Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Inhibition of AMPK activity by venetoclax in AML cells. ( A ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with a dose range of venetoclax during 24 h. Cell viability was then measured using the ATPlite luminescent reagent, and results were analyzed with the nonlinear regression module and plotted using log(inhibitor) versus response (three parameters) function of the Prism software. ( B ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with 100 nM venetoclax during 0 h o 8 h. Then, ATP content was measured using the ATP lite luminescent reagent. Results of ATP content (Y-axis) were plotted on incubation time (X-axis) and a linear regression was performed using the Prism software. Time for achieving a 50% reduction of ATP content is indicated by vertical dashed lines for each cell lines following their respective color code. ( C ) AML cell lines were seeded at 5 × 10 5 cells/mL and incubated with 100 nM venetoclax for the indicated times. Western blots were performed using the anti-phospho-AMPKα T172, -AMPKα, -Bcl-2, and -β-actin antibodies. ( D ) AML cell lines were incubated with vehicle (DMSO) or 100 nM venetoclax (VEN) for 6 h, and Western blots were performed using anti-phospho-ACC S79, -phospho-ULK-1 S555, -phospho-AMPKα T172, -AMPKα, and -β-actin antibodies.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Inhibition, Activity Assay, Incubation, Software, Western Blot

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Decreased amount of AMPK subunits by venetoclax in AML. ( A ) Western blots were done from in vitro kinase assay protein mix using anti-phospho-ACC S79 and anti-phospho-AMPK T172 antibodies. ( B , C ) AML cell lines were incubated without or with 100 nM venetoclax (VEN), and then submitted to a cycloheximide pulse during the indicated times. ( B ) Western blots were done using anti-AMPKα, β, γ, -Bcl-2, and -β-actin antibodies. ( C ) Quantification of the Western blot signals from three independent experiments using ImaJ software for AMPKα detection in the control (CTR) or venetoclax (VEN) conditions.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Western Blot, In Vitro, Kinase Assay, Incubation, Software, Control

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: AMPK degradation is due to on-target caspase activation by venetoclax. ( A ) AML cell lines were incubated with 100 nM venetoclax during the indicated times and processed for flow cytometry using annexin V and DAPI staining. Annexin V-positive and DAPI-negative cells are those in early apoptosis, while double positivity indicates either late apoptosis or necrotic cells. The experiment was repeated three times separately. Vertical bars indicate standard deviations. * p < 0.05, *** p < 0.001. ( B , C ) AML cell lines were incubated with vehicle (CTR), 100 nM venetoclax (VEN), 50 µM Z-VAD (pan-caspase inhibitor) or a combination of 50 µM Z-VAD (added 24 h before VEN) and 100 nM venetoclax for 4 h. ( B ) Flow cytometry measurement of annexin V binding done in three separate experiments and plotted in a heat-map format. ( C ) Western blots done using anti-phospho-AMPK T172, -AMPK α, β, γ, -cleaved caspase 3, and –β-actin.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Activation Assay, Incubation, Flow Cytometry, Staining, Binding Assay, Western Blot

Journal: Cancer Biomarkers

Article Title: Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells

doi: 10.3233/CBM-230454

Figure Lengend Snippet: GLS1 gene expression and activity of venetoclax under hypoxic conditions. (A) SKM-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS with or without hypoxia for 24 h. Gene expressions of GLS1 and GLS2 were evaluated by RT-PCR. ** p< 0.01 was compared to control. ns, not significant. (B) SKM-1 cells were incubated with RPMI 1640 medium with or without glutamine and/or hypoxia condition for 72 h. Cell viability was analyzed by cell counting kit-8. * p< 0.05 was compared to normoxia. ns, not significant. (C) SKM-1 cells were cultured with or without hypoxia for 24 h. 20S proteasome activity was analyzed. ** p< 0.01 was compared to normoxia. (D) MDS and AML cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS with the indicated concentrations of venetoclax for 72 h in normoxia. Cell growth was evaluated using the CellTiter-Glo ™ Luminescent Cell Viability Assay Kit or Cell Counting Kit-8. (E) MDS and AML cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS with the indicated concentrations of venetoclax for 72 h in normoxia. Cell growth was evaluated using the Cytotoxicity LDH Assay Kit. (F) SKM-1 cells were treated with the indicated concentrations of venetoclax for 48 h. Caspase 3/7 activity was analyzed using the Caspase Glo 3/7 Assay Kit. **** p< 0.0001 were compared to normoxia condition. ns, not significant.

Article Snippet: The MDS cell line SKM-1 (monoblastic leukemia following MDS) and the AML cell lines MOLM-14 (acute monocytic leukemia: AML-M5a) and Kasumi-1 (8;21 chromosome translocation) were obtained from the Japan Research Bioresource Cell Bank (Ibaraki, Osaka, Japan).

Techniques: Gene Expression, Activity Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control, Incubation, Cell Counting, Cell Viability Assay, Lactate Dehydrogenase Assay, Caspase-Glo Assay

Journal: Cancer Biomarkers

Article Title: Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells

doi: 10.3233/CBM-230454

Figure Lengend Snippet: Activity of GLS1 inhibitors in MDS and AML cells. (A, B) MDS and AML cell lines were cultured with the indicated concentrations of CB839 or IPN60090 for 72 h under hypoxia. Cell growth was evaluated using the CellTiter-Glo ™ Luminescent Cell Viability Assay Kit or Cell Counting Kit-8. (C, D, E, F) MDS and AML cell lines were cultured with the indicated concentrations of CB838 or IPN60090 for 24 h. The total amount of NADPH or NADP + was analyzed by using NADP/NADPH Assay Kit-WST. (G) SKM-1 cells were incubated with the indicated concentration of CB839 or IPN60090 for 72 h. Intracellular ATP levels were determined using the “Cell” ATP Assay Reagent Ver. 2 Kit. *** p< 0.001, **** p< 0.0001 were compared to control. ns, not significant. (H) SKM-1 cells were incubated with the indicated concentration of CB839 for 24 h. The extracellular OCR and the extracellular acidification were analyzed.

Article Snippet: The MDS cell line SKM-1 (monoblastic leukemia following MDS) and the AML cell lines MOLM-14 (acute monocytic leukemia: AML-M5a) and Kasumi-1 (8;21 chromosome translocation) were obtained from the Japan Research Bioresource Cell Bank (Ibaraki, Osaka, Japan).

Techniques: Activity Assay, Cell Culture, Cell Viability Assay, Cell Counting, Incubation, Concentration Assay, ATP Assay, Control

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a) Heatmap of inhibitors of upstream regulators of S6 activity and the pearson correlation of their area under curve (AUC) with DUSP6 expression in AML cell lines. b) CRISPR dropout screen showing RPS6KA1 as an essential gene in AML. Candidates were identified if meeting criteria of FDR < 10% and whose inhibition affected # of AML lines but neither of non-AML lines. Data retrieved from Tzelepis et al. c) RPS6KA1 expression across 10,071 patient samples representing 31 distinct cancer subtypes from the TCGA Pan-Cancer cohort. Expression values provided as log2 (value +1). See additional information in methods. Boxplots represent min to max ranges with median, 25th, and 75th percentiles. d) Immunoblot analysis of RPS6KA1 knockdown by shRNA or control vector in HEL cells. Immunoblot representative of 5 independent experiments. e) Cell viability assay of HEL cells after RPS6KA1 knockdown relative to control vector. Cells were grown for 96 hours and viability was normalized to the pLKO control vector. n = 6 independently treated cell cultures pooled from two independent experiments per construct. Mean and standard deviation presented. Statistics were assessed by two-tailed Student’s t test. f) Heatmap of altered signaling pathways of lin- CD34 + cells from unique normal bone marrow donors and peripheral blood of MF patients by mass cytometry. Patient samples were treated with 5 μM BI-D1870 for 4 hours, 20 ng/mL TPO for 1 hour, or combination. Signals were normalized to the control treatment of each individual patient sample and reported as 90th percentile Arcsinh ratio. g) Ridge plot of RPS6KA1 expression from CD34 + scRNA-seq of N34, N39, and 381812 at MF and sAML stages. h) RPS6KA1 expression from CD34 + cells from NBM (n = 5), MF (n = 14), and sAML (n = 6) patient samples. RPS6KA1 values represent RMA from microarray. Statistics were assessed by two-tailed Student’s t test. i) Downregulation of HES1 expression from RNA sequencing of HEL cells treated for 4 hours with 1 μM BCI vs DMSO control. n = 2 independently treated cell cultures. j) qRT-PCR of HES1 in HEL cells treated with 1 μM BCI for 24 hours. HES1 mRNA expression normalized to ACTB. n = 3 independent experiments. Statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. k) Immunoblot of HES1 in HEL cells after DUSP6 knockdown by shRNA or control vector. Immunoblot representative of 3 independent experiments. l) qRT-PCR of HES1 in HEL cells after DUSP6 knockdown by shRNA or control vector. HES1 mRNA expression normalized to ACTB for each group and then normalized to pLKO vector. n = 3 independent experiments. Statistics were assessed by two-tailed Student’s t test. Data are presented as mean values + /− s.d. m) Immunoblot of HEL cells after HES1 knockdown by shRNA or control vector. Immunoblot representative of 2 independent experiments. n) Cell viability curve of HEL cells after ectopic expression of RPS6KA1 or GFP control treated with increasing concentrations of BI-D1870. Cells were treated for 96 hours and viability was normalized to the control treatment from each group. n = 6 independently treated cell cultures pooled from two independent experiments per construct. Mean and standard deviation presented. o) Cell viability assay of HEL cells treated with 1 μM BI-D1870, 300 μM BCI or combination, and UKE-1 cells treated with 2 μM BI-D1870, 200 μM BCI, or combination. Cells were treated for 72 hours and viability was normalized to the control treatment. n = 6 independently treated cell cultures pooled from two independent experiments at each drug dose. Mean and standard deviation presented. p) Hallmark gene set enrichment analysis showing top altered pathways from RNA-seq of HEL cells treated for 24 hours with 10 μM BI-D1870 + 1 μM BCI compared to DMSO control (left) and 10 μM BI-D1870 + 1 μM BCI compared to 10 μM BI-D1870 alone (right). q) Dot plot of mass cytometry analysis of lin- CD34 + cells from MF103 treated with 1 μM BCI for 4 hours, 5 μM BI-D1870 for 4 hours, 20 ng/mL TPO for 1 hour, or combination. Signals of key phosphorylated proteins were normalized to the control treatment and reported as 90th percentile Arcsinh ratio.

Article Snippet: Gene expression of DUSP family genes across AML cell lines and RPS6KA1 expression (public 21Q1; https://depmap.org/portal/download/ ) were accessed from the CCLE by the Broad Institute.

Techniques: Functional Assay, Activity Assay, Expressing, CRISPR, Inhibition, Western Blot, Knockdown, shRNA, Control, Plasmid Preparation, Viability Assay, Construct, Standard Deviation, Two Tailed Test, Protein-Protein interactions, Mass Cytometry, Microarray, RNA Sequencing, Quantitative RT-PCR

Journal: Nature cancer

Article Title: DUSP6 mediates resistance to JAK2 inhibition and drives leukemