the operation of a motor vehicle. The identification and quantitation of carfentanil using liquid chromatography with tandem mass spectrometry was performed in blood samples obtained through impaired driving-related investigations from 2017 to 2019. In a series of 66 cases submitted to the Centre of Forensic Sciences in Ontario, Canada, blood concentrations of carfentanil ranged from <0.04 to 2.1 ng/mL in the population studied. Driving behaviors frequently came to the attention of concerned citizens, decreased levels of consciousness were commonly reported and a variety of medical interventions were required, in some cases, to preserve life due to apparent opioid toxicity.

Abstract 3207: Notch decoys as potential anti-angiogenic biotherapeutics

Abstract Notch signaling is activated by ligands Delta-like 4 (Dll4) and Jagged1 in the tumor microenvironment to promote tumor angiogenesis and perfusion. Notch activation is associated with poor outcomes in several cancers, particularly triple negative breast cancer (TNBC), and affects both tumor angiogenesis and metastasis. The development of therapeutics targeting angiogenesis, such as the Notch pathway, has again attracted attention. Previous approaches to globally inhibit the Notch pathway or block Dll4/Notch1 activation, such as γ-secretase inhibitors (GSI), have raised safety concerns due to gastrointestinal toxicity due to accumulation of secretory goblet cells in the intestine. Similarly, anti-Dll4 therapy resulted in pathological changes in the liver as well as severe vascular neoplasms when evaluated using preclinical animal models. Development of new approaches for targeting the Notch pathway remains a critical clinical problem currently unaddressed. Our lab has previously developed ligand-specific inhibitors of Notch signaling, called Notch decoys, which are comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain. These Notch decoys bind ligand non-productively and interfere with ligand function. Jagged-specific Notch1 decoys inhibit angiogenesis in vitro and significantly impair tumor growth, tumor angiogenesis, and perfusion without apparent toxicity in mouse models of TNBC. However, this previous work utilized Notch decoys produced via viral expression vectors, which preclude dosage control and limit clinical applicability. We have developed a new generation of Notch “mini” decoys that contain fewer EGF-like repeats. These mini decoys show improved secretion characteristics and can be purified as active proteins in clinically relevant quantities. Using a variety of binding assays, we observed that these Notch mini decoys demonstrate strong but distinct binding to Notch ligands Dll4 and Jagged1 and block Notch signaling when evaluated in cultured cells. Our newly generated Notch decoys can now be sufficiently purified for use in a dose-dependent manner to test the therapeutic role of Notch inhibition on tumor angiogenesis using preclinical animal models. Citation Format: Timothy Sargis, Seock-Won Youn, Hyun Lee, L.A. Naiche, Jan Kitajewski. Notch decoys as potential anti-angiogenic biotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3207.

