Metabolic Drug Survey Highlights Cancer Cell Dependencies and Vulnerabilities

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 26-27
Author(s):  
Tea Pemovska ◽  
Johannes Bigenzahn ◽  
Ismet Srndic ◽  
Alexander Lercher ◽  
Andreas Bergthaler ◽  
...  
Keyword(s):  
Cell Lines ◽  
Drug Testing ◽  
Fatty Acid Synthase ◽  
Structural Information ◽  
Leukemia Cell ◽  
Area Under The Curve ◽  
Mtor Inhibitors ◽  
Nucleotide Metabolism ◽  
Response Curves ◽  
Metabolic Drug

Here we assembled a novel metabolic drug library covering 243 compounds allowing to systematically identify metabolic dependencies in high-throughput phenotypic screens. The CeMM Library of Metabolic Drugs (CLIMET) was compiled in a stepwise fashion, starting from 8000 candidate compounds, after a survey of public drug-target databases, and ending with 243 highly-curated compounds including extensive crosschecking for approval status, structural information, compound's potency and selectivity for the intended target, pathway/target redundancy, and commercial availability. To assess the potency of the compounds in CLIMET, we screened the full collection against a panel of 15 diverse myeloid leukemia cell lines. Each compound was tested for its effect on cell growth and survival in a 10,000-fold concentration range (1nM to 10uM) enabling the generation of dose response curves and calculation of area under the curve values (AUC) for each drug. We, further, functionally grouped the cell lines and drugs based on metabolic drug efficacy patterns and associated them with distinct genomic and metabolic attributes. Analysis of the metabolic drug response profiles revealed that 77 compounds (32%) affected cell viability with the top effective compounds targeting nucleotide metabolism, oxidative stress, and the PI3K/mTOR pathway. Unsupervised hierarchical clustering of the drug sensitivity profiles stratified the cell lines in 5 functional taxonomic groups, with the activity of 19 compounds significantly contributing to the cell line grouping (e.g. PF-02545920, GW 4064, mTOR inhibitors, daporinad). Comparison of the oxygen consumption rate and extracellular acidification rate showed that the examined cell lines have analogous baseline metabolic phenotypes, suggesting that the mitochondrial function of the cells as assessed by Seahorse analysis did not significantly influence the clustering. Genotype to phenotype associations were identified between FLT3mutations and sensitivity to 5-FU, lestaurtinib, and PF-02545920. Moreover, RAS mutations negatively correlated to mTOR and mitochondrial respiration inhibitor sensitivity, whereas TP53 mutations conferred a resistance phenotype to PI3K pathway inhibitors and antineoplastic agents. Selective sensitivities were detected to the lactate transporter (SLC16A1) inhibitor AZD3965, the PI3K inhibitor pictilisib, and the fatty acid synthase inhibitor GSK2194069, which could be explained by varied gene expression in sensitive cell lines and target/process dependency. CLIMET allows for identification of metabolic susceptibilities, grouping of cancer cells based on metabolic dependencies, as well as understanding of context-dependent mechanism of action of drugs. Functional drug testing may provide a rapid and robust approach to identify metabolic vulnerabilities, responding patients, and prioritize compounds for clinical evaluation as illustrated with our study. Disclosures Staber: Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene/ BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; msd: Consultancy, Honoraria.

scholarly journals Metabolic drug survey highlights cancer cell dependencies and vulnerabilities

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tea Pemovska ◽  
Johannes W. Bigenzahn ◽  
Ismet Srndic ◽  
Alexander Lercher ◽  
Andreas Bergthaler ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput Screening ◽  
Drug Response ◽  
Fatty Acid Synthase ◽  
Leukemia Cell ◽  
Cellular Metabolism ◽  
Acid Synthesis ◽  
Metabolic Drug ◽  
Response Profiles ◽  
Synthase Inhibitor

AbstractInterrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.

Dasatinib (BMS-354825), a Multi-Targeted Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane and Activation Loop Mutant Kit Isoforms Associated with Human Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3358-3358
Author(s):  
Marcus M. Schittenhelm ◽  
Sharon Shiraga ◽  
Arin Schroeder ◽  
Amie S. Corbin ◽  
Diana Griffith ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Mtor Inhibitors ◽  
Clinical Activity ◽  
Activation Loop ◽  
Proliferative Effect

Abstract Activating mutations of the activation loop (AL) of KIT are associated with certain human neoplasms, including a subset of patients with AML,systemic mast cell disorders (SM), seminoma, and Gastrointestinal Stromal Tumors (GIST). KIT AL mutations such as D816V that are typically found in AML and SM are resistant to imatinib (IM, IC50 > 5–10 μM). Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC. Due to its potent inhibition of these kinases, dasatinib is currently being tested in clinical trials of patients with imatinib resistant/intolerant CML/Ph+ ALL. Based on previous observations of the ability of certain SRC/ABL inhibitors to also inhibit KIT kinase, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. The inhibitory potential of dasatinib against WT KIT, KIT mutant isoforms and KIT-dependent downstream pathways was evaluated by immunoblotting. In addition, we evaluated the effects of dasatinib on cellular proliferation and induction of apoptosis. Dasatinib potently inhibited WT, juxtamembrane- (JM) and AL-mutant KIT autophosphorylation. Based on the ability of dasatinib to bind to BCR-ABL irrespective of the ATP AL conformation (inactive versus active), dasatinib was expected to be insensitive to KIT AL conformation. In contrast, we found that the IC50 for KIT autophosphorylation varied significantly among the various KIT mutant isoforms: WT KIT, D816Y, V560G (JM mutation) [IC50 1–10 nM] <D816F [IC50 100 nM] <D816V [IC50 200–250 nM]. These results indicate that the conformation of the KIT AL does influence dasatinib potency. Inhibition of KIT kinase activity by dasatinib reduced cellular proliferation and induced apoptosis in mast cell/leukemia cell lines expressing mutant KIT isoforms. In these cell lines, KIT activates downstream pathways important for cell viability and cell survival such as RAS/MAPK, JAK/STAT and PI3K/AKT. Dasatinib potently blocked activation of MAPK1/2 and STAT3. Inhibition of KIT by dasatinib abrogated phosphorylation of AKT S473, but not AKT T308. This partial inhibition of AKT activation was insufficient to inhibit phosphorylation of p70S6K, a kinase downstream of AKT and mTOR. Combining dasatinib with rapamycin, a known mTOR inhibitor, had an additive to synergistic anti-proliferative effect on cells expressing KIT D816V. Our studies suggest that dastatinib may have clinical activity against human neoplasms that are associated with gain-of-function KIT mutations such as AML, SM, seminoma, and GIST. Combining dasatinib with mTOR inhibitors may further increase efficacy against KIT-driven malignancies.

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1067-1074 ◽  
Author(s):  
Marianne G. Rots ◽  
Rob Pieters ◽  
Gert-Jan L. Kaspers ◽  
Christina H. van Zantwijk ◽  
Paul Noordhuis ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Mtt Assay ◽  
Thymidylate Synthase ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Inhibition Assay ◽  
Response Curves ◽  
Methotrexate Resistance

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P = .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P = .001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = −.58, P < .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Intra-Pathway Inhibition of Upstream (PI3K) and Downstream (mTOR) Kinases Synergistically Induces Apoptosis in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2477-2477
Author(s):  
Zhihong Zeng ◽  
Zeev Estrov ◽  
David Harris ◽  
Frank Giles ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Mammalian Target Of Rapamycin ◽  
Leukemia Cell ◽  
Mtor Inhibitors ◽  
Phosphatidylinositol 3 Kinase ◽  
Growth And Survival ◽  
Downstream Target ◽  
Pathway Inhibition ◽  
Induced Apoptosis

