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Principal Investigator: Naris Nilubol
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2024
Award: $802,871
Funding agency: National Cancer Institute
Clinical trial development of MELK and CDK1-2 inhibitor is pending as we need to find a substitute CDK1/2 inhibitor because Merck did not want to provide their inhibitor. OncoTherapy Science has agreed to provide OTS167. We currently have three manuscripts in preparation. We are also working on the novel synergistic combination of PI3K and HSP90 inhibitors in ACC, and our manuscript is almost ready for submission. Our aim is to identify and validate the effective novel synergistic drug combinations using PI3K inhibitor and Heat-shock protein 90 inhibitors and to study the molecular mechanisms of synergy identified by the computerized drug combination matrix screening in ACC cells. We analyzed publicly available databases and found that ACC overexpressed several isoforms of HSP90 and its clients involved in ACC initiation and progression. We validated the synergy in two ACC cell lines in a cell proliferation assay. ACC 3D tumor spheroid assays showed increased efficacy in the combination treatment group Because PIK75 is not yet available for a clinical trial, we identified and validated the synergy between BGT226 and STA9090 or with HSP990 in vitro, clonogenic assays, and invasion/migration assays with corresponding decreased epithelial-to-mesenchymal markers than single-drug groups at clinically achievable concentrations. We discovered that the synergistic mechanisms of cell death were completely different between PIK75 and BGT226. PIK75 combination induced G2M cell cycle arrest, followed by caspase-3/7 dependent apoptosis. We validated the treatment effects that decreased p-AKT, mTOR, and 4EP1 which was more pronounced in the combination treatments. However, the combination of BGT226 and STA9090 did not cause cell cycle arrest nor it induced apoptosis. RNA-seq data of ACC cells treated with these combinations showed BGT226 combination induced autophagy. We successfully validated the efficacy of BGT26 and STA9090 in patient-derived organoids (n=5). The in vivo efficacy in ACC xenografts confirmed that the tumor burden of mice treated with BGT226 and STA9090 combination was significantly lower than monotherapy groups. we are also working on evaluating the effects and mechanism of action of auranofin in combination with radiotherapy (RT) in ACC. Most patients with ACC develop locoregional recurrence after surgery. After identifying auranofin from a quantitative high-throughput drug screening in two ACC cell lines as a novel radiosensitizer with a potent cytotoxic effect. The analyses of the independent databases of ACC samples showed the mRNAs of several genes involved in oxidative stress response including the TXNRD pathway, targetable by auranofin, are differentially expressed in ACC.TXNRD is the pro-survival reducing enzyme of TXN. In addition, patients with low TXNRD1 mRNA expression in ACC had shorter overall survival and disease-free survival (OS and DFS). We showed downregulation of several anti-oxidative stress-related genes was inversely correlated with overexpression of MKi67 and CCNB1 (poor prognostic markers). We confirmed the synergistic activity of auranofin and RT in monolayer culture. The combination treatment of auranofin and RT was more effective than single treatments in 3D ACC spheroids and clonogenic assays in two cell lines at clinically achievable concentrations. We completed the 3rd in vivo study after optimizing the dosing scheme of RT and auranofin and found that the tumor burden in mice treated with RT and auranofin was significantly lower than that of monotherapy. We confirmed that the effective concentrations inhibited thioredoxin reductase activity in ACC. We completed the mechanistic studies and confirmed that auranofin increased