Cell Line U937 Research Articles

Investigation of Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG)

Natural products are appealing as anticancer agents because of their low toxicity profiles. Cinnamon is a naturally occurring component with a variety of pharmacological properties, including anti-oxidant, anti-microbial, and anti-cancer properties for cancer cell apoptosis. Therefore, the current studies were performed to investigate Anticancer Properties of Cinnamon Phytochemicals on Protein expression in Glioblastoma Multiforme Cell Lines (U87-MG) since the proteins are directly involved in cell survival and function.Hot and cold extraction for Cinnamon performed from Cinnamon verum followed by phytochemicals tests. The Glioblastoma Multiforme Cell Lines U87 was subjected to cell viability by MTT Assay with Addition of cinnamon extract. The protein content was determined by spectrometric assay and its expression was investigated by SDS PAGE.The aqueous Cinnamon extract found to be positive for Alkaloids, Tannins, Saponins, Flavonoids, Terpenoids (only for Hot extract), Phenols and Reducing sugars (only for Cold extract). The cell viability at cinnamon extract concentration of 0.051 mg/100μl is shown to be highest and cell death at maximum cinnamon extract concentration of 1.65 mg/100μl is highest. Further these phytochemicals together showed less protein content on cultured U-87 cells and displayed lower protein expression in gel band pattern when compared to their control. The Cinnamon extract phytochemicals showed significant anticancer properties for reduced U87 cell viability with IC50 0.729. Thus positive phytochemicals result has shown to play an important role in inhibiting cancer properties.by showing less protein content and its expression for cinnamon phytochemicals treated U87 cell lines.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

Estrogen α and β Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model.

Glioblastoma multiforme (GBM) is a malignant tumor with a higher prevalence in men and a higher survival rate in transmenopausal women. It exhibits distinct areas influenced by changing environmental conditions. This study examines how these areas differ in the levels of estrogen receptors (ERs) which play an important role in the development and progression of many cancers, and whose expression levels are often correlated with patient survival. This study utilized two research models: an in vitro model employing the U87 cell line and a second model involving tumors resected from patients (including tumor core, enhancing tumor region, and peritumoral area). ER expression was assessed at both gene and protein levels, with the results validated using confocal microscopy and immunohistochemistry. Under hypoxic conditions, the U87 line displayed a decrease in ERβ mRNA expression and an increase in ERα mRNA expression. In patient samples, ERβ mRNA expression was lower in the tumor core compared to the enhancing tumor region (only in males when the study group was divided by sex). In addition, ERβ protein expression was lower in the tumor core than in the peritumoral area (only in women when the study group was divided by sex). Immunohistochemical analysis indicated the highest ERβ protein expression in the enhancing tumor area, followed by the peritumoral area, and the lowest in the tumor core. The findings suggest that ER expression may significantly influence the development of GBM, exhibiting variability under the influence of conditions present in different tumor areas.

Urease and human glioblastoma (U87) cells growth inhibitory iridoid glycoside acetates from Nyctanthes arbor-tristis Linn.

The present study was undertaken on the chemical constituents of ethanol extract of aerial parts of Nyctanthes arbor-tristis Linn., and their determination of growth inhibitory activity against glioblastoma multiforme cell line (U87) and urease inhibitory activity. Six constituents were isolated including two new arbortristoside F tetraacetate (1) and arbortristoside G heptaacetate (2) and four known arborside A tetraacetate (3), arborside C pentaacetate (4), 6,7-di-O-acetyl-6β-hydroxyloganin hexaacetate (5) and nyctanthoside heptaacetate (6) iridoid glycoside acetates. Their structures were elucidated using spectroscopic methods, including 1D and 2D NMR and mass analyses. Compounds 3 and 6 showed significant growth inhibition of U87 cell line (76.41 and 87.62% inhibition) respectively while 2, 4 and 5 showed moderate inhibition. 3 and 6 showed notable urease inhibition (IC50 = 17.2 ± 0.4 and 17.2 ± 0.7 µM) respectively, and IC50 of 2, 4 and 5 were 23.8 − 56.3 µM. Compound 1 was inactive.