Abstract Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway regulates the growth and survival of acute myeloid leukemia (AML). We hypothesized that targeting this pathway with both PI3K and mTOR inhibitors may greatly enhance the effectiveness of these two inhibitors in the treatment of AML. PI3KI1 is a novel PI3K inhibitor that induced apoptosis in AML cell lines and primary AML cells at an IC50 of 5μM. It directly inhibited AKT at Ser473, however had limited effects on pGSK3b and on the mTOR downstream target p70S6K at Thr389. Colony-forming assays demonstrated that PI3KI1 decreased the viability of primary AML samples but spared normal bone marrow progenitor cells. mTOR inhibitor CCI779 inhibited phosphorylation of downstream mTOR targets p70S6K and 4EBP, however showed only minor cytotoxicity to AML cell lines and primary samples, suggesting that inhibition of mTOR signaling is not sufficient to cause growth inhibition in the majority of AML. Combined use of PI3KI1 and CCI779 synergistically induced apoptosis in U937 cells, with a combination index of 0.061±0.02. Western blot analysis demonstrated enhanced suppression of pP70S6K, pAKT and p4EBP1(Thr70) when PI3KI1 and CCI779 were used in combination. In primary AML samples, combined inhibition of PI3K and mTOR pathways enhanced apoptosis induction in 8/12 samples, with true synergistic responses in 3 samples. Importantly, the combination, but not PI3KI1 or CCI779 alone, was able to overcome the growth advantage conferred to AML cell lines or primary AML samples by adherence to bone marrow stromal cells. Taken together, our results indicate that PI3K and mTOR are relevant molecular targets in AML and that intra-pathway inhibition of both, upstream and downstream proteins may be required for maximal inhibition of leukemia cell growth.

Differential Methotrexate Resistance in Childhood T- Versus Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1067-1074 ◽  
Author(s):  
Marianne G. Rots ◽  
Rob Pieters ◽  
Gert-Jan L. Kaspers ◽  
Christina H. van Zantwijk ◽  
Paul Noordhuis ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Mtt Assay ◽  
Thymidylate Synthase ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Inhibition Assay ◽  
Response Curves ◽  
Methotrexate Resistance

Abstract Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P = .46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P = .001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = −.58, P &lt; .001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Antioxidant effects and in vitro cytotoxicity on human cancer cell lines of flavonoid-rich Flamboyant (Delonix regia (Bojer) Raf.) flower extract

2020 ◽  
Vol 21 ◽  
Author(s):  
Putthiporn Khongkaew ◽  
Phanphen Wattanaarsakit ◽  
Konstantinos I. Papadopoulos ◽  
Watcharaphong Chaemsawang
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Flower Extract ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Murine Leukemia Cell ◽  
Dose Dependent

Background: Cancer is a noncommunicable disease with increasing incidence and mortality rates both worldwide and in Thailand. Its apparent lack of effective treatments is posing challenging public health issues. Introduction: Encouraging research results indicating probable anti-cancer properties of the Delonix regia flower extract (DRE) have prompted us to evaluate the feasibility of developing a type of product for future cancer prevention or treatment. Methods and Results: In the present report, using High Performance Liquid Chromatography (HPLC), we demonstrate in the DRE, the presence of high concentrations of three identifiable flavonoids, namely rutin 4.15±0.30 % w/w, isoquercitrin 3.04±0.02 %w/w, and myricetin 2.61±0.01 % w/w respectively while the IC50 of DPPH and ABTS assay antioxidation activity was 66.88±6.30 µg/ml and 53.65±7.24 µg/ml respectively. Discussion: Our cancer cell line studies using the MTT assay demonstrated DREs potent and dose dependent inhibition of murine leukemia cell line (P-388: 35.28±4.07% of cell viability remaining), as well as of human breast adenocarcinoma (MCF-7), human cervical carcinoma (HeLa), human oral cavity carcinoma (KB), and human colon carcinoma (HT-29) cell lines in that order of magnitude. Conclusion: Three identifiable flavonoids (rutin, isoquercitrin and myricetin) with high antioxidation activity and potent and dose dependent inhibition of murine leukemia cell line and five other cancer cell lines were documented in the DRE. The extract’s lack of cytotoxicity in 3 normal cell lines is a rare advantage not usually seen in current antineoplastic agents. Yet another challenge of the DRE was its low dissolution rate and long-term storage stability, issues to be resolved before a future product can be formulated.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

Sign in / Sign up

Export Citation Format

Share Document