oxidative stress in depleting GSH which caused ferroptosis as demonstrated by reduced GPX4. However, we did not observe an increased lipid peroxidation, a hallmark of ferroptosis, when NCI-H295R cells were treated with auranofin and RT but we confirmed that the combination treatment induced lipid peroxidation in SW-13. We also found that RT depleted more GSH in NCI-295R than SW13, consistent with a higher sensitivity to RT in NCI-H295R. The combination treatments induced a higher level of gH2AX, activated p-CHK1, and induction of p-BRCA1 in both cell lines consistent with double-strand DNA break and repair, respectively. We observed that NCI-H295R (cortisol-producing cells with activating CTNNB1 mutation) was more sensitive to RT and auranofin than SW-13 (SMARCA4 mutations, non-steroid producing cell line). While we observed cleaved-PARP in NCI-H295R treated with the combination, we did not observe cleaved-caspase or cleaved PARP in SW13 treated with auranofin and RT. Auranofin and RT induced p21 and p27 in SW-13 cells, but not NCI-H295R. To further explore the mechanism of action in both cell lines, we found that SW-13 effectively overexpressed NRF2 and KEAP1 nuclear translocation as well as TXNRD1 and HMOX1 as compared to that of NCI-H295R, suggesting SW-13 had a more robust anti-oxidative stress response. In addition, the oxidative stress from auranofin and RT treatments effectively induced mitotic catastrophe and our results showed that auranofin prolonged the duration of mitotic catastrophe causing more cell death in combination treatments. We excluded the role of corticosteroids in radiosensitivity in ACC using various treatments. Another work in the lab is our Multi-Omic approach to identify genes and pathways involved in radioresistance in ACC. We completed bulk RNA-sequencing, total and phosphoproteomics studies of NCI-H295R receiving 2-week fractionated RT with a dosing scheme similar to patients to understand the genes and pathways. Selected candidate genes were selected to create a custom, focused CRISPR-KO library for 3D ACC tumor spheroids. Further validation of these candidate gens is in-progress. CAS-9 Stable ACC cells were created pending CAS-9 activity validation. We also aim to elucidate the mechanism of action of TNF-a in reducing tumor interstitial pressure leading to improved drug delivery efficiency. We found that TGF-b signaling played a central role in TNF-a-related improved drug delivery by downregulating LOX, decreasing hyaluronan (HA) synthesis, and increasing HA degradation. We confirmed the role of TGF-b and LOX in regulating vascular permeability via VEGF-R and VEGF-A secretion. To confirm the efficacy of the treatments using recombinant TNF-a and TGFb inhibition, we combined the TME treatments with paclitaxel in two iodine-resistant poorly differentiated (PDTC) (TPC1) and anaplastic thyroid cancer (ATC) (8505C) 3D tumor spheroids and found that the anti-tumor efficacy was higher in the combination treatment groups than single-treatment groups. Furthermore, we demonstrated the higher intratumoral penetration of fluorescent-labeled paclitaxel when thyroid cancer spheroids were treated with TGFb inhibitors and LOX inhibition (siLOX and BAPN which inhibits LOX enzymatic activity) and the intratumoral paclitaxel concentration was severalfold higher than the paclitaxel-only group. We validated the enhanced intratumoral paclitaxel delivery in vivo using HPLC by treating 8505C xenografts with nanogold particles carrying TNF-a and the TGFb inhibitor. Similar improvements in drug delivery efficiency and treatment efficacy can be seen in much denser co-cultured thyroid cancer spheroids with cancer-associated fibroblasts.