Has-miR-134-5p inhibits the proliferation and migration of glioma cells by regulating the BDNF/ERK signaling pathway.

Our research investigated the effects of hsa-miR-134-5p on glioma progression, focusing on its interaction with the BDNF/ERK signaling pathway. U251 and U87 cell lines were analyzed post-transfection with hsa-miR-134-5p mimics and inhibitors, confirming the miRNA's binding to BDNF using dual luciferase assays. Q-PCR was employed to measure expression changes, revealing that hsa-miR-134-5p markedly inhibited glioma cell proliferation, migration, and invasion, as evidenced by CCK8, monoclonal formation, and Transwell assays. Scratch tests and Western blotting demonstrated hsa-miR-134-5p's modulation of the BDNF/ERK pathway and associated decrease in MMP2/9 protein levels. Flow cytometry suggested that hsa-miR-134-5p might also block the G0/S phase transition. In vivo studies using nude mice corroborated the tumor-suppressing effects of hsa-miR-134-5p, which were negated by elevated BDNF levels. Comparative protein analysis across groups confirmed the pathway's significance in tumorigenesis. Our findings identify hsa-miR-134-5p as a key molecule impeding glioma cell growth by curtailing the BDNF/ERK pathway, with the reversal by BDNF upregulation pointing to the potential of therapeutically exploiting the hsa-miR-134-5p/BDNF axis in glioma care.

Relevance of Thymic Stromal Lymphopoietin on the Pathogenesis of Glioblastoma: Role of the Neutrophil

Glioblastoma multiforme (GBM) is the most predominant and malignant primary brain tumor in adults. Thymic stromal lymphopoietin (TSLP), a cytokine primarily generated by activated epithelial cells, has recently garnered attention in cancer research. This study was aimed to elucidate the significance of TSLP in GBM cells and its interplay with the immune system, particularly focused on granulocyte neutrophils. Our results demonstrate that the tumor produces TSLP when stimulated with epidermal growth factor (EGF) in both the U251 cell line and the GBM biopsy (GBM-b). The relevance of the TSLP function was evaluated using a 3D spheroid model. Spheroids exhibited increased diameter, volume, and proliferation. In addition, TSLP promoted the generation of satellites surrounding the main spheroids and inhibited apoptosis in U251 treated with temozolomide (TMZ). Additionally, the co-culture of polymorphonuclear (PMN) cells from healthy donors with the U251 cell line in the presence of TSLP showed a reduction in apoptosis and an increase in IL-8 production. TSLP directly inhibited apoptosis in PMN from GBM patients (PMN-p). Interestingly, the vascular endothelial growth factor (VEGF) production was elevated in PMN-p compared with PMN from healthy donors. Under these conditions, TSLP also increased VEGF production, in PMN from healthy donors. Moreover, TSLP upregulated programed death-ligand 1 (PDL-1) expression in PMN cultured with U251. On the other hand, according to our results, the analysis of RNA-seq datasets from Illumina HiSeq 2000 sequencing platform performed with TIMER2.0 webserver demonstrated that the combination of TSLP with neutrophils decreases the survival of the patient. In conclusion, our results position TSLP as a possible new growth factor in GBM and indicate its modulation of the tumor microenvironment, particularly through its interaction with PMN.Graphical Protumoral activity of TSLP. Neutrophils (derived from GBM patients) and GBM cells (under EGF stimulus) not only produce TSLP but also express its receptor. TSLP induces PDL1 expression and decreases apoptosis on both GBM cells and neutrophils. TSLP also increases proliferation and satellite development on GBM cells, whereas favors more neutrophil infiltration by increasing IL8 production.

Folic Acid Functionalized AQ4N/Gd@PDA Nanoplatform with Real-Time Monitoring of Hypoxia Relief and Enhanced Synergistic Chemo/Photothermal Therapy in Glioma.