Abstract 3955: Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy

Abstract Introduction Clinical relapse following CD19 CAR T therapy after failure to BTK inhibitors (BTKi) is a novel and fast growing medical challenge in treating patients with mantle cell lymphoma (MCL). Thus, developing a novel therapy to overcome this BTKi-CAR T dual resistance (Dual-R) is an urgent need. Our unpublished data revealed MYC targets and cyclin-dependent kinase 9 (CDK9) are highly upregulated in the Dual-R compared to BTKi-R samples. CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex. Its inhibition induces acute decline of short-lived mRNA and proteins, especially MYC and MCL-1, in acute myeloid leukemia and diffuse large B-cell lymphoma. Therefore, we hypothesize that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. BAY-1251152 is a novel selective CDK9 inhibitor with nanomolar potency. However, whether it has the potential to overcome BTKi-CAR T dual resistance has not been assessed. Methods To validate the correlation of MYC and CDK9 with drug resistance and clinical outcome, we performed whole transcriptomic profiling using primary patient samples. To assess the in vitro efficacy of BAY-1251152, we performed cell viability assay and cell apoptosis assay using MCL cell lines and primary patient samples and followed up with functional studies. furthermore, we assessed its in vivo efficacy using patient-derived xenograft (PDX) models derived from MCL patients including one with Dual-R. Results The expression of MYC oncogene associates with ibrutinib resistance and Dual-R, and poor clinical outcome. CDK9 expression does not correlates with ibrutinib resistance but it does associate with Dual-R and poor clinical outcome in CAR T-relapsed patients. CDK9 inhibition by BAY-1251152 is highly potent in anti-MCL activity in MCL cell lines with low nanomolar range of IC50 (59.6-172.3 nM) by inducing robust cell apoptosis. BAY-1251152 induces dose and time-dependent CDK9 inhibition. A rapid decline of phosphorylation of RNA polymerase II, MYC, MCL-1, and Cyclin D1 can be observed as early as 2 hours. Furthermore, BAY-1251152 (10mg/kg, QW) significantly inhibited tumor growth (p = 0.000003) in a PDX model derived from a Dual-R patient without causing apparent toxicity in NSG mice. In addition, BAY-1251152 also significantly suppressed tumor growth in the PDX models derived from a BTKi-R patient (p=0.00015) and a BTKi-venetoclax dual-resistant patient (p=0.009). Conclusion Our findings showed that targeting CDK9 by its specific inhibitor BAY-1251152 led to potent in vitro anti-MCL activity. BAY-1251152 induces fast CDK9 inhibition and rapid decline of MYC, MCL-1 and Cyclin D1 to induce robust cell death. BAY-1251152 is also potent in inhibiting tumor growth in PDX models. These data support that CDK9 is a promising target to overcome BTKi-CAR T dual resistance in MCL, which is in urgent need. Citation Format: Vivian Changying Jiang, Lingzhi Li, Alexa Jordan, Yu Xue, Fangfang Yan, Joseph McIntosh, Yang Liu, Yuxuan Che, Yijing Li, Qingsong Cai, Angela Leeming, Lukas Simon, Zhongming Zhao, Jia Zhou, Michael Wang. Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3955.

Abstract LB083: BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index

Abstract BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical BRAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF (i.e. “BRAF monomer-selective”) providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric BRAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. We identified and characterized a novel class of BRAF inhibitors that preferentially bind and inhibit dimeric over monomeric BRAF(V600E) (i.e. “BRAF dimer-selective inhibitors”). Biochemical analysis using BRAF dimer-selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of BRAF monomer- plus BRAF dimer selective inhibitor plus a MEK inhibitor that disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical BRAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the BRAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher Therapeutic Index. Finally, off-label use of the triple combination in patients with advanced BRAF(V600E) cancers that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective BRAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Overall, our work supports the notion that for most tumors a “3-drug” combinatorial strategy will potentially be most effective: one drug targeting MAPK signaling directly, such as a MEK inhibitor (“pathway” inhibitor), one drug targeting mutated BRAF or RAS selectively in the tumor (“therapeutic index” inhibitor, e.g. BRAF monomer-selective inhibitor, RAS(G12C) inhibitors, etc) and one drug targeting components of the feedback loop that is responsible for adaptive resistance (“feedback” inhibitor, e.g. BRAF dimer-selective inhibitors, SHP2 or SOS inhibitors, etc). Our data contribute to the development of a roadmap for the treatment of MAPK-driven tumors using drug combinations tailored to the specific driver oncoprotein. Citation Format: Christos Adamopoulos, Tamer Ahmed, Peter M. Ung, Min Xiao, Stuart Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Poulikos I. Poulikakos. BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB083.

Abstract 2807: HER3 is an actionable target in advanced prostate cancer

Abstract Background: ERBB signaling is implicated in castration resistant prostate cancer (CRPC), but so far clinical trials of ERBB targeting drugs failed to demonstrate antitumor activity. We elected to re-investigate ERBB receptors in endocrine treatment-resistant lethal PC, hypothesizing that targeting ERBB receptors merits further evaluation in metastatic CRPC (mCRPC). Design: We analyzed matching, same-patient, formalin-fixed paraffin-embedded (FFPE) treatment-naïve, castration-sensitive PC (CSPC) samples (n=88), and mCRPC biopsies (n=51), from patients treated at The Royal Marsden Hospital, UK. Samples were stained for HER2 and HER3 protein, by immunohistochemistry (IHC), data was generated through digital image analysis and results were analyzed against clinical characteristics and outcome data. Moreover, we treated HER3 high (CP50) and low (CP142) expressing patient derived xenograft (PDX) models with anti-HER3 antibody-drug conjugate (HER3-ADC) U3-1402 (10mg/Kg), IgG-ADC (MAAA-9289, 10mg/Kg), anti-HER3 antibody Patritumab (U3-1287, 10mg/Kg) and 10mM acetate buffer-5% sorbitol-pH 5.5 as vehicle control, in vitro and in vivo. In vitro cell growth inhibitory activity was monitored for 7-days with endpoint assay luminescence. In vivo efficacy was evaluated comparing tumor volumes, measured every 2-3 days. Statistical significance was analyzed using ANOVA with Dunnett’s multiple comparisons correction test. Results: Membranous HER2 (mHER2) and HER3 (mHER3) proteins were detectable in both CSPC and mCRPC biopsies, with HER3 being highly expressed in many tumors. The median optical density (OD) for mHER3 expression at diagnosis was 2958.0; PC with high mHER3 expression (&amp;gt; median OD; n=44) had a significantly shorter median time to CRPC (20.3 vs 14.2 months; p=0.016) and worse overall survival (OS) (79.0 vs 48.8 months; p=0.04) compared to CSPC with low mHER3 (≤ median; n=44). mHER2 staining did not associate with outcome. U3-1402 demonstrated in vivo potent and sustained antitumor activity in CP50, without inducing any body weight loss or apparent toxicity. Additionally, no tumor regrowth was observed up to 60-days following the end of dosing. This anti-HER3-ADC had minimal antitumor activity in CP142, highlighting the relevance of high HER3 expression as a functional therapeutic target. Conclusion: HER3 is commonly expressed in advanced PC and has clinical relevance in this setting. Our data indicate that HER3 is a valid target for clinical trials for men suffering from high HER3 expressing advanced PC. Citation Format: Susana Miranda, Veronica Gil, Ruth Riisnaes, Bora Gurel, Mariantonietta D’Ambrosio, Alessandro Vasciaveo, Mateus Crespo, Ana Ferreira, Daniela Brina, Martina Troiani, Adam Sharp, Beshara Sheehan, Rossitza Christova, George Seed, Ines Figueiredo, Maryou Lambros, David Dolling, Jan Rekowski, Abdullah Alajati, Matthew Clarke, Rita Pereira, Penny Flohr, Gemma Fowler, Gunther Boysen, Semini Sumanasuriya, Diletta Bianchini, Pasquale Rescigno, Caterina Aversa, Nina Tunariu, Christina Guo, Alec Paschalis, Claudia Bertan, Lorenzo Buroni, Jian Ning, Suzanne Carreira, Paul Workman, Amanda Swain, Andrea Califano, Michael M. Shen, Andrea Alimonti, Antje Neeb, SU2C/PCF International Prostate Cancer Dream Team, Jonathan Welti, Wei Yuan, Johann de Bono. HER3 is an actionable target in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2807.

Huaier Inhibits Gastric Cancer Growth and Hepatic Metastasis by Reducing Syntenin Expression and STAT3 Phosphorylation

Gastric cancer (GC) is a common malignant tumor worldwide and poses a serious threat to human health. As a traditional Chinese medicine, Huaier (Trametes robiniophila Murr.) has been used in the clinical treatment of GC. However, the mechanism underlying the anticancer effect of Huaier remains poorly understood. In this study, we used in vivo imaging technology to determine the anticancer effect of the Huaier n-butanol extract (HBE) on orthotopic and hepatic metastasis of GC mouse models. We found that HBE suppressed tumor growth and metastasis without causing apparent host toxicity. Proteomic analysis of GC cells before and after HBE intervention revealed syntenin to be one of the most significantly downregulated proteins after HBE intervention. We further demonstrated that HBE suppressed the growth and metastasis of GC by reducing the expression of syntenin and the phosphorylation of STAT3 at Y705 and reversing the epithelial-mesenchymal transition (EMT). In addition, we confirmed that syntenin was highly expressed in GC tissue and correlated with metastasis and poor prognosis. In conclusion, our results suggest that Huaier, a clinically used anticancer drug, may inhibit the growth and liver metastasis of GC by inhibiting the syntenin/STAT3 signaling pathway and reversing EMT.