Terms: <(TNF)-α><1-Phosphatidylinositol 3-Kinase><3-D><3-Dimensional><3D><AKT><Adherent Culture><Adrenal Cortex Carcinoma><Adrenal Cortex Hormones><Adrenal Cortical Carcinoma><Adrenocortical Carcinoma><Aeroseb-HC><Agreement><Akt protein><Anzatax><Apopain><Apoptosis><Apoptosis Pathway><Apoptosis-Related Cysteine Protease Caspase 3><Asotax><Assay><Auranofin><Autophagocytosis><BRCA1><BRCA1 Gene Product><BRCA1 Protein><BRCA1 gene><BRG-1><BRG-1 Gene><BRG1><BRG1 Gene><BRM/SWI2-Related Gene-1><Bioassay><Biological Assay><Bone-Derived Transforming Growth Factor><Breast Cancer 1 Gene><Breast Cancer 1 Gene Product><Breast Cancer Type 1 Susceptibility Gene><Breast Cancer Type 1 Susceptibility Protein><Breast-Ovarian Cancer Protein><Bristaxol><CASP-3><CASP3><CASP3 gene><CCNB><CCNB1><CCNB1 gene><CDC2><CDC2 Protein Kinase><CDC2 gene><CDK1><CGB1><CHEK1><CHEK1 gene><CHK1><CPP-32><CPP32><CPP32 protein><CPP32B><CPP32beta><CRISPR><CRISPR/Cas system><CTNNB><CTNNB1><CTNNB1 gene><Cachectin><Cancer Patient><Cancers><Candidate Disease Gene><Candidate Gene><Caspase><Caspase Gene><Cell Body><Cell Communication and Signaling><Cell Cycle Arrest><Cell Cycle Controller CDC2 Gene><Cell Cycle Controller cdc2><Cell Death><Cell Division Control Protein 2 Homolog><Cell Division Cycle 2><Cell Division Cycle 2 Protein><Cell Growth in Number><Cell Line><Cell Migration Assay><Cell Multiplication><Cell Proliferation><Cell Signaling><Cell-Cycle Checkpoint Kinase><Cell-Death Protease><CellLine><Cells><Cellular Proliferation><Cetacort><Checkpoint kinase 1><Client><Clinical><Clinical Trials><Clustered Regularly Interspaced Short Palindromic Repeats><Co-culture><Cocultivation><Coculture><Coculture Techniques><Combined Modality Therapy><Cort-Dome><Cortef><Cortenema><Corticoids><Corticosteroids><Cortisol><Cortispray><Cortril><Custom><Cyclin B1 Gene><Cyclin-Dependent Kinase 1><Cysteine Endopeptidases><Cysteine Protease><Cysteine Protease CPP32><Cysteine Protease CPP32 Gene><Cysteine Proteinases><DNA Double Strand Break><Data><Data Bases><Databases><Dermacort><Development><Disease-Free Survival><Dose><Down-Regulation><Drug Combinations><Drug Delivery><Drug Delivery Systems><Drugs><Early Onset Gene Breast Cancer 1><Early Onset Protein Breast Cancer 1><Eldecort><Endocrine Cancer><Endocrine Gland Cancer><Enzyme Gene><Enzymes><Epithelium><Event-Free Survival><Exclusion><FK506 Binding Protein 12-Rapamycin Associated Protein 1><FKBP12 Rapamycin Complex Associated Protein 1><FRAP1><FRAP1 gene><FRAP2><Family><Fibroblasts><Fractionated radiotherapy><Genes><Genetic Alteration><Genetic Change><Genetic defect><Goals><HPLC><HSP-90><HSP90><Heat-Shock Proteins 90><Hereditary Breast Cancer 1><Heterograft><Heterologous Transplantation><High Performance Liquid Chromatography><High Pressure Liquid Chromatography><High Speed Liquid Chromatography><Hyaluronan><Hydrocortisone><Hydrocortone><Hytone><ICE-like protease><In Vitro><Induction of Apoptosis><Intracellular Communication and Signaling><Invaded><Iodine><Isoforms><LOX><LOX gene><Label><Libraries><Lipid Peroxidation><Macrophage-Derived TNF><Malignant Endocrine Neoplasm><Malignant Endocrine Tumor><Malignant Neoplasms><Malignant Thyroid Gland Neoplasm><Malignant Tumor><Malignant Tumor of the Thyroid><Malignant Tumor of the Thyroid Gland><Malignant neoplasm of thyroid><Manuscripts><Mechanistic Target of Rapamycin><Medication><Mesenchymal><Messenger RNA><Mice><Mice Mammals><Migration Assay><Milk Growth Factor><Mitotic><Molecular><Monocyte-Derived TNF><Monolayer culture><Multimodal Therapy><Multimodal Treatment><Murine><Mus><Mutation><Nuclear Translocation><Nutracort><Operative Procedures><Operative Surgical Procedures><Organoids><Oxidative Stress><PARP Cleavage Protease><PARP Cleavage Protease Gene><PI-3 Kinase><PI3-Kinase><PI3CG><PI3KGamma><PI3k><PIK3><PIK3CG><PIK3CG gene><Paclitaxel><Paclitaxel (Taxol)><Pathway interactions><Patients><Penetration><Pharmaceutical Preparations><Phosphatidylinositol 3-Kinase><Phosphatidylinositol-3-OH Kinase><Phosphoinositide 3-Hydroxykinase><Platelet Transforming Growth Factor><Play><Praxel><Preparation><Proctocort><Prognostic Marker><Programmed Cell Death><Protein Isoforms><Protein