Hypoxia is often associated with glioma chemoresistance, and alleviating hypoxia is also crucial for improving treatment efficacy. However, although there are already some methods that can improve efficacy by alleviating hypoxia, real-time monitoring that can truly achieve hypoxia relief and efficacy feedback still needs to be explored. AQ4N/Gd@PDA-FA nanoparticles (AGPF NPs) were synthesized using a one-pot method and were characterized. The effects of AGPF NPs on cell viability, cellular uptake, and apoptosis were investigated using the U87 cell line. Moreover, the effectiveness of AGPF NPs in alleviating hypoxia was explored in tumor-bearing mice through photoacoustic imaging. In addition, the diagnosis and treatment effect of AGPF NPs were evaluated by magnetic resonance imaging (MRI) and bioluminescent imaging (BLI) on orthotopic glioma mice respectively. In vitro experiments showed that AGPF NPs had good dispersion, stability, and controlled release. AGPF NPs were internalized by cells through endocytosis, and could significantly reduce the survival rate of U87 cells and increase apoptosis under irradiation. In addition, we monitored blood oxygen saturation at the tumor site in real-time through photoacoustic imaging (PAI), and the results showed that synergistic mild-photothermal therapy/chemotherapy effectively alleviated tumor hypoxia. Finally, in vivo anti-tumor experiments have shown that synergistic therapy can effectively alleviate hypoxia and inhibit the growth of orthotopic gliomas. This work not only provides an effective means for real-time monitoring of the dynamic feedback between tumor hypoxia relief and therapeutic efficacy, but also offers a potential approach for the clinical treatment of gliomas.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening

BackgroundGlioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM.MethodsWe conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells.ResultsWe established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy.ConclusionsOur study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Network pharmacology-based investigation of the effects of Shenqi Fuzheng injection on glioma proliferation and migration via the SRC/PI3K/AKT signaling pathway

Ethnopharmacological relevanceIn the clinic, Shenqi Fuzheng Injection (SFI) is used as an adjuvant for cancer chemotherapy. However, the molecular mechanism is unclear. Aim of the studyWe screened potential targets of SFI action on gliomas by network pharmacology and performed experiments to validate possible molecular mechanisms against gliomas. Materials and methodsWe consulted relevant reports on the SFI and glioma incidence from PubMed and Web of Science and focused on the mechanism through which the SFI inhibits glioma. According to the literature, two primary SFI components—Codonopsis pilosula (Franch.) Nannf. and Astragalus membranaceus (Fisch.) Bunge—have been found. All plant names have been sourced from "The Plant List" (www.theplantlist.org). The cell lines U87, T98G and GL261 were used in this study. The inhibitory effects of SFI on glioma cells U87 and T98G were detected by CCK-8 assay, EdU, plate cloning assay, scratch assay, Transwell assay, immunofluorescence, flow cytometry and Western blot. A subcutaneous tumor model of C57BL/6 mice was constructed using GL261 cells, and the SFI was evaluated by HE staining and immunohistochemistry. The targets of glioma and the SFI were screened using network pharmacology. ResultsA total of 110 targets were enriched, and a total of 26 major active components in the SFI were investigated. There were a total of 3,343 targets for gliomas, of which 79 targets were shared between the SFI and glioma tissues. SFI successfully prevented proliferation and caused cellular S-phase blockage in U87 and T98G cells, thus decreasing their growth. Furthermore, SFI suppressed cell migration by downregulating EMT marker expression. According to the results of the in vivo tests, the SFI dramatically decreased the development of tumors in a transplanted tumour model. Network pharmacological studies revealed that the SRC/PI3K/AKT signaling pathway may be the pathway through which SFI exerts its anti-glioma effects. ConclusionsThe findings revealed that the SRC/PI3K/AKT signaling pathway may be involved in the mechanism through which SFI inhibits the proliferation and migration of glioma cells.

