Potent Antiproliferative Effects Research Articles

Efficacy of GluN2B-Containing NMDA receptor antagonist for antitumor and antidepressant therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer. Evidence suggests that the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA) receptor, a critical molecule in the central nervous system, is expressed in NSCLC. However, the specific expression patterns, subcellular localization, functional modulation, and pathological implications of NMDA receptor subtypes in NSCLC have not been fully elucidated. In this study, we employed a multi-disciplinary approach, combining biochemical and molecular biology with electrophysiological recordings and behavioral assays, to investigate these aspects. We reveal the expression of GluN2B-containing NMDA receptors in A549 and H460 NSCLC cell lines and the induction of NMDA receptor-mediated currents by glutamate in A549 cells. Furthermore, the GluN2B-specific inhibitors ifenprodil and Ro 25–6981 significantly reduced cell viability and migration, while promoting apoptosis. Importantly, intraperitoneal administration of ifenprodil in nude mice inhibited the growth of subcutaneous tumors derived from A549 and H460 cells and ameliorated depression-like behaviors. These findings underscore the potential antiproliferative effects of ifenprodil and Ro 25–6981 and suggest that GluN2B-containing NMDA receptors may represent novel therapeutic targets for NSCLC, with the added benefit of potential antidepressant action.

Bufadienolide-Fatty Acid Conjugates from the Fertilized Eggs of Toad Bufo gargarizans: Isolation, Characterization, Toxicity, and Antiproliferative Evaluation.

Bufadienolides (BDs) are a class of naturally occurring toxins present in amphibian toads. Serving as the chemical weapons, they exist not only in the adult toads but also in toad eggs. Guided by mass spectrometry (MS)-based component analysis and feature-based molecular networking (FBMN), 30 bufadienolide-fatty acid conjugates (BDFs) were isolated from the fertilized eggs of toad Bufo gargrizans, including 25 previously undescribed compounds (1-25). Their chemical structures were elucidated by extensive spectroscopic analysis, chemical methods, and GC-MS. The toxicities of all BDFs and their corresponding free BDs were assessed using the zebrafish model. The structure-toxicity relationship analysis showed that the modification of BDs by hydroxy fatty acids can cause a significant increase of the toxicity. Furthermore, all the isolated compounds were evaluated for their antiproliferative activities in pancreatic cancer cell lines ASPC-1 and PANC10.05. The structure-activity relationship (SAR) analysis revealed that BDFs with hellebrigenin as the bufogenin moiety (6 and 7) exhibited the most potent antiproliferative effect. Further investigation into their functional mechanism demonstrated that 6 and 7 induced apoptosis in pancreatic cancer cells PANC10.05 and significantly suppressed the expression of the apoptosis-related gene c-MYC. In addition, 6 and 7 effectively inhibited the expression of the PI3K/Akt/mTOR pathway in PANC10.05. Moreover, we assessed the efficacy of 6 and 7 on cancer cells from various tissues and observed their broad-spectrum antiproliferative activity.

Synthesis, characterization, and anti-cancer potential of novel p53-mediated Mdm2 and Pirh2 modulators: an integrated In silico and In vitro approach.

Introduction: Leukemia is a global health concern that requires alternative treatments due to the limitations of the FDA-approved drugs. Our focus is on p53, a crucial tumor suppressor that regulates cell division. It appears possible to stabilize p53 without causing damage to DNA by investigating dual-acting inhibitors that target both ligases. The paper aims to identify small molecule modulators of Mdm2 and Pirh2 by using 3D structural models of p53 residues and to further carry out the synthesis and evaluation of hit candidates for anti-cancer potency by in vitro and in silico studies. Methods: We synthesized structural analogues of MMs02943764 and MMs03738126 using a 4,5-(substituted) 1,2,4-triazole-3-thiols with 2-chloro N-phenylacetamide in acetone with derivatives of PAA and PCA were followed. Cytotoxicity assays, including MTT, Trypan Blue Exclusion, and MTS assays, were performed on cancer cell lines. Anti-proliferation activity was evaluated using K562 cells. Cell cycle analysis and protein expression studies of p53, Mdm2, and Pirh2 were conducted using flow cytometry. Results: As for results obtained from our previous studies MMs02943764, and MMs03738126 were selected among the best-fit hit molecules whose structural analogues were further subjected to molecular docking and dynamic simulation. Synthesized compounds exhibited potent anti-proliferative effects, with PAC showing significant cytotoxicity against leukemia cells. PAC induced cell cycle arrest and modulated p53, Mdm2, and Pirh2 protein expressions in K562 cells. Molecular docking revealed strong binding affinity of PAC to p53 protein, further confirmed by molecular dynamics simulation. Discussion: The study presents novel anticancer compounds targeting the p53 ubiquitination pathway, exemplified by PAC. Future perspectives involve further optimization and preclinical studies to validate PAC's potential as an effective anticancer therapy.