Kinase B><Proto-Oncogene Proteins c-akt><PtdIns 3-Kinase><QOL><Quality of life><RAC-PK protein><RAFT1><RNA Seq><RNA sequencing><RNAseq><RNF53><Radiation Sensitivity><Radiation Sensitizers><Radiation Tolerance><Radiation therapy><Radiation-Sensitizing Agents><Radiation-Sensitizing Drugs><Radioresistance><Radiosensitivity><Radiosensitizing Agents><Radiosensitizing Drugs><Radiotherapeutics><Radiotherapy><Radiotherapy sensitizer><Recombinants><Recurrence><Recurrent><Resistance><Role><SCA-1><SCA-1 Gene><SMARCA4><SMARCA4 gene><SNF2-Beta><SREBP Cleavage Activity 1><SREBP Cleavage Activity 1 Gene><SWI/SNF-Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily A, Member 4 Gene><Sampling><Scheme><Science><Signal Transduction><Signal Transduction Systems><Signaling><Strains Cell Lines><Strategic Planning><Surgical><Surgical Interventions><Surgical Procedure><Survivors><TGF B><TGF-beta><TGF-β><TGFbeta><TGFβ><TNF><TNF A><TNF Alpha><TNF gene><TNF-α><TNFA><TNFα><Taxol><Taxol A><Taxol Konzentrat><Thyroid Cancer><Transforming Growth Factor beta><Transforming Growth Factor-Beta Family Gene><Treatment Efficacy><Tumor Burden><Tumor Load><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><Type I Phosphatidylinositol Kinase><Type III Phosphoinositide 3-Kinase><VEGF><VEGFs><Validation><Vascular Endothelial Growth Factors><Vascular Permeabilities><Work><Xenograft><Xenograft procedure><Xenotransplantation><Yama><Yama protein><anaplastic thyroid cancer><anaplastic thyroid carcinoma><autophagy><biological adaptation to stress><biological signal transduction><brca 1 gene><c-akt protein><cancer care><candidate selection><candidate validation><caspase-3><cdc2 gene product><cdc2+ Protein><cdk1 Kinase><cell-cycle check point kinase><check point kinase 1><chk1 kinase><chk1 protein kinase><combination therapy><combined modality treatment><combined treatment><computerized><cultured cell line><customs><cystein protease><cystein proteinase><cysteine endopeptidase><cysteine protease P32><cytotoxic><data base><developmental><differential expression><differentially expressed><drug/agent><efficacy validation><genome mutation><gold nano particle><gold nanoparticle><high-throughput drug screening><hsp90 Family><improved><in vivo><inhibitor><interstitial><intervention efficacy><mRNA><mRNA Expression><mTOR><malignancy><malignant endocrine gland neoplasm><malignant endocrine gland tumor><mammalian target of rapamycin><molecular targeted therapeutics><molecular targeted therapies><molecular targeted treatment><multi-modal therapy><multi-modal treatment><multiomics><multiple omics><nano gold><nanoGold><necrocytosis><neoplasm/cancer><new anti-cancer agent><new anticancer agent><new anticancer drug><new antineoplastic><new cancer drug><novel><novel anti-cancer agent><novel anti-cancer drug><novel anticancer agent><novel anticancer drug><novel antineoplastic><novel cancer drug><overexpress><overexpression><p34 Protein Kinase><p34 Protein Kinase Gene><p34(CDC2) Gene><p34CDC2><panomics><pathway><phospho-proteomics><phosphoproteomics><preparations><pressure><prognostic biomarker><proto-oncogene protein RAC><proto-oncogene protein akt><public data base><public database><publicly accessible data base><publicly accessible database><publicly available data base><publicly available database><rac protein kinase><radiation resistance><radiation treatment><radio resistance><radio-sensitivity><radiosensitive><radiosensitizer><reaction; crisis><related to A and C-protein><repair><repaired><resistant><screening><screenings><social role><spheroids><stress response><stress; reaction><surgery><synergism><therapeutic efficacy><therapeutic target><therapy efficacy><thioredoxin reductase><three dimensional><transcriptional differences><transcriptome sequencing><transcriptomic sequencing><treatment effect><treatment group><treatment with radiation><tumor><validate efficacy><validations><xeno-transplant><xeno-transplantation>