Abstract 5073: A novel in-situ nanoparticle self-assembly for combination delivery of therapeutics to non-small cell lung cancer

Abstract Background: Lung cancer is a predominant cause of morbidity and mortality across the world. Among all types of cancers, lung cancer is the leading cause of cancer mortalities in the United States. Non-small cell lung cancer is one of the dominant divisions of lung cancer that solely represents 85-90% of all lung cancer cases. Up to the present time, primary treatment regimens for this cancer include radiation therapy, chemotherapy, and surgery, with chemotherapy being the best option thus far. Despite the eminent benefits of chemotherapy, its value is offset by severe side effects such as renal and/or hepatic toxicity or insufficient amounts of drug available at the target site. Such pitfalls can be handled by employing natural compounds (polyphenols, phytochemicals, xanthonoid, etc.,) as chemopreventives or chemosensitizers which can improve chemotherapy activity while reducing its systemic side effects and drug resistance. To this end, our recent efforts demonstrated a synergistic therapeutic benefit of gambogic acid (GA) and gemcitabine (Gem) against lung cancer. However, simultaneous delivery of these two drugs at the tumor site is highly challenging. Therefore, development of an injectable nanoformulation that can effectively deliver both hydrophobic (GA) and hydrophilic (Gem) drugs in one formulation is a clinically unmet need. Methods: Pursuing the novel nanotherapy approach, our lab has developed an in-situ biodegradable and biocompatible human serum albumin (HSA) and tannic acid (TA) mediated complexed GA and Gem nanoparticles (G-G@HTA NPs) using the solvent evaporation method. G-G@HTA NPs formation was confirmed by particle size, FT-IR, and H-NMR. A superior therapeutic activity of G-G@HTA NPs was demonstrated by multiple in vitro functional assays as well as in an animal mouse model. Results: Our results confirmed that G-G@HTA NPs have the ideal particle size and surface charge for cancer cell/tissue delivery which can clearly be evident by preferential uptake of these nanoparticles in lung cancer cells. Further, G-G@HTA NPs superiorly inhibited cell proliferation and clonogenicity of NSCLC cells. Additionally, G-G@HTA NPs revealed an obvious and precise targeting of tumors in vivo. The promoted and more synergistic anti-tumor efficacy of G-G@HTA NPs was attained than that of combined treatments and single drugs treatments. These events were resulted with no apparent systemic and organ toxicities. Conclusion: In summary, this study details the design of a novel nanocarrier and provides an optimized strategy for constructing dual-loaded nanoparticles using a biodegradable, non-toxic, human serum albumin-tannic acid-based platform for GA and Gem co-delivery in the treatment of NSCLC which confirms a strong feasibility to implement such synergistic nanomedicine regimen in pre-clinical and clinical translations in future. Citation Format: Elham Hatami, Prashanth K. Nagesh, Neeraj Chauhan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. A novel in-situ nanoparticle self-assembly for combination delivery of therapeutics to non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5073.

Abstract 6247: Evaluation of AS-10, identified based on SAR studies of newly generated organoselenium compounds, as a potential colorectal cancer therapeutic acting potentially by acetylating histones

