Panel Of Cell Lines Research Articles

Evaluation of cytotoxic effect of siphonochilone from African ginger: an in vitro analysis.

Plants provide a wide array of compounds that can be explored for potential anticancer properties. Siphonochilone, a furanoterpene that represents one of the main components of the African plant Siphonochilus aethiopicus, shows numerous health benefits. However, to date, its antiproliferative properties have not been tested. The aim of this study was to analyze the cytotoxic effects of siphonochilone on a panel of cancer cell lines and its underlying mechanism of action. Our results demonstrated that siphonochilone exhibited significant cytotoxic effects on pancreatic, breast, lung, colon, and liver cancer cell lines showing a IC50 ranging from 22 to 124 μM at 72 h of treatment and highlighting its cytotoxic effect against MCF7 and PANC1 breast and pancreas cancer cell lines (22.03 and 39.03 μM, respectively). Cell death in these tumor lines was mediated by apoptosis by the mitochondrial pathway, as evidenced by siphonochilone-induced depolarization of the mitochondrial membrane potential. In addition, siphonochilone treatment involves the generation of reactive oxygen species that may contribute to apoptosis induction. In this work, we described for the first time the cytotoxic properties of siphonochilone and provided data about the molecular processes of cell death. Although future studies will be necessary, our results support the interest in this molecule in relation to their clinical application in cancer, and especially in breast and pancreatic cancer.

Molecular characteristics of early-onset compared with late-onset colorectal cancer: A case controlled study.

Early-Onset Colorectal Cancer (EOCRC) is associated with a poorer prognosis relative to Late-Onset Colorectal Cancer (LOCRC), and its incidence has witnessed a gradual escalation in recent years. This necessitates a comprehensive examination of the underlying pathogenesis and the identification of therapeutic targets specific to EOCRC patients. The present study aimed to delineate the distinct molecular landscape of EOCRC by juxtaposing it with that of LOCRC. A total of 11,344 colorectal cancer patients, diagnosed between 2003 and 2022, were enrolled in this study, comprising 578 EOCRC cases and 10,766 LOCRC cases. Next-generation sequencing technology was employed to assess the tumor-related mutation and tumor mutation burden (TMB) in these patients. PD-L1 expression was quantified using immunohistochemistry. Microsatellite instability (MSI) was determined via capillary electrophoresis (2B3D NCI Panel). Upon comparing LOCRC with EOCRC patients, the latter group demonstrated a tendency towards advanced TNM stage, lower tumor differentiation, and less favorable histological types. Among LOCRC patients, those with MSI-H status were found to have an earlier TNM stage compared to those with MSI-L/MSS status. Significantly, the incidence of MSI-H was notably higher in EOCRC (10.2%) compared to LOCRC (2.2%). Mutations in the 7-gene panel (ARID1A, FANCI, CASP8, DGFRA, DPYD, TSHR, and PRKCI) were more prevalent in LOCRC. Within the EOCRC cohort, patients with the MSI-H subtype displayed an earlier TNM stage but concurrently exhibited poorer tissue differentiation and a higher frequency of mucinous adenocarcinoma. Among EOCRC patients, FBXW7, FAT1, ATM, ARID1A, and KMT2B mutations were significantly enriched in the MSI-H subgroup. A comparative analysis of MSI-H patients revealed heightened mutation frequencies of FGFBR2, PBRM1, RNF43, LRP1B, FBXW7, ATM, and ARID1A in the EOCRC group. Furthermore, EOCRC patients demonstrated a higher overall TMB, particularly in the MSI-H subtype. PD-L1 expression was elevated in EOCRC and positively associated with MSI status. This study revealed a significantly higher MSI-H distribution rate in early-onset colorectal cancer, and EOCRC exhibits a distinct mutational signature coupled with higher PD-L1 expression. These findings hold promise in guiding personalized therapeutic strategies for improved disease management in EOCRC patients.

IRX4204 Induces Senescence and Cell Death in HER2-positive Breast Cancer and Synergizes with Anti-HER2 Therapy.