Gedatolisib shows superior potency and efficacy versus single-node PI3K/AKT/mTOR inhibitors in breast cancer models

The PI3K, AKT, and mTOR (PAM) pathway is frequently dysregulated in breast cancer (BC) to accommodate high catabolic and anabolic activities driving tumor growth. Current therapeutic options for patients with hormone receptor (HR) + / HER2- advanced BC (ABC) include PAM inhibitors that selectively inhibit only one PAM pathway node, which can lead to drug resistance as cells rapidly adapt to maintain viability. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, may be more effective in BC cells than single-node PAM inhibitors by limiting adaptive resistances. By using multiple functional assays, a panel of BC cell lines was evaluated for their sensitivity to four different PAM inhibitors: gedatolisib (pan-PI3K/mTOR inhibitor), alpelisib (PI3Kα inhibitor), capivasertib (AKT inhibitor), and everolimus (mTORC1 inhibitor). Gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects regardless of the PAM pathway mutational status of the cell lines compared to the single-node PAM inhibitors. The higher efficacy of gedatolisib was confirmed in three-dimensional culture and in BC PDX models. Mechanistically, gedatolisib decreased cell survival, DNA replication, cell migration and invasion, protein synthesis, glucose consumption, lactate production, and oxygen consumption more effectively than the other PAM inhibitors tested. These results indicate that inhibition of multiple PAM pathway nodes by a pan-PI3K/mTOR inhibitor like gedatolisib may be more effective at inducing anti-tumor activity than single-node PAM inhibitors. A global Phase 3 study is currently evaluating gedatolisib plus fulvestrant with and without palbociclib in patients with HR+/HER2− ABC.

Design, synthesis, biological evaluation of novel piperidine-based derivatives as potent poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors

Poly(ADP-ribose) polymerase-1 (PARP-1) is a crucial member of DNA repair enzymes responsible for repairing DNA single-strand breaks. Developing PARP inhibitors based on synthetic lethality strategies is an effective approach for treating breast cancer and other diseases. In this study, a series of novel piperidine-based benzamide derivatives were designed and synthesized using structure-based drug design principles. The anticancer activities of these compounds were evaluated against five human cancer cell lines (MDA-MB-436, CAPAN-1, SW-620, HepG2, SKOV3, and PC3) and the preliminary structure–activity relationships were delineated. Among the compounds, 6a and 15d demonstrated potent antiproliferative effects against MDA-MB-436 cells with IC50 values of 8.56 ± 1.07 μM and 6.99 ± 2.62 μM, respectively. Furthermore, both compounds exhibited excellent inhibitory activity against PARP-1, with IC50 values of 8.33 nM and 12.02 nM, respectively. Mechanistic investigations revealed that 6a and 15d effectively inhibited colony formation and cell migration of HCT116 cells. Moreover, they induced apoptosis by upregulating the expression of Bax and cleaved Caspase-3, while downregulating the expression of Caspase-3 and Bcl-2 in HCT116 cells. Based on its impressive pharmacodynamic data in vitro, we conducted a study to evaluate the efficacy of 15d in a xenograft tumor model in mice when used in combination with cytotoxic agents. Collectively, these findings suggest that 15d could be promising drug candidates worthy of further investigation.