Benzimidazole-Platinum Complex and Its Cytotoxic activity on U87 Cell Lines

Çalışmanın amacı, güçlü sitotoksik aktiviteye ve düşük yan etkilere sahip olabilen yeni bir benzimidazol-platin(II) kompleksinin sentezi, karakterizasyonu ve sitotoksik aktivitelerinin değerlendirilmesidir. Bir benzimidazol ligandı, 2-(3-fenoksifenil)-1-fenil-1H-benzimidazol (L) ve bunun yeni platin kompleksi (Pt-L), sentezlenmiş yapıları farklı spektroskopik yöntemlerle karakterize edilmiştir. Çalışmada, sentezlenen kompleksin öncelikle U87 kanser hattındaki aktivitesi incelenmiş, daha sonra sağlıklı Vero hatları üzerinde çalışılmıştır.

Phytochemical Characterization, Antioxidant, and Anti-Proliferative Activities of Wild and Cultivated Nigella damascena Species Collected in Sicily (Italy).

The use of Nigella damascena seeds in the culinary field or as aerial parts infusions in the pharmaceutical and cosmetic fields is widely reported. The biological activity of this plant, as demonstrated over the years, is closely related to its phytochemical content. This investigation focused on the comparative study of the same plants of N. damascena, one totally wild (WND), while the other two, one with white flowers (CWND) and the other with blue flowers (CBND), were subject to cultivation, irrigation, and manual weeding. Using the potential of 1D and 2D-NMR spectroscopy, coupled with MS/MS spectrometric studies, the three methanolic extracts of N. damascena were investigated. Chemical studies have highlighted the presence of triterpene saponin compounds and various glycosylated flavonoids. Finally, the in vitro antiproliferative and antioxidant activities of the three individual extracts were evaluated. The antiproliferative activity performed on U-937, HL-60, and MCF-7 tumor cell lines highlighted a greater anticancer effect of the CBND and CWND extracts compared to the data obtained using WND. The antioxidant activity, however, performed to quantify ROS generation is comparable among the extracts used.

Investigation of the Effects of PFKFB3 Small Molecule Inhibitor KAN0438757 on Cell Migration and Expression Level of N-cadherin Protein in Glioblastoma Cell Lines

PFKFB3 (6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3) is a enzyme involved in glycolysis, the process by which cells convert glucose into energy. PFKFB3 is known to be overexpressed in many types of cancer, including glioblastoma, a highly aggressive brain tumour. The epithelial-mesenchymal transition (EMT) is a biological mechanism linked to cancer growth and enhanced invasion and metastasis. Inhibition of PFKFB3 in glioblastoma cells is seen as a potential therapeutic strategy to target EMT and inhibit cancer progression. Various small molecule PFKFB3 inhibitors have been created and tested in preclinical trials. The purpose of this study is to look into the possible effect of KAN0438757, a very efficient PFKB3 inhibitor, on glioblastoma cells. KAN0438757's impact on viability of cells, cell migration and cell death in glioblastoma cancer cell lines U373 and U251 were investigated by WST-1 Cell viability, AO/EtBr staining western blotting and wound healing-cell migration assays. Glioblastoma cells showed decreased cell viability and dose-dependent apoptotic morphological changes after KAN0438757 treatment. In addition, it was determined that N-cadherin protein level decreased and cell migration was suppressed. In conclusion, KAN0438757, a PFKFB3 inhibitor, can be considered as a valid approach to target cell death and EMT in glioblastoma cancer cell lines.

Bulk and single cells transcriptomes with experimental validation identify USP18 as a novel glioma prognosis and proliferation indicator.