Rexinoids, agonists of nuclear retinoid X receptor (RXR), have been used for the treatment of cancers and are well-tolerated in both animals and humans. However, the usefulness of rexinoids in treatment of breast cancer remains unknown. This study examines the efficacy of IRX4204, a highly specific rexinoid, in breast cancer cell lines and preclinical models to identify a biomarker for response and potential mechanism of action. IRX4204 effects on breast cancer cell growth and viability were determined using cell lines, syngeneic mouse models and primary PDX tumors. In vitro assays of cell cycle, apoptosis, senescence, and lipid metabolism were used to uncover a potential mechanism of action. Standard anti-HER2 therapies were screened in combination with IRX4204 on a panel of breast cancer cell lines to determine drug synergy. IRX4204 significantly inhibits the growth of HER2-positive breast cancer cell lines, including trastuzumab and lapatinib resistant JIMT-1 and HCC1954. Treatment with IRX4204 reduced tumor growth rate in the MMTV-ErbB2 mouse and HER2-positive PDX model by 49% and 44% respectively. Mechanistic studies revealed IRX4204 modulates lipid metabolism and induces senescence of HER2-positive cells. In addition, IRX4204 demonstrates additivity and synergy with HER2 targeted monoclonal antibodies, tyrosine kinase inhibitors, and antibody drug conjugates. These findings identify HER2 as a biomarker for IRX4204 treatment response and demonstrate a novel use of RXR agonists to synergize with current anti-HER2 therapies. Furthermore, our results suggest that RXR agonists can be useful for the treatment of anti-HER2 resistant and metastatic HER2-positive breast cancer.

Different contractility modes control cell escape from multicellular spheroids and tumor explants.

Cells can adapt their active contractile properties to switch between dynamical migratory states and static homeostasis. Collective tissue surface tension, generated among others by the cortical contractility of single cells, can keep cell clusters compact, while a more bipolar, anisotropic contractility is predominantly used by mesenchymal cells to pull themselves into the extracellular matrix (ECM). Here, we investigate how these two contractility modes relate to cancer cell escape into the ECM. We compare multicellular spheroids from a panel of breast cancer cell lines with primary tumor explants from breast and cervical cancer patients by measuring matrix contraction and cellular spreading into ECM mimicking collagen matrices. Our results in spheroids suggest that tumor aggressiveness is associated with elevated contractile traction and reduced active tissue surface tension, allowing cancer cell escape. We show that it is not a binary switch but rather the interplay between these two contractility modes that is essential during this process. We provide further evidence in patient-derived tumor explants that these two contractility modes impact cancer cells' ability to leave cell clusters within a primary tumor. Our results indicate that cellular contractility is an essential factor during the formation of metastases and thus may be suitable as a prognostic criterion for the assessment of tumor aggressiveness.

Abstract PO2-14-10: A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model

Abstract Background Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer diagnoses but up to 30% of breast cancer-related deaths. Similarly, Inflammatory breast cancer (IBC) accounts for 2-4% of breast cancer diagnosis but causes 8%–10% of all breast cancer-related deaths. The myeloid cell leukemia-1 (MCL-1) is a member of the BCL-2 family of proteins, which is highly amplified in numerous human cancers and associated with cell immortalization, transformation, and chemoresistance. In fact, MCL-1 is amplified in 55% of TNBC after preoperative chemotherapy. Amongst the MCL-1 inhibitors that have been developed, AZD5991 showed high selectivity and affinity for MCL-1. In myeloma and acute myeloid leukemia cells AZD5991 induces apoptosis and showed anti-tumor effects in in vivo models. In the present study, we evaluated the biological role of the MCL-1 inhibitor AZD5991 in IBC and TNBC in vitro and in vivo models. Materials and methods The expression of the MCL-1 gene was analyzed in the World Consortium IBC patient dataset. Next, we screened a panel of TNBC and IBC cell lines for MCL-1 expression by western blot analysis. The effect of AZD5991 on TNBC and IBC cells was analyzed via cell titer-blue proliferation, clonogenic assays and soft agar assay. To further validate these findings, we used siRNA to knockdown MCL-1 and performed cell proliferation, clonogenic and migration assays. To identify signaling pathways involved in MCL-1 Inhibitor’s effects we performed a proteome profiler human apoptosis array in TNBC cells in the presence of AZD5991. The anti-tumor effect of the MCL-1 inhibitor was evaluated using a SUM-149 (TN-IBC) orthotopic xenograft mouse model. The mice were treated with AZD5991 at doses of 15 and 30mg/kg once weekly via tail vein injection for a duration of 4 weeks. Results The mean expression level of MCL-1 was higher in patients with TNBC than in those with non-TNBC. Further, compared to the patients with ER positive, HER-2 negative tumors those with TNBC showed an increased in the mean expression of MCL-1 gene (p value= 0.05). We screened a panel of TNBC and IBC cell lines in which most of the cell lines showed high levels of MCL-1. The cell proliferation assay showed IC50 ranging from 0.27 – 19.6 μM. The SUM149, SUM190, FCIBC02, MDA-IBC3 and MDA-MB-468 cells showed high sensitivity and showed significant decrease in the proliferation rate while BCX010, KPL4, MDA-MB-231, 4T1.2 and E0771 cells as well as the normal breast cells MCF10A exhibited resistance with no significant decrease in proliferation. The clonogenic and soft agar assays with these sensitive and resistant cell lines showed similar results. Further, these findings were validated via genetic knockdown of MCL-1 which resulted in a significant decrease in cell proliferation, colony formation and migration capability of the TNBC cells. To identify signaling pathways involved in MCL-1 Inhibitor’s phenotypic changes, we performed a proteome profiler human apoptosis array which showed a decreased in BCLx expression, and an increase in expression of BAX and phospho-Rad17 proteins in the AZD5991 treated TN-IBC cells. Finally, in a SUM-149 (TN-IBC) orthotopic xenograft mouse model, mice injected with 30mg/kg showed a significant tumor regression compared to the vehicle-treated mice (vehicle vs 30 mg/kg, p value=0.0006). Conclusions and Future directions Our data demonstrates that AZD5991 inhibits tumorigenesis in an inflammatory breast cancer xenograft model. We are currently validating several significant targets to understand the biological mechanisms related to our candidate drug. Our long-term goal is to develop MCL-1 targeted therapy in combination with standard-of-care treatment for inflammatory and triple-negative breast cancers. Citation Format: Mohd Mughees, Moises Tacam, Alex Tan, Michael White, Bisrat Debeb, Emilly Villodre, Xiaoding Hu, Debu Tripathy, Wendy Woodward, Rachel Layman, Geoffrey Bartholomeusz, Chandra Bartholomeusz. A selective MCL-1 inhibitor AZD5991 showed tumor regression in a triple-negative inflammatory breast cancer xenograft model [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 PO2-14-10.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched >4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index < 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [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 PO4-14-12.