Abstract PO1-24-04: Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models

Abstract Background: The PI3K, AKT, and mTOR (PAM) pathway is frequently activated in breast cancer (BC). Current standard-of-care therapy options for patients with advanced BC (ABC) include PAM inhibitors (PAMi), such as everolimus and alpelisib, and hormonal therapy, such as letrozole and fulvestrant. Most PAMi selectively inhibit one or only a few PAM pathway components, which can lead to drug resistance. To minimize drug resistance, a more comprehensive inhibition of the multiple PI3K isoforms and downstream mTOR complexes may be required. We hypothesized that gedatolisib, which potently inhibits all Class I PI3K isoforms, as well as mTORC1 and mTORC2, can be more effective in BC cells than a PAMi targeting single PAM pathway components. Methods: A panel of BC cell lines with mutated or non-mutated PAM pathway genes (PIK3CA, PTEN, AKT) were evaluated for their sensitivity to gedatolisib and other PAMi (PI3K-α: alpelisib; AKT: capivasertib; mTORC1: everolimus; pan-PI3K/mTOR: gedatolisib). Cell viability, growth rate inhibition, and cell death were evaluated by luciferase- and fluorescence-based assays, both in monolayer cultures and three-dimensional (3D) cultures on reconstituted basement membrane. Flow cytometry analytical assays were conducted to assess DNA synthesis (EdU incorporation), protein synthesis (OPP incorporation), and PAM pathway activity (RPS6 and 4EBP1 phosphorylation). BC xenograft studies evaluating gedatolisib in vivo were also performed. Results: Gedatolisib strongly inhibited PAM pathway activity and reduced cell viability and growth rate in the cell lines tested. Compared to the other PAMi, gedatolisib exhibited more potent and efficacious anti-proliferative and cytotoxic effects, regardless of the cell lines’ PI3K pathway mutational status. Gedatolisib effects were confirmed in 3D culture on reconstituted basement membrane, where gedatolisib inhibited tumor cell spheroid growth and induced regression. Mechanistically, gedatolisib decreased both DNA and protein synthesis more effectively than the other PAMi tested. Gedatolisib also exerted superior anti-proliferative and cytotoxic effects relative to the other PAMi tested in estrogen receptor-positive BC cell lines concomitantly treated with fulvestrant. BC xenografts confirmed growth inhibitory effects of gedatolisib in vivo. Conclusions: The pan-PI3K/mTOR inhibitor, gedatolisib, may more effectively address potential drug resistance mechanisms associated with narrowly targeted PAM inhibitors. Gedatolisib has previously demonstrated promising preliminary clinical efficacy and safety data in ABC in combination with hormonal therapy. A Phase 3 study (VIKTORIA-1) evaluating gedatolisib plus fulvestrant with and without palbociclib is underway in patients with ABC. Citation Format: Stefano Rossetti, Aaron Broege, Adrish Sen, Salmaan Khan, Ian MacNeil, Jhomary Molden, Igor Gorbatchevsky, Brian F. Sullivan, Lance Laing. Gedatolisib, a pan-PI3K/mTOR inhibitor, shows superior potency and efficacy relative to other PI3K/AKT/mTOR pathway inhibitors in breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-04.

Abstract PS15-06: Results of the window-of-opportunity PIONEER trial evaluating addition of the progesterone receptor (PR) agonist megestrol to letrozole for early stage estrogen receptor (ER) positive breast cancer: exploiting ER-PR interaction

Abstract Background: Pre-clinical data suggest that combining anti-estrogen treatment with a progesterone receptor agonist leads to greater inhibition of tumor proliferation, due to molecular interactions between ER and PR [1]. A high dose of the PR agonist megestrol (160mg daily) is approved as monotherapy for the treatment of ER positive metastatic breast cancer. A lower dose of megestrol (20-40mg daily) can be an effective treatment for severe hot flashes associated with endocrine therapy [2] but whether this dose has anti-tumor activity is unknown. The PIONEER trial evaluated the potential anti-proliferative effect of low and high dose megestrol in combination with letrozole, relative to letrozole alone, using a short-term preoperative ‘window’ trial design assessing the direct effects of the trial treatment on tumor tissue before and after treatment. Methods: Eligible patients were post-menopausal women with histologically confirmed ER+ (Allred ≥ 3) HER2 negative breast cancer at least 10mm in size, with an ECOG performance status ≤ 2, planned for primary surgery or endocrine therapy. Enrolled patients were randomised 2:3:3 to Arm A: letrozole alone, Arm B: letrozole + lower-dose megestrol (40mg) or Arm C: letrozole + higher-dose megestrol (160mg). Treatment was given for 15 (13-19) days prior to surgery or end of treatment (EOT) core biopsy. The primary endpoint was change in tumor proliferation between baseline and EOT in Arm A vs (Arms B+C combined), measured by Ki67 immunohistochemistry (IHC). Secondary endpoints were comparison of Ki67 change in high versus low dose megestrol arms, absolute Ki67 at EOT, and change in tumor apoptosis (cleaved caspase 3 IHC), proliferation (Aurora Kinase A IHC), PR and androgen receptor expression. Exploratory analysis of ER chromatin binding (ChIP-Seq) was conducted on paired fresh-frozen samples from a subset of patients. Results: A total of 243 patients were randomised from July 2017 to October 2022 with recruitment paused for 3 months at onset of the COVID pandemic. 198 patients completed treatment and had evaluable tissue samples at baseline and EOT (Arm A: n = 51, Arm B: n = 74, Arm C: n = 73). Baseline mean Ki67 values were well balanced. Therapy was well tolerated and adverse events ≥ grade 3 were similarly rare across arms (A: 3.3%, B+C: 3.5%). The mean % reduction in Ki67 for each arm was: Arm A (letrozole): 71%, Arm B (letrozole + 40mg megestrol): 79%, Arm C (letrozole + 160mg megestrol): 80%. There was a statistically significantly greater reduction in Ki67 with megestrol combinations (B+C) versus letrozole alone (A) (P = 0.013). Analyses of secondary IHC endpoints and ER ChIP-Seq are ongoing and will be presented. Conclusion: Addition of the PR agonist megestrol enhanced the anti-proliferative effect of letrozole in this window-of opportunity trial. Megestrol combinations were well tolerated, and the anti-proliferative effect was observed in both low and high dose arms. These data support the potential use of low-dose megestrol as an inexpensive and well-tolerated means of improving aromatase inhibitor efficacy. Low dose megestrol can also ameliorate hot flashes and therefore might be a strategy to improve both treatment adherence and clinical outcomes for patients taking adjuvant endocrine therapy.