Abstract Colorectal cancer (CRC) is the third most common form of cancer diagnosed in the United States. Standard treatment for CRC utilizes a combination therapy composed of 5-fluorouracil, oxaliplatin, and folinic acid, known as FOLFOX. It has been established that patients with CRC tend to develop drug resistance to FOLFOX leading to a need for new chemotherapeutic treatments. Recent work has established that AS-10, a small molecule established for its promising role in treating pancreatic cancer, to be capable of inhibiting the growth of a range of CRC cell lines, including FOLFOX resistant CRC cells. Twenty-four cyclic and open chain analogs of AS-10 were synthesized and evaluated for their activity in inhibiting the viability of different cancer cells in vitro. The cell viability of these compounds was compared to AS-10 in the cell lines of pancreatic carcinoma (MiaPaCa2), lung carcinoma (H460), colorectal adenocarcinoma (HT29), colorectal carcinoma (HCT116), and FOLFOX resistant HT29 and HCT116 cells. Based on the IC50 values observed from these initial studies, only ASA-7, an open-chain alkyl selenocyanate, could treat all cancer cell lines in a similar concentration range of AS-10. Dose-response curves completed in 6 CRC cell lines indicated IC50 values in the range of 1-2 µM for AS-10 and 3.3-6.6µM for ASA-7. Cell viability evaluation in normal kidney (HEK293T) and colon (FHC) cell lines indicated AS-10 to be less toxic as compared to ASA-7. A similar trend was observed in vivo - AS-10 was 4 times more tolerable in mice as compared to ASA-7. A HCT116 xenograft study demonstrated the ability of AS-10 to significantly inhibit tumor growth in vivo without any apparent systemic toxicity. Moreover, AS-10 was assessed for its ability to act as an acetyl donor for histone acetyltransferase (HAT) and was found to initiate HAT activity in the absence of Acetyl-CoA in pancreatic epithelioid carcinoma (Panc-1) and colorectal carcinoma (HCT116) cells. In conclusion, AS-10 was identified as the best among the 24 analogs, in vitro and in vivo, when treating CRC and found to be capable of inducing HAT activity in both pancreatic and colorectal carcinoma elucidating a possible mechanism of action. Citation Format: Hannah Johnson, Amandeep Singh, Asif Raza, Deepkamal Karelia, Daniel Plano, Shantu Amin, Arun Sharma. Evaluation of AS-10, identified based on SAR studies of newly generated organoselenium compounds, as a potential colorectal cancer therapeutic acting potentially by acetylating histones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6247.

Design, synthesis and bioactivity evaluation of favorable evodiamine derivative scaffold for developing cancer therapy

Natural product evodiamine is one of the most privileged scaffolds in drug discovery and is suitable for derivatization, which can be conducted quickly for structure optimization and structure-activity relationship research. In this work, a comprehensive SAR study on evodiamine scaffold with N14-3′-fluorophenyl substituted was completed, and compounds with high anti-tumor activity and good inhibitory effect on Top1 and Top2 were screened out. Tested evodiamine derivatives exhibited excellent broad-spectrum anti-tumor activity. Among them, compound 8b revealed 55.15% and 55.50% inhibition for Top1 and Top2 at 25 μM, as well as 0.16 and 0.13 μM IC50 value for MGC-803 and SGC-7901 cells, respectively; compound 9a revealed 70.50% and 71.81% inhibition for Top1 and Top2 at 25 μM, as well as 0.22 and 0.27 μM IC50 value for MGC-803 and SGC-7901 cells, respectively. The further biological evaluation showed that they could functionally induce apoptosis, significantly arrest the cell cycle at the G2/M phase, and markedly inhibit cell proliferation, migration and invasion. In addition, compound 9a performed a tumor inhibitory rate of 36.35% and showed no apparent toxicity in vivo. Overall, these optimized protocols will advance the progression of cancer chemotherapy and can be used to expand the options for screening therapeutic cancer drugs.

Abstract 3439: An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts

Abstract Carcinoembyronic antigen (CEA) is a cell surface protein normally found at low levels in adult tissues but over-expressed in numerous solid tumors. CEA is constitutively released from tumor cells and reaches detectable levels in peripheral blood, making CEA targeting particularly challenging. Granzyme B is a key effector in immune-mediated killing of target cells, with cell death through both caspase-dependent and -independent mechanisms. We developed a fusion protein composed of granzyme B and a human scFv targeting the juxta-membrane epitope of CEA. The construct also incorporates a fragment of human IgG heavy chain to cause dimerization and provide increased in vivo circulation and antitumor efficacy. The GrB-Fc-CEA construct was cloned into a mammalian expression vector, transiently expressed by HEK-293E suspension cells, and secreted into the culture media. The final yield after purification and activation was 10-15 mg/L. The enzymatic activity of granzyme B in GrB-Fc-CEA was comparable to that of commercially available human granzyme B. In vitro cytotoxicity against CEA-positive cell lines demonstrated cytotoxicity in the nanomolar range, compared to micromolar range cytotoxicity against control (CEA-negative) cell lines. Importantly, GrB-Fc-CEA cytotoxicity against tumor cells in vitro was unaffected by exogenously-added CEA (200 ng/ml) extracellular domain, suggesting that the construct is not competitively absorbed by soluble CEA released by tumors and present in the circulation of patients. GrB-Fc-CEA internalized rapidly (within one hour) into the target cell lines A-431 and HT-1080/CEA whereas internalization was not observed with untargeted granzyme B, clearly demonstrating receptor-mediated internalization. GrB-Fc-CEA induced mitochondrial depolarization and caspase activation, consistent with the well-described granzyme B mechanism of action. Treatment of female nude mice bearing A-549 tumors with GrB-Fc-CEA (80 mg/kg over 5 doses) resulted in a significant growth inhibition of the established tumors compared to vehicle controls (1200 mm3 vs &amp;lt;40 mm3). Complete tumor regression was observed in seven of ten tumors in mice treated with GrB-Fc-CEA. Mouse weight was unaffected in both treated and control groups for the duration of the study, suggesting no apparent toxicity of the construct. Thus, GrB-Fc-CEA appears to have significant potential in targeting a large category of CEA-expressing solid tumors and is a promising candidate for further pre-clinical development. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Lawrence H. Cheung, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. An internalizing anti-CEA construct for targeted delivery of granzyme B results in significant antitumor activity in human lung carcinoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3439.