Abstract PO1-04-07: Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2. DV consists of an anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable vedotin linker. DV has multimodal antitumor mechanisms of action that include direct cytotoxicity of HER2-expressing cancer cells and bystander effect-based cytotoxicity of neighboring cells, both of which are mediated by the intracellular release of MMAE. Released MMAE can also induce immunogenic cell death, which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity, which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth, survival, and proliferation. Previously, we showed the cytotoxic activity of DV against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range. Here, we sought to further characterize the preclinical antitumor activity of DV in patient-derived 3D models and patient-derived xenographs (PDX). In cultured breast cancer cells, DV was more potent and internalized to a greater magnitude than the HER2-directed ADCs trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In patient-derived 3D models, DV had superior activity compared to T-DXd. We further explored whether dual HER2 targeting with DV in combination with the HER2-selective oral TKI tucatinib improved the antitumor outcomes. In PDX models with varying HER2 IHC levels and models refractory to T-DXd, DV as a monotherapy and in combination with tucatinib showed significant tumor growth inhibition. This result is consistent with previous in vitro results that showed increased cytotoxicity of the combination of DV and tucatinib in cell lines with a wide range of HER2 expression, which may be mechanistically attributed to elevated HER2 cell surface levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast cancer patients as a monotherapy or in combination with tucatinib. Citation Format: Kelsi Willis, Renee Hein, Katie Snead, Robert Thurman, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical 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 PO1-04-07.

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 PO3-26-07: Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer

Abstract The sodium iodide symporter (NIS, encoded by SLC5A5) is the sole transporter of iodide, and successfully exploited in thyroid cancer to uptake radioiodide (RAI). NIS is essentially undetected in normal mammary epithelial cells but elevated during lactation, and has a heterogeneous expression profile in breast cancer (BRCA), as evidenced by TCGA cohort and also in a ~120 sample TMA we have profiled. Furthermore, pre-clinical studies support an epigenetic mechanisms that involves Retinoic Acid Receptor alpha (RARa) to regulate SLC5A5 expression. Therefore, we have applied an orthogonal approach to both elucidate and target mechanisms that control NIS expression and function to deliver a therapeutic strategy that targets this transporter as a novel radiotherapy in BRCA. To understand the epigenetic control of expression we segregated samples in the TCGA-BRCA cohort into lower and upper quartile SLC5A5 expression and identified ~600 significant differentially expressed of genes (DEG). Interestingly, restricting analyses to either triple negative (TN) or tumors from African American (AA) patients doubled the number of DEGs. These DEGs were significantly associated with several transcription factors including LHX3, a pituitary development transcription factor regulated by thyroid signaling. The most significantly altered ~70 DEGs significantly clustered tumors by genomic ancestry and ERBB2/HER2 IHC status. The DEGs also supported a footprint of RARa signaling include PRAME, a known RARa co-repressor associated with histone deacetylase, and reflects the finding that NIS expression is highly responsive to SAHA treatment. To investigate if PRAME expression suppresses RARa regulation of SLC5A5 we have established a panel of BRCA cell lines (AU565, SKB3 SUM52 and MDA-MB-231) stably transfected with either CRISPR-VP64 (activator) and CRISPR-KRAB (repressor) to modulate PRAME and measure the epigenomic impact, the expression of SLC5A5 and the ability to transport RAI. In parallel we have applied a drug screen approach that targets non-canonical approaches to govern NIS expression, for example by targeting valosin-containing protein (VCP) that induces NIS proteolysis. In the first instance we have developed a therapeutic strategy to target NIS by inhibiting VCP. Specifically, we are screening a range of VCP inhibitors in combination with the HDAC inhibitor SAHA or the transcriptional potentiator, disulfiram metabolite Cu(DDC)2 in BRCA cell lines by testing the ability to augment RAI uptake. Remarkably, in MDA MB 231 cells, Cu(DDC)2 combined with the VCP inhibitor CB5339, resulted in a significant additive increase in RAI uptake and a prominent decrease in cell proliferation and survival. These studies combined with future murine and PDX testing of CRISPR-edited tumors and drug exposures will develop a comprehensive analyses of the mechanisms that control SLC5A5 expression across breast cancer cell types, how this can be most effectively targeted to restore NIS expression, and exploited to promote RAI as a novel targeted BRCA radiotherapy. Citation Format: Katie Brookes, Martin Read, Sajad Wani, Sissy Jhiang, Daniel Stover, Mathew Ringel, Christopher McCabe, Moray Campbell. Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer [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 PO3-26-07.

Dysregulation of a Subset of Circulating and Vesicle-Associated miRNA in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

Chemical Constituents From the Leaves of Grewia bulot Gagn. and Their Cytotoxic Activity

Objective: The ultimate objective of this study was to isolate bioactive compounds from Grewia bulot Gagn. leaves and evaluate their cytotoxicity against a panel of human cancer cell lines. Methods: Compounds were isolated by column chromatography, whereas their chemical structures were elucidated by NMR spectroscopic data, and literature comparison. The cytotoxic activity of the isolated compounds was assessed using SRB assay against 4 cancer cell lines, including MCF-7 (human breast carcinoma), Hep-G2 (human hepatocellular carcinoma), KB (human carcinoma in the mouth), and SK-LU-1 (human lung carcinoma). Results: From the n-hexane extract, 4 compounds were isolated and identified as taraxerol (1), 3-( E)-coumaroyltaraxerol (2), 3-( Z)-coumaroyltaraxerol (3), and lupeol (4). Additionally, 6 compounds were isolated from the ethyl acetate extract and identified as daucosterol (5), trans-tiliroside (6), inugalactolipid A (7), (3 S,5 R,6 S,7 E,9 R)-7-megastigmene-3,6,9-triol (8), biphenyl-3,3′,4,4′-tetrol (9), and smiglabrone B (10). Among these isolates, only compound 9 exhibited moderate cytotoxic activity against all tested cancer cell lines with the IC50 values ranging from 31.67 to 63.15 µM. Conclusion: All these compounds have been detected from G bulot for the first time, biphenyl-3,3′,4,4′-tetrol (9) emerged as a promising candidate for cancer drug development. This study contributes to the growing body of knowledge regarding natural compounds with anticancer properties.

HDAC inhibitors activate lipid peroxidation and ferroptosis in gastric cancer

Gastric cancer remains among the deadliest neoplasms worldwide, with limited therapeutic options. Since efficacies of targeted therapies are unsatisfactory, drugs with broader mechanisms of action rather than a single oncogene inhibition are needed. Preclinical studies have identified histone deacetylases (HDAC) as potential therapeutic targets in gastric cancer. However, the mechanism(s) of action of HDAC inhibitors (HDACi) are only partially understood. This is particularly true with regard to ferroptosis as an emerging concept of cell death.In a panel of gastric cancer cell lines with different molecular characteristics, tumor cell inhibitory effects of different HDACi were studied. Lipid peroxidation levels were measured and proteome analysis was performed for the in-depth characterization of molecular alterations upon HDAC inhibition. HDACi effects on important ferroptosis genes were validated on the mRNA and protein level.Upon HDACi treatment, lipid peroxidation was found increased in all cell lines. Class I HDACi (VK1, entinostat) showed the same toxicity profile as the pan-HDACi vorinostat. Proteome analysis revealed significant and concordant alterations in the expression of proteins related to ferroptosis induction. Key enzymes like ACSL4, POR or SLC7A11 showed distinct alterations in their expression patterns, providing an explanation for the increased lipid peroxidation. Results were also confirmed in primary human gastric cancer tissue cultures as a relevant ex vivo model.We identify the induction of ferroptosis as new mechanism of action of class I HDACi in gastric cancer. Notably, these findings were independent of the genetic background of the cell lines, thus introducing HDAC inhibition as a more general therapeutic principle.