PUMC-MB1 is a novel group 3 medulloblastoma preclinical model, sensitive to PI3K/mTOR dual inhibitor.

Medulloblastoma (MB), a common and heterogeneous posterior fossa tumor in pediatric patients, presents diverse prognostic outcomes. To advance our understanding of MB's intricate biology, the development of novel patient tumor-derived culture MB models with necessary data is still an essential requirement. We continuously passaged PUMC-MB1 in vitro in order to establish a continuous cell line. We examined the in vitro growth using Cell Counting Kit-8 (CCK-8) and in vivo growth with subcutaneous and intracranial xenograft models. The xenografts were investigated histopathologically with Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC). Concurrently, we explored its molecular features using Whole Genome Sequencing (WGS), targeted sequencing, and RNA sequecing.Guided by bioinformatics analysis, we validated PUMC-MB1's drug sensitivity in vitro and in vivo. PUMC-MB1, derived from a high-risk MB patient, displayed a population doubling time (PDT) of 48.18h and achieved 100% tumor growth in SCID mice within 20 days. HE and Immunohistochemical examination of the original tumor and xenografts confirmed the classification of PUMC-MB1 as a classic MB. Genomic analysis via WGS revealed concurrent MYC and OTX2 amplifications. The RNA-seq data classified it within the Group 3MB subgroup, while according to the WHO classification, it fell under the Non-WNT/Non-SHH MB. Comparative analysis with D283 and D341med identified 4065 differentially expressed genes, with notable enrichment in the PI3K-AKT pathway. Cisplatin, 4-hydroperoxy cyclophosphamide/cyclophosphamide, vincristine, and dactolisib (a selective PI3K/mTOR dual inhibitor) significantly inhibited PUMC-MB1 proliferation in vitro and in vivo. PUMC-MB1, a novel Group 3 (Non-WNT/Non-SHH) MB cell line, is comprehensively characterized for its growth, pathology, and molecular characteristics. Notably, dactolisib demonstrated potent anti-proliferative effects with minimal toxicity, promising a potential therapeutic avenue. PUMC-MB1 could serve as a valuable tool for unraveling MB mechanisms and innovative treatment strategies.

TEMPO-Mediated Dehydrogenative Hydroxyfluoroalkylation of Arylamines with Polyfluorinated Alcohols.

An efficient 2,2,6,6-tetramethylpiperidinooxy (TEMPO)-mediated hydroxyfluoroalkylation of arylamines with polyfluorinated alcohols via a radical-triggered C(sp2)-H/C(sp3)-H dehydrogenative cross-coupling process was developed. This transformation features simple operation, high atom economy, broad substrate compatibility, and excellent regioselectivity, leading to a series of hydroxyfluoroalkylated arylamine derivatives. Importantly, these synthetic products were further used to evaluate the antitumor activity in cancer cell lines by Cell Counting Kit-8 assay and the outcomes indicated that some compounds show a potent antiproliferative effect.

Therapeutic potential of LINS01 histamine H3 receptor antagonists as antineoplastic agents for triple negative breast cancer

The aims of this work were to evaluate the expression of histamine H3 receptor (H3R) in triple negative breast cancer (TNBC) samples and to investigate the antitumoral efficacy and safety of the LINS01 series of H3R antagonists, through in silico, in vitro, and in vivo approaches. Antitumor activity of LINS01009, LINS01010, LINS01022, LINS01023 was assayed in vitro in 4T1 and MDA-MB-231 TNBC cells (0.01–100 μM), and in vivo in 4T1 tumors orthotopically established in BALB/c mice (1 or 20 mg/kg). Additionally, H3R expression was assessed in 50 human TNBC samples. We have described a higher H3R mRNA expression in basal-like/TNBC tumors vs. matched normal tissue using TCGA Pan-Cancer Atlas data, and a higher H3R expression in human tumor samples vs. peritumoral tissue evidenced by immunohistochemistry associated with poorer survival. Furthermore, while all the essayed compounds showed antitumoral properties, LINS01022 and LINS01023 exhibited the most potent antiproliferative effects by: i) inducing cell apoptosis and suppressing cell migration in 4T1 and MDA-MB-231 TNBC cells, and ii) inhibiting cell growth in paclitaxel-resistant 4T1 cells (potentiating the paclitaxel antiproliferative effect). Moreover, 20 mg/kg LINS01022 reduced tumor size in 4T1 tumor-bearing mice, exhibiting a safe toxicological profile and potential for druggability estimated by ADME calculations. We conclude that the H3R is involved in the regulation of TNBC progression, offering promising therapeutic potential for the novel LINS01 series of H3R antagonists.

Antioxidant Activity and Antitumor Potential of Hedyotis chrysotricha Extract in Human Colorectal Cancer and Breast Cancer Cell Lines

Background: Cancer is a serious disease severely endangering human health and life in the world in the world, and conventional anticancer drugs have frequently suffered from severe side effects and resistance. Therefore, developing novel and effective therapeutic strategies is urgently needed. As a Chinese herbal medicine with important medical values, Hedyotis chrysotricha has been suggested to be useful for the treatment of many diseases, especially cancer. Objective: Our aim was to investigate the antioxidant and antitumor activities of the phytochemical extracts of H. chrysotricha. Methods: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay was applied to determine antioxidant property. 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay, wound healing assay, transwell matrix assay, flow cytometry and cell morphology were established to observe anti-proliferative, anti-migration, anti-invasion and apoptosis induction effects. The intracellular reactive oxygen species generation was detected by reactive oxygen species detection assay. Results: The ethyl acetate fraction showed promising antioxidant activity with IC50 of 98.67±0.49 μg/ml, and petroleum ether fraction exhibited the most potent antiproliferative effect on various human cancer cell lines, especially MDA-MB-231 (IC50 = 8.90±1.23 μg/ml) and HCT-116 (IC50 = 9.69±3.69 μg/ml) cancer cells. Further investigation revealed that the petroleum ether fraction suppressed the proliferation, migration and invasion of the cancer cells significantly. Additionally, it promoted cell apoptosis by increasing reactive oxygen species levels. Conclusion: H. chrysotricha extract possessed excellent antioxidant and antitumor activities. Therefore, it could be useful as a source of antioxidants and compounds for cancer therapy.

Irradiated riboflavin over nonradiated one: Potent antimigratory, antiproliferative and cytotoxic effects on glioblastoma cells.

Riboflavin is a water-soluble yellowish vitamin and is controversial regarding its effect on tumour cells. Riboflavin is a powerful photosensitizer that upon exposure to radiation, undergoes an intersystem conversion with molecular oxygen, leading to the production of ROS. In the current study, we sought to ascertain the impact of irradiated riboflavin on C6 glioblastoma cells regarding proliferation, cell death, oxidative stress and migration. First, we compared the proliferative behaviour of cells following nonradiated and radiated riboflavin. Next, we performed apoptotic assays including Annexin V and caspase 3, 7 and 9 assays. Then we checked on oxidative stress and status by flow cytometry and ELISA kits. Finally, we examined inflammatory change and levels of MMP2 and SIRT1 proteins. We caught a clear antiproliferative and cytotoxic effect of irradiated riboflavin compared to nonradiated one. Therefore, we proceeded with our experiments using radiated riboflavin. In all apoptotic assays, we observed a dose-dependent increase. Additionally, the levels of oxidants were found to increase, while antioxidant levels decreased following riboflavin treatment. In the inflammation analysis, we observed elevated levels of both pro-inflammatory and anti-inflammatory cytokines. Additionally, after treatment, we observed reduced levels of MMP2 and SIRT. In conclusion, radiated riboflavin clearly demonstrates superior antiproliferative and apoptotic effects on C6 cells at lower doses compared to nonradiated riboflavin.

The Combined Effect of Vulpinic Acid and Doxorubicin in Breast Cancer MCF-7 Cells

Breast cancer is a major global health problem that imposes a significant social and economic burden on individuals and societies. Chemotherapy, a common treatment approach, often leads to resistance and unwanted side effects, especially in the advanced stages of the disease. This has led to a search for more effective and less toxic anticancer agents. Lichens, which are associations of fungi and algae, are gaining attention for their potential in cancer therapy. Lichens are rich sources of secondary metabolites with diverse biological effects, including anti-tumor properties. In this study, we summarise the combined effects of vulpinic acid (VA), a lichen acid, with doxorubicin, a chemotherapeutic drug, on breast cancer MCF-7 cells. The results of the XTT assay and subsequent Compsyn analysis showed that VA and doxorubicin (Dox), a chemotherapeutic drug, alone exhibited potent anti-proliferative effects in a dose- and time-dependent manner, and interestingly, when used in combination, they produced an antagonistic effect in the same cancer line. These results provide the first example of a study to show what effect the combination of VA and Dox will have on other breast cancer cell lines.