Abstract 2608: Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors

Abstract Background: PARP inhibitors are widely used for BRCA-mutant cancers, but often tumors become resistant to PARP inhibitors. The resistance develops through various mechanisms, including BRCA1/2 reversion mutation. Several publications have suggested that CHK2 inhibition could help overcome the resistance to PARP inhibitors. We discovered a novel small molecule CHK2 inhibitor, ART-122, and conducted a series of experiments to explore the rationale for combining a PARP inhibitor and ART-122 to treat the BRCA wild-type ovarian and breast tumors. Results: In the LanthaScreen࣪ Kinase assay, ART-122 was shown to have the IC50 values of 0.57 nM and 10.85 nM, for CHK2 and CHK1, respectively. We observed the strong synergy between ART-122 and olaparib in the mouse xenograft model using the MDA-MB-231 cancer cells (BRCA1/2 wild type, triple-negative breast cancer cells). Animals were treated with 50 mg/kg olaparib on days 1-5, 30 mg/kg ART-122 on days 1-2 each week for 3 weeks, or both therapies. The result demonstrated approximately 41% reduction in tumor volume in mice treated with the combination therapy compared to the vehicle group. Animals treated with either of the single-agent therapies showed tumor growth similar to the vehicle group. We did not observe apparent toxicity or significant body weight differences among the animals. Additionally, the synergism between ART-122 and olaparib was confirmed in other cancer cell lines, including several BRCA wild-type and BRCA mutant cells lines such as CaOV3, SK-OV-3, UWB1.289, UWB1.289 with BRCA overexpression, HCC1937, MDA-MB-436, and MCF7. Conclusion: The combination of ART-122 and Olaparib demonstrated a synergistic effect in BRCA wild-type ovarian and breast tumor cell lines. In the mouse xenograft model, the CHK2 inhibitor ART-122 sensitized BRCA1/2 wild-type tumor cells MDA-MB-231 to olaparib treatment. The data provide a strong rationale for the further development of ART-122 as a therapeutic agent. Citation Format: Taegon Baik, Chaeun Park, Young Woo Kang, Danny Joh, Ha-Young Lee, Sojung Park, Supyong Hwang, In Ki Kim, Jong Oh Kim. Pre-clinical evaluation of small molecule CHK2 inhibitor, ART-122 combined with olaparib in BRCA wild type tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2608.

An irreversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 inhibits estradiol synthesis in human endometriosis lesions and induces regression of the non-human primate endometriosis

Endometriosis is a gynecological disorder affecting about 10% of women and can lead to invalidating painful symptoms and infertility. Since there is no current definitive cure for this disease, new therapeutic options are necessary. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the production of estradiol (E2), the most potent estrogen in women, and of 5-androstene-3β,17β-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. 17β-HSD1 is therefore a pharmacological target of choice for the treatment of estrogen-dependent diseases such as endometriosis. We developed a targeted-covalent (irreversible) and non-estrogenic inhibitor of 17β-HSD1, a molecule named PBRM, and herein evaluated its efficiency for the treatment of endometriosis. In a cell-free assay containing estrone (E1), the natural substrate of 17β-HSD1, PBRM was able to block the formation of E2 in a collection of 50 human endometriosis lesions from a different clinical feature type, location, and phase. When given orally by gavage at 15 mg/kg to baboons, the resulting plasmatic concentration of PBRM was found to be sufficiently high (up to 125 ng/mL) for an efficacy study in a non-human primate (baboon) endometriosis model. After 2 months of treatment, the number of lesions/adhesions decreased in 60% of animals (3/5) in the PBRM-treated group, compared to the placebo group which showed an increase in the number of lesion/adhesions in 60% (3/5) of animals. Indeed, the total number of lesions/adhesions decreased in treated group (−6.5 or −19% when excluding one animal) while it increased in the control group receiving a placebo (+11%). Analysis of specific endometriotic lesions revealed that PBRM decreased the number of red lesions (−67%; 8/12) and white lesions (−35%; 11/31), but not of blue-black lesions. Similarly, PBRM decreased the surface area of dense adhesions and filmy adhesions, as compared to placebo. Also, PBRM treatment did not significantly affect the number of menstrual days. Finally, this targeted covalent inhibitor showed no adverse effects and no apparent toxicity for the duration of the treatment. These data indicate that 17β-HSD1 inhibitor PBRM is a promising candidate for therapy targeting endometriosis and supports the need of additional efforts toward clinical trials.

Conjugation with 8-arm PEG and CRM197 enhances the immunogenicity of SARS-CoV-2 ORF8 protein

Safe and effective vaccines are urgently needed to combat the COVID-19 pandemic. However, the SARS-CoV-2 variants raise concerns about the effectiveness of vaccines. As a SARS-CoV-2 antigen target, ORF8 strongly inhibits the IFN-β and NF-κB-responsive promoter, and can be potentially used for the development of SARS-CoV-2 vaccine. However, it is necessary to improve the immunogenicity of ORF8 by adjuvants or delivery systems. CRM197 was a carrier protein with the ability to activate T helper cells for antigens. Eight-arm PEG could conjugate multiple antigen molecules in one entity with inherent adjuvant effect. In the present study, ORF8 was conjugated with CRM197 and 8-arm PEG, respectively. The cellular and humoral immune responses to the conjugates (ORF8-CRM and ORF8-PEG) were evaluated in the BALB/c mice. As compared with ORF8-CRM and ORF8 administrated with aluminum adjuvant (ORF8/AL), ORF8-PEG induced a higher ORF8-specific IgG titer (2.6 × 104), higher levels of cytokines (IFN-γ, TNF-α, IFN-β, and IL-5), stronger splenocyte proliferation. Thus, conjugation with 8-arm PEG was an effective method to improve the immune response to ORF8. Moreover, ORF8-PEG did not lead to apparent toxicity to the cardiac, liver and renal functions. ORF8-PEG was expected to act as an effective vaccine to provide the immune protection against SARS-CoV-2.

Autophagy is involved in Ficus carica fruit extract-induced anti-tumor effects on pancreatic cancer.

Pancreatic cancer (PaCa), a common and highly lethal malignant cancer, is often insensitive to radio- and/or chemotherapy. Therefore, effective treatment regiments are still lacking. Herein, we found that an extract of Ficus carica fruit (EFCF) exerted anti-tumor effects on PaCa cells. EFCF induced cell viability inhibition and apoptotic cell death in two PaCa cell lines in a dose- and time dependent manner. EFCF effectively suppressed the migration, metastasis, invasion, and colony formation of PaCa cells. Mechanistically, EFCF stimulated an increase in intracellular ROS to promote cell death and senescence. EFCF treatment also triggered autophagy, and autophagy inhibition enhanced EFCF-induced cell death. We found that EFCF decreased mitochondrial membrane potential and promoted lipid peroxidation. Moreover, intragastric administration of EFCF effectively suppressed xenograft PaCa growth inhibition by activating cell death. EFCF had no apparent toxicity to normal pancreatic epithelial cells. Together, these findings suggest that EFCF may be a potential treatment for PaCa.