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10μM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8μM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

MYTX-011: a pH-dependent anti-cMET antibody-drug conjugate designed for enhanced payload delivery to cMET expressing tumor cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Globospiramine from Voacanga globosa Exerts Robust Cytotoxic and Antiproliferative Activities on Cancer Cells by Inducing Caspase-Dependent Apoptosis in A549 Cells and Inhibiting MAPK14 (p38α): In Vitro and Computational Investigations.

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.

Inhibition of SRC-3 as a potential therapeutic strategy for aggressive mantle cell lymphoma.

Mantle cell lymphoma (MCL) has a poor prognosis and high relapse rates despite current therapies, necessitating novel treatment regimens. Inhibition of SRC-3 show effectiveness in vivo and in vitro in other B cell lymphomas. Additionally, previous studies have shown that SRC-3 is highly expressed in the lymph nodes of B cell non-Hodgkin's lymphoma patients, suggesting SRC-3 may play a role in the progression of B cell lymphoma. This study aimed to investigate novel SRC-3 inhibitors, SI-10 and SI-12, in mantle cell lymphoma. The cytotoxic effects of SI-10 and SI-12 were evaluated in vitro and demonstrated dose-dependent cytotoxicity in a panel of MCL cell lines. The in vivo efficacy of SI-10 was confirmed in two ibrutinib-resistant models: an immunocompetent disseminated A20 mouse model of B-cell lymphoma and a human PDX model of MCL. Notably, SI-10 treatment also resulted in a significant extension of survival in vivo with low toxicity in both ibrutinib-resistant murine models. We have investigated SI-10 as a novel anti-lymphoma compound via the inhibition of SRC-3 activity. These findings indicate that targeting SRC-3 should be investigated in combination with current clinical therapeutics as a novel strategy to expand the therapeutic index and to improve lymphoma outcomes.

Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

Structure-based drug design of DNA minor groove binders and evaluation of their antibacterial and anticancer properties

Antimicrobial and chemotherapy resistance are escalating medical problem of paramount importance. Yet, research for novel antimicrobial and anticancer agents remains lagging behind. With their reported medical applications, DNA minor groove binders (MGBs) are worthy of exploration. In this study, the approach of structure-based drug design was implemented to generate 11 MGB compounds including a novel class of bioactive alkyne-linked MGBs. The NCI screening protocol was utilized to evaluate the antitumor activity of the target MGBs. Furthermore, a variety of bactericidal, cytopathogenicity, MIC90, and cytotoxicity assays were carried out using these MGBs against 6 medically relevant bacteria: Salmonella enterica, Escherichia coli, Serratia marcescens, Bacillus cereus, Streptococcus pneumoniae and Streptococcus pyogenes. Moreover, molecular docking, molecular dynamic simulations, DNA melting, and isothermal titration calorimetry (ITC) analyses were utilized to explore the binding mode and interactions between the most potent MGBs and the DNA duplex d(CGACTAGTCG)2. NCI results showed that alkyne-linked MGBs (26 & 28) displayed the most significant growth inhibition among the NCI-60 panel. In addition, compounds MGB3, MGB4, MGB28, and MGB32 showed significant bactericidal effects, inhibited B. cereus and S. enterica-mediated cytopathogenicity, and exhibited low cytotoxicity. MGB28 and MGB32 demonstrated significant inhibition of S. pyogenes, whereas MGB28 notably inhibited S. marcescens and all four minor groove binders significantly inhibited B. cereus. The ability of these compounds to bind with DNA and distort its groove dimensions provides the molecular basis for the allosteric perturbation of proteins-DNA interactions by MGBs. This study shed light on the mechanism of action of MGBs and revealed the important structural features for their antitumor and antibacterial activities, which are important to guide future development of MGB derivatives as novel antibacterial and anticancer agents.