Methods for High-Throughput Drug Combination Screening and Synergy Scoring

Background: Osteosarcoma (OS) is a malignant bone tumor that often develops during the period of rapid growth associated with adolescence. Despite successful primary tumor control accompanied by adjuvant chemotherapy, death from pulmonary metastases occurs in approximately 30% of patients within 5 years. As overall survival in patients remains unchanged over the last 30 years, urgent needs for novel therapeutic strategies exist. Cancer metastasis is characterized by complex molecular events which result from alterations in gene and protein expression/function. Recent studies suggest that metabolic adaptations, or "metabolic reprogramming," may similarly contribute to cancer metastasis. The goal of this study was to specifically interrogate the metabolic vulnerabilities of highly metastatic OS cell lines in a series of in vitro and in vivo experiments, in order to identify a tractable metabolically targeted therapeutic strategy for patients. Methods: Nutrient deprivation and drug treatment experiments were performed in MG63.3, 143B, and K7M2 OS cell lines to identify the impact of glutaminase-1 (GLS1) inhibition and metformin treatment on cell proliferation. We functionally validated the impact of drug treatment with extracellular flux analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. 13 C-glucose and 13 C-glutamine tracing was employed to identify specific contributions of these nutrients to the global metabolic profiles generated with GLS1 inhibition and metformin treatment in vivo. Results: Highly metastatic OS cell lines require glutamine for proliferation, and exposure to CB-839, in combination with metformin, induces both primary tumor growth inhibition and a distinct reduction in metastatic outgrowth in vivo. Further, combination-treated OS cells showed a reduction in cellular mitochondrial respiration, while NMR confirmed the pharmacodynamic effects of glutaminase inhibition in tumor tissues. We observed global decreases in glycolysis and tricarboxylic acid (TCA) cycle functionality, alongside an increase in fatty acid oxidation and pyrimidine catabolism. Conclusions: This data suggests combination-treated cells cannot compensate for metformin-induced electron transport chain inhibition by upregulating glutaminolysis to generate TCA cycle intermediates required for cell proliferation, translating into significant reductions in tumor growth and metastatic progression. This therapeutic approach could be considered for future clinical development for OS patients presenting with or at high risk of developing metastasis.

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“…Twenty-five combinations of CB-839 and metformin were tested. The data analysis was performed employing the method previously described in [27].…”

Section: Synergy Combination Index (Ci) Assaymentioning

confidence: 99%

Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma

et al. 2020

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“…Multi-dimensional two drug synergy studies were also completed with the screened inhibitors. The drug combination effect was assessed using the Synergyfinder package [22] in R Studio to determine the Bliss independence model score [23]. In quantifying the response to drug interactions, the Bliss independence model works on the assumption that the combined drugs are acting independently (mutually non-exclusive) of one another.…”

Section: Cell Proliferation Assaysmentioning

confidence: 99%

Olaparib Combined with an ATR or Chk1 Inhibitor as a Treatment Strategy for Acquired Olaparib-Resistant BRCA1 Mutant Ovarian Cells

et al. 2020

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Objective: Despite the promise of PARP inhibitors (PARPi) for treating BRCA1/2 mutated ovarian cancer (OC), drug resistance invariably develops. We hypothesized rationale drug combinations, targeting key molecules in DNA repair pathways and the cell cycle may be synergistic and overcome acquired PARPi resistance. Methods: Drug sensitivity to PARPi alone and in combination with inhibitors of key DNA repair and cell cycle proteins, including ATR (VE-821), Chk1 (MK-8776), Wee1 (MK-1775), RAD51 (RI-1) was assessed in PARPi-sensitive (UWB1) and -resistant (UWB1-R) gBRCA1 mutant OC cell lines using a cell proliferation assay. The Bliss synergy model was used to estimate the two-drug combination effect and pharmacologic synergy (Bliss score ≥ 0) or antagonistic (Bliss score ≥ 0) response of the PARPi in combination with the inhibitors. Results: IC50 for olaparib alone was 1.6 ± 0.9 µM compared to 3.4 ± 0.6 µM (p = 0.05) for UWB1 and UWB1-R cells, respectively. UWB1-R demonstrated increased sensitivity to ATRi (p = 0.04) compared to UWB1. Olaparib (0.3–1.25 µM) and ATRi (0.8–2.5 µM) were synergistic with Bliss scores of 17.2 ± 0.2, 11.9 ± 0.6 for UWB1 and UWB1-R cells, respectively. Olaparib (0.3–1.25 µM) and Chk1i(0.05–1.25 µM) were synergistic with Bliss scores of 8.3 ± 1.6, 5.7 ± 2.9 for UWB1 and UWB1-R cells, respectively. Conclusions: Combining an ATRi or Chk1i with olaparib is synergistic in both PARPi-sensitive and -resistant BRCA1 mutated OC cell models, and are rationale combinations for further clinical development.

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“…After 72 hours of incubation for TP53-KO hESCs and 96 hours of incubation for TP53-KO RKO cells, cells were stained with 35 mmol/L resazurin (Sigma), then quantification of fluorescent signal intensity was performed on Thermo Fluorskan Ascent plate reader at excitation and emission wavelengths of 544/590 nm. The drug-synergy score was evaluated using the ZIP model (Zero Interaction Potency model) in the synergyfinder package (27).…”

Section: Drug-synergy Experiments and Analysismentioning

confidence: 99%

Spindle Assembly Checkpoint Inhibition Can Resensitize p53-Null Stem Cells to Cancer Chemotherapy

2019

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TP53 mutations are common in most human cancers, but few therapeutic options for TP53-mutant tumors exist. To identify potential therapeutic options for cancer patients with TP53 mutations, we profiled 127 FDAapproved chemotherapy drugs against human embryonic stem cells (hESC) in which we engineered TP53 deletion by genome editing. We identified 27 cancer therapeutic drugs for which TP53 mutations conferred resistance; most of these drugs target DNA synthesis or topoisomerase and cause DNA damage. We then performed a genome-wide CRISPR/Cas9 knockout screen in the TP53-null hESC in the presence and absence of sublethal concentrations of cisplatin and identified 137 genes whose loss selectively resensitized the p53-null cells to this chemotherapeutic agent. Gene ontology classification of the resensitizing loci revealed significant overrepresentation of spindle checkpoint pathway genes. Moreover, we confirmed that targeting ZNF207/BuGZ sensitizes p53-null hESC to cisplatin. These data indicate that targeted inhibition of spindle assembly checkpoints (SAC) and chromosomal organizing centers may provide a way to treat p53-deficient cancer cells with standard chemotherapy drugs. Development of small-molecule inhibitors of SAC proteins may be a useful strategy for rescuing DNA-damaging chemotherapeutics in TP53mutant cancers. Significance: These findings show that inhibition of spindle assembly checkpoints and chromosomal organizing centers may provide a new way to treat p53-deficient cancer cells with standard chemotherapy drugs.

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Methods for High-Throughput Drug Combination Screening and Synergy Scoring

Cited by 44 publications

References 19 publications

Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma

Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma

Olaparib Combined with an ATR or Chk1 Inhibitor as a Treatment Strategy for Acquired Olaparib-Resistant BRCA1 Mutant Ovarian Cells

Spindle Assembly Checkpoint Inhibition Can Resensitize p53-Null Stem Cells to Cancer Chemotherapy

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