Abstract 2083: Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress

Abstract DNA damage response (DDR) is an important cellular process that is regulated by poly ADP ribosylation (PARylation). PARP1 binds to DNA break sites and then catalyzes PARylation, which leads to recruitment of repair factors and initiation of base-excision and single-strand break (SSB) repair. Removal of PAR chains is performed by PARG and is also critical for successful completion of DNA repair. Suppression of PAR chain hydrolysis via PARG depletion or inhibition leads to defective single-strand break repair, reduced kinetics of repair, and hypersensitivity to certain DNA damaging agents. Cancer cells demonstrate high levels of replication stress, characterized as the slowing or stalling of replication forks during DNA replication. Replication stress generates DNA SSBs and exposes unligated Okazaki fragments which activate PARP1. PARP1 catalyzed PARylation slows replication fork progression as a protective mechanism, initiates damage repair, and promotes fork reversal. PAR chain removal, via PARG activity, enables completion of damage repair, replication fork restart, and recovery from replication stress. Under conditions of replication stress, PARP inhibition can result in unrestrained fork progression and the occurrence of DNA double strand breaks from fork runoff. In contrast, PARG inhibition causes prolonged slowing or stalling of replication fork progression, which can lead to replication catastrophe and cancer cell death. Because of these opposing roles, PARG inhibitors show distinct pharmacology as compared to PARP inhibitors in certain cancer cells including cancer cells that are BRCA wild type, homologous recombination proficient, and PARP inhibitor insensitive or resistant. ETX-19477 is a novel inhibitor of PARG that demonstrates low nM, on-target activity in cells as measured by the accumulation of PAR chains. ETX-19477 induces death and/or inhibits the proliferation of multiple cancer cell types including ER+HER2- breast, serous and mucinous ovarian, lung, and gastric cancers and induces pan-nuclear γH2AX expression, a hallmark of replication catastrophe. Preclinical studies demonstrate that ETX-19477 is orally bioavailable with favorable pharmacokinetics. Oral treatment of mice with ETX-19477 is well tolerated and shows robust dose-dependent in vivo anti-tumor activity in breast and ovarian cell-line derived and patient-derived xenograft models. Efficacy is observed with either once or twice daily dosing and with a discontinuous dosing regimen. In mouse xenograft models, induction of γH2AX expression in tumor tissue correlates with tumor growth inhibition. Preclinical data show ETX-19477 demonstrates potent and specific anti-proliferative effects by exposing underlying existing replication stress vulnerabilities in cancer cells across multiple tumor types. ETX-19477 will be advanced into clinical trials in 2024. Citation Format: John P. Holleran, Tamara S. Rodems, Shweta Sharma, Angelina M. Santini, Cindy Wuerz, Juping Liu, Lu Tian, Joseph Dosch, Joy Nader, Richard Rivera, Shyama Herath, Joseph Marakovits, Christopher McBride, Ryan Stansfield, Amy Salyards, James M. Veal, Jeffrey A. Stafford, Gretchen Bain. Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress [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 2083.

Abstract 5795: Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems

Abstract Phenotypic plasticity, or the ability of cancer cells to switch between cell states, has recently been recognized as a key hallmark of cancer. Cancer cells can hijack normal developmental pathways, preventing terminal differentiation or causing de-differentiation or transdifferentiation to a more proliferative, plastic cell state similar to those found in early development. Using our proprietary AI/ML platform AURIGIN, we created a high-resolution atlas to define the drivers of normal cell states during human development. After mapping primary human tumors to the atlas, we identified the histone acetyltransferase KAT2A as a key driver of tumor cell plasticity in a subset of acute myeloid leukemias (AML), small cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC). Targeting KAT2A was genetically validated in MOLM-13 (AML) cells, where knockdown resulted in significant cell growth inhibition and induction of cell state changes consistent with a more mature differentiation phenotype. Similarly, an early tool heterobifunctional degrader of KAT2A and KAT2B (AUR101) resulted in potent growth inhibition and granulocytic differentiation in MOLM-13 cells and in 5/12 primary AML patient samples grown ex vivo. Treatment of NCI-H1048 SCLC cells with AUR101 potently degraded KAT2A/B, induced a significant shift from a dedifferentiated cell state to a more differentiated epithelial cell state and potently inhibited cell growth both in vitro and in vivo. The strong biological validation of KAT2A/B in SCLC and AML tumors, led us to develop AUR1545, a novel sub-nanomolar degrader of KAT2A and KAT2B. AUR1545, with its improved drug like properties, was tested across representative models of AML, SCLC and NEPC. In vitro, treatment with AUR1545 inhibited proliferation and promoted profound cell state changes in MOLM-13 (AML) cells. In NCI-H1048 (SCLC), LASPC-01 (NEPC) cell lines as well as a primary NEPC organoid model, AUR1545 demonstrated potent antiproliferative effects. In vivo, AUR1545 significantly inhibited tumor growth in an NCI-H1048 xenograft model. Taken together, these data genetically and pharmacologically validate KAT2A and KAT2B as key drivers of cell state plasticity in SCLC, NEPC and AML tumors and point to their utility as novel targets in these aggressive, metastatic and drug resistant cancers. Citation Format: Kimberly S. Straley, James Neef, Ileana Antony-Debre, Maulasri Bhatta, Sambad Sharma, Sara Sinicropi-Yao, Joe DeBartolo, Andrew McRiner, Betty Chan, Henry Wilson, Christina S. Lee, Zied Boudhraa, Mohamed El Ezzy, Umar Sharif, Mark Bittinger, Laura Antipov, Thomas G. Graeber, Stephane de Botton, Katharine E. Yen, David S. Millan. Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems [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 5795.

Abstract 5707: Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies

Abstract KRLS-017 is a potent, selective, and reversible inhibitor of Cyclin Dependent Kinase 7 (CDK7). Its molecular formula is C24H35N5O2Si and its molecular weight is 453.65 Daltons. KRLS-017 is being developed for the treatment of patients with advanced solid tumor malignancies. To differentiate KRLS-017 from other CDK7 inhibitors, we created a reversible inhibitor to allow for treatment schedule optimization and maximization of therapeutic index in clinic. High selectivity of KRLS-017 for CDK7 was confirmed using a panel of 313 recombinant human kinases. The IC50 of KRLS-017 against recombinant human CDK7 was determined to be 16 nM, and no other kinases in the panel showed IC50 values less than 300 nM. Antiproliferative activity of KRLS-017 was investigated in vitro using a broad panel of human tumor cell lines. KRLS-017 showed potent antiproliferative effect across a wide variety of both solid and hematologic tumor types, producing GI50 values <200 nM in 258/302 human tumorcell lines tested. Antitumor activity of KRLS-017 was investigated in vivo using multiple mouse xenograft tumormodels at dose levels ranging from 25 mg/kg to 200 mg/kg using both continuous (daily) andintermittent dosing schedules. Statistically significant tumor growth inhibition was seen at doselevels of 50 mg/kg and higher, with tumor regression observed at 100 mg/kg and 200 mg/kg. Better in vivo tolerability at efficacious doses was observed using intermittent dosing schedules in mouse xenograft models, which led to adoption of an intermittent schedule in the proposed first-in-human phase 1 trial. KRLS-017 is orally bioavailable in mice, rats, and dogs and plasma drug exposure increased in a proportional manner with dose. Comparing first dose and 28th dose in the dog, minimal plasma accumulation was observed, suggesting once daily dosing in humans would be initially tested. Repeat dose 28 day GLP toxicology studies in rat and dog showed adverse effects in highly proliferative bone marrow and gastrointestinal tissues which is consistent with the known mechanism of action for CDK7 and is considered to be monitorable and manageable in the setting of oncology. The clinical development plan for KRLS-017 includes an initial Phase 1 dose escalation study (KRLS-017-101) in patients with refractory solid tumor malignancies to assess pharmacokinetics/pharmacodynamics and to determine an optimal dose level and treatment schedule to test in expansion cohorts. Given the potential of CDK7 inhibitors in the treatment of hormone receptor positive breast cancer, combination work with other agents (such as selective estrogen receptor degraders) is on-going. Citation Format: Alison L. Hannah, Steve Ritland, Thomas Steele, Steven Smith, Ashwin Ram, BT Slingsby, Kapil Dhingra, James Chrisman, Rajesh Dua, Yasunori Tokunaga, Shigeyuki Kono, Yasuhiro Aga. Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies [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 5707.

Alpha-Pinene and Tannic Acid Inhibit Trichomonas vaginalis Protozoan Cells by Inducing Apoptosis.

Trichomoniasis is one of the most common sexually transmitted infections worldwide. The growing concern of drug resistance of this infection has cautioned the need for new drug development. We evaluated the potential antiproliferative and apoptotic effect of α-pinene and tannic acid (TA) on Trichomonas vaginalis cells. In addition, the cytotoxicity of agents on Vero cells was investigated. Trichomonas cells were axenically cultured in TYI-S-33 medium. In vitro antiproliferative activity of α-pinene, TA, and metronidazole was investigated against Trichomonas cells. The assays were carried out in triplicate using microtiter plate and trypan blue staining method. Annexin V/PI staining with flow cytometry was used to evaluate apoptosis induction. In addition, the cytotoxic effect was measured by MTT assay. α-Pinene and TA exhibited significant inhibition of the Trichomonas cells and the lowest IC50 values were 22.9 μg/ml and 140 μg/ml at 48 hours' incubation, respectively. The CC50 was found at 116 μg/ml for α-pinene and 473 μg/ml for TA, after 48 hours of treatment. The flow cytometry study demonstrated that the natural compounds induced apoptosis in Trichomonas cells. After 24 hours of treatment, the induction of apoptosis was 5.2% - 36.6% at concentrations of 3.9 - 62.5 μg/ml for α-pinene and TA induced-apoptosis was 6.1% - 53.8% at concentrations of 125-2000 μg/ml. Although the results show the antiproliferative and apoptotic effect of α-pinene and TA on Trichomonas cells, in vivo studies are needed to further clarify the effects of these compounds.

Antiproliferative and Apoptosis-Inducing Effects of Ethanolic Extract of Morus Alba Leaves on Human Chronic Myeloid Leukaemia K-562 Cell Lines

The rate of occurrence and prevalence of chronic myeloid leukaemia (CML) are increasing globally, despite it being a rare type of cancer. Morus alba also known as mulberry, has been used in Chinese traditional medicine for a long time to treat various conditions and diseases like hypertension and arthritis. Modern scientific studies have also recognized its medicinal properties, including antioxidant, antidiuretic, antidiabetic, antimicrobial and anticancer. However, its antiproliferative effects on specifically human CML K-562 cell lines remain poorly elucidated. This study aims to determine the antiproliferative effects of M. alba on K-562 cells. In vitro cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Acridine orange and propidium iodide (AO/PI) dual staining were then employed to assess the method of cell death. The ethanolic extract of M. alba leaves exhibited significant antiproliferative activity, with the lowest maximal half inhibitory concentration (IC50) value observed at 72 hours being 23 µg/mL. Notably, AO/PI staining revealed significant morphological changes, such as membrane blebbing, nuclear fragmentation, and indicator of early apoptosis, and late apoptosis. These results demonstrated that ethanolic extract of M. alba leaves induces apoptotic cell death in K-562 cells. Overall, these findings suggested that ethanolic extract of M. alba leaves exerts potent antiproliferative effects on human CML K-562 cells a in time- and dose-dependent manner.

Abstract PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Abstract PR-002: Uncovering novel therapeutic avenues for glioma by exploring the functional interplay of FGFR1, p53, and ribosome biogenesis

Exploring the therapeutic potential of SGLT2 inhibitors in cancer treatment: integrating in silico and in vitro investigations.

The present study aimed to investigate the anti-cancer mechanism of canagliflozin (CANA) and dapagliflozin (DAPA), sodium-glucose co-transporter-2 (SGLT2) inhibitors, using in silico and in vitro approaches. Network pharmacology was employed to predict the targets of the inhibitors and GO gene enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation conducted to explore the interacting pathways. Molecular docking and molecular dynamic (MD) simulation studies were performed to confirm the important targets and assess conformational stability. In vitro cytotoxicity assays, MIA-PaCa-2 and DU-145 cell lines CANA and DAPA was performed. Protein-protein interaction (PPI) network analysis indicated that CANA and DAPA exert anticancer effects through MAPK, mTOR, EGFR-KRAS-BRAF, FGFR, and PI3KA pathways. KEGG analysis revealed that these inhibitors could be used in the treatment of various cancers, including breast, prostate, pancreatic, chronic myeloid leukemia, thyroid, small cell lung, gastric, and bladder cancer. Docking results showed highest affinity for MAPK1 for CANA (- 9.60kcal/mol) and DAPA (- 9.58kcal/mol). MD simulation results showed that RMSD values for the MAPK1-compound exhibit remarkable stability over a timeframe of 100ns. In cytotoxicity assays using MIA-PaCa-2 and DU-145 cell lines, CANA demonstrated a potential antiproliferative effect on the pancreatic cell line MIA-PaCa-2 after 48h of treatment at a concentration of 100µg/ml. Furthermore, CANA arrested the cell cycle in the sub-G1 phase and induced late apoptosis and necrosis in MIA-PaCa-2 cell line. Based on these findings, CANA shows promise as a potential novel treatment strategy for pancreatic cancer.