The mechanism by which ubiquitin-specific protease 18 (USP18) (enzyme commission: 3.4.19.12) inhibition in cancer promotes cell pyroptosis via the induction of interferon (IFN)-stimulated genes has been recently demonstrated. It is also known that USP18 influences the epithelial-mesenchymal transition of glioma cells. In the present study, the upregulation of USP18 in glioma was revealed through bulk transcriptome analysis, which was associated with poor prognosis in patients with glioma. Furthermore, USP18 levels affected the response to immunotherapy in patients with glioma. Single-cell transcriptome and enrichment analyses demonstrated that USP18 was associated with type 1 IFN responses in glioma T cells. To demonstrate the effect of USP18 expression levels on glioma cells, USP18 expression was knocked down in U251 and U87MG ATCC cell lines. A subsequent Cell Counting Kit-8 assay revealed that glioma cell viability was significantly decreased 4 days after USP18 knockdown. In addition, the knockdown of USP18 expression significantly inhibited the clonogenicity of U251 and U87MG ATCC cells. In conclusion, the present study demonstrated that knockdown of USP18 expression inhibited the proliferation of glioma cells, which may be mediated by the effect of USP18 on the IFN-I response.

Cytotoxic potential of selenium nanoparticles (SeNPs) derived from leaf extract of Mentha longifolia L.

The search for alternative methods in cancer treatments has been going on for many years. In the current study conducted for this purpose, selenium nanoparticles (ML-SeNPs) were produced from the aqueous leaf extract of Mentha longifolia L. easily and inexpensively, without harming the environment. The anticancer potential of ML-SeNPs on glioblastoma cell (U373), osteosarcoma cell (U2OS), and healthy retinal pigment epithelial cell (RPE-1) lines was determined by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromid) test. For the test, ML-SeNPs were applied at 100, 300, and 600 µg/mL levels and interaction was provided for 24 and 48 hours. The survival rates (%) in RPE-1, U373, and U2OS cell lines in the 24-hour application were 107.49-98.89, 97.66-86.82, and 87.81-83.37, respectively. The viability rates (%) of the cells in 48 hours of application were 72.27-87.39, 68.17-73.48, and 81.00-84.67, respectively. In general, it was discovered that the cytotoxic effect of ML-SeNPs on RPE-1, U373, and U2OS cell lines was greater at low doses and increased over time. In-vivo studies that support the antiproliferative action of ML-SeNPs may boost the prospect of using them as therapeutic agents in potential cancer treatment procedures in the following years.

Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 μM and 19.92±1 μM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.

Abstract 1996: Novel mechanism of temozolomide resistance in glioblastoma

Abstract Intrinsic expression of PD-1 has been discovered in different types of tumurs, exhibiting either oncogenic or tumor-suppressing function. In glioblastoma (GBM), intrinsic PD-1 has been found to play a role in promoting the proliferation and self-renewal of brain tumor-initiating cells through nuclear factor κB without involving PD-L1 ligation, with unknown factors activating this signalling pathway. Meanwhile, TMZ resistance significantly contributes to treatment failure and an extremely poor prognosis for GBM. This study investigated whether intrinsic PD-1 is also expressed in differentiated human GBM cell lines and its role in GBM proliferation and temozolomide (TMZ) resistance. In vitro studies demonstrated the presence of PD-1 in both U87 and U251 TMZ-sensitive and -resistant cell lines, with a higher expression on TMZ-resistant cell lines. PDCD1 knockdown led to decreased GBM progression, including cell proliferation, colony formation and cell migration, in TMZ-resistant U87 and U251 cell lines using cell viability assays. It also resensitizes these cell lines to TMZ. Overexpression of PDCD1 in the PDCD1-knockdown cell lines increases their proliferation and restores their resistance to TMZ. Treatment of U87 and U251 TMZ-resistant cell lines with TMZ upregulated PD-1 expression. An orthotopic xenograft mouse model is further deployed by injecting previously cultured U87 and U251 cell lines into the mouse brains. A smaller tumour size is observed in the model with PDCD1 knockdown model than the one without PDCD1 knockdown when treated with TMZ. This study reveals that intrinsic PD-1 expression contributes to TMZ resistance in GBM and TMZ may be a factor activating the PD-L1-independent PD-1 signalling. PD-1 and its downstream signalling pathway, such as SHP-2 and IKK, may be potential therapeutic targets for GBM patients with TMZ resistance. Co-administration of TMZ and agents inhibiting the intrinsic PD-1 signalling pathway can potentially produce synergistic therapeutic effects. The higher expression of PD-1 on TMZ-resistant GBM than on TMZ-sensitive GBM also suggests that the intrinsic PD-1 level can be a predictor of TMZ outcome. Citation Format: Yuet Yi Charmaine Hung, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Novel mechanism of temozolomide resistance in glioblastoma [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 1996.

Abstract 3179: Spindle assembly checkpoint as a therapeutic target for TP53-mutated myeloid neoplasms

Abstract Introduction TP53-mutated (TP53mut) myeloid neoplasms (MN) are a distinct entity of myeloid neoplasms with no effective therapies and poor survival (Grob et al, Blood 2022). The TP53-deficient state facilitates the downstream dysfunction of the spindle assembly checkpoint (SAC), predisposing to genomic instability. Recently, a SAC protein, threonine tyrosine kinase (TTK), has emerged as an attractive therapeutic target. Upregulation of TTK has been described in various solid tumors. It is stipulated that targeting SAC proteins might result in extreme level of genomic instability that even cancer cells could not tolerate. We hypothesized that inhibition of TTK with a small molecular inhibitor—TTKi may offer a promising therapeutic strategy for TP53mut MN. Method We evaluated relative TTK mRNA expression and its association with TP53mut and complex karyotypes (CK) in the BEAT AML cohort (Tyner et al., 2018). We used myeloid cell lines, OCI-AML-3, THP-1, and U937 cell lines that harbor-near diploid, moderately aneuploid, and highly aneuploid karyotype, respectively. The efficacy of TTKi CFI-402257 (AbMole) was tested using cell viability, apoptosis, and colony-forming unit (CFU) assays using the standard protocol. Finally, we tested the efficacy of CFI-402257 using the diagnostic bone marrow mononuclear cell (BMMC) cells from TP53mut MN patients. Results Among 591 AML patients in the BEAT AML cohort (41.6% diploid, 35.9% non-CK, 22.7% CK), TTK mRNA expression was progressively higher with increasing chromosomal abnormality burden. TTK expression was 1.96-fold higher in TP53mut AML compared to TP53wt AML (P=0.0008). Further stratified by TP53mut VAF, there was a trend towards higher expression of TTK in TP53mut VAF≥50 compared to the cases with VAF&amp;lt;50 (P=0.06). Next, the treatment with TTKi (CFI-402257) led to a dose-dependent reduction in cell viability across all cell lines, with TP53mut cells (U937 and THP-1) being more sensitive than TP53-wildtype (TP53wt) OCI_AML-3 cells. Similarly, TTKi inhibited the CFU potential of THP-1, U937, and OCI-AML-3 cells. Finally, TTKi induced dose-dependent apoptosis in TP53mut MN patient BMMC but not in healthy donors, resulting in a significant reduction in colony-forming potential. Conclusion Overexpression of TTK was strongly associated with TP53mut and CK in AML. Inhibition of TTK-induced dose-dependent apoptosis in all myeloid cell lines tested with TP53mut highly aneuploid cell line (U937)—traditionally the most chemorefractory phenotype—being the most sensitive. TTKi induced preferential and dose-dependent inhibition of viability and cell proliferation potential in TP53mut MN compared to healthy donor cells, implying a therapeutic window. Further pre-clinical and clinical studies are indicated to evaluate SAC inhibition as a therapeutic strategy for TP53mut MN. Citation Format: Arini Arsana, Huixing Huang, Arief Al-Kali, Mithun Shah. Spindle assembly checkpoint as a therapeutic target for TP53-mutated myeloid neoplasms [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 3179.

Abstract 3883: Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia

Abstract The AAA ATPase p97, also known as valosin-containing protein (VCP), serves to regulate protein homeostasis by facilitating the translocation of ubiquitinated proteins from membranes or chromatin to the proteasome for degradation. Certain cancers, including multiple myeloma (MM) and acute myeloid leukemia (AML) are known for the intracellular overexpression of proteins, and are thus susceptible to the inhibition of p97 and resulting proteotoxic stress. A known p97 inhibitor, CB-5083 entered human phase 1 clinical trials in 2015, but trials were terminated due to side effects that were later traced to strong interaction of the compound with the phosphodiesterase PDE6. To address this problem, we devised a subtractive approach that leveraged high resolution structures of CB-5083 bound to PDE6 and p97. Using this information, we designed a p97 inhibitor that no longer displayed significant off-target binding but retained strong on-target binding. The optimization process started when we identified the 7-azaindole core from an in silico screen as a suitable hit pharmacophore and confirmed the binding mode of several variants of this scaffold in p97 by cryo-EM. Through a structure-guided medicinal chemistry program, we identified GND-135, a potent, on-target binder through the synthesis of less than 100 compounds. The design was supported with about 50 cryo-EM structures throughout the optimization process. Compared to CB-5083, our compound GND-135 displays greatly reduced PDE6 activity (&amp;lt; 1% inhibition at 10 μM versus 89% inhibition for CB-5083) and more potent p97 ATPase inhibition (3 nM IC50 versus 12 nM for CB-5083). We also evaluated the pharmacokinetic profile of GND-135 in mice; the half-life and clearance of 6.6 h and 15.9 mL/min/kg respectively were deemed suitable for daily administration. Based on the initial profiling, we carried out an efficacy study in a cell derived xenograft mouse model of AML using a U937 cell line with subcutaneous implantation. In this model, where GND-135 was administered IP (40 mg/kg QD) and CB-5083 was administered orally (40 mg/kg QD) the compounds showed statistically comparable efficacy, which was differentiated from untreated control. Thus, GND-135 is a novel p97 inhibitor lead compound with on-target selectivity powered by cryo-EM driven rational drug design. Citation Format: Jason Crawford, Ravi Munuganti, Charles Leung, Kriti Singh, Ellen Gates, Xing Zhu, Marcel Bally, Nancy Dos Santos, Maryam Sharifiaghdam, Zeynab Nosrati, Peter Axerio-Cilies, Alison Berezuk, Spencer Cholak, Sriram Subramaniam. Cryo-EM-guided enhancement of target selectivity of a novel p97 inhibitor for treating multiple myeloma and acute myeloid leukemia [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 3883.

Abstract 7207: Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma

Abstract The study aims to investigate whether the Hippo signaling pathway contributes to temozolomide chemoresistance in glioblastoma, and to validate whether the combinational treatment of temozolomide and YAP inhibitor is effective against glioblastoma. Temozolomide-sensitive and temozolomide-resistant U87 and U251 GBM human cell lines were previously established. Temozolomide-resistant cells were maintained in low-dose temozolomide. Here, we investigate whether Hippo signaling contributes to temozolomide chemoresistance. By modulating the Hippo signaling pathway through YAP gene knockdown, the outcome on cell proliferation was studied by cell viability assay (in vitro) and mouse orthotopic xenograft (in vivo). Finally, the therapeutic effect of YAP inhibitor is studied in vitro and in vivo respectively. Our data showed that YAP is overexpressed in temozolomide-resistant glioblastoma cells. Nuclear translocation of YAP (which becomes its active state) is increased in temozolomide-resistant cells. In fact, YAP inhibition show decreased cell viability and higher susceptibility to temozolomide. In addition, mice with tumor injection of the YAP knockdown cells reduced tumor size with temozolomide treatment. Similarly, YAP inhibitor resensitizes temozolomide-resistant cells to temozolomide. Combinational treatment of YAP inhibitor and temozolomide is effective in shrinking tumor size in mouse xenograft models. In conclusion, our data suggest dysregulated Hippo signaling pathway contributes to temozolomide chemoresistance, thus targeting the Hippo signaling pathway resensitizes GBM cells to temozolomide. This research provides pre-clinical evidence on using YAP inhibitor for combinational therapy with temozolomide for the treatment of glioblastoma. Citation Format: Cheuk Lun Ethan Wong, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Targeting hippo signaling pathway to overcome chemoresistance in glioblastoma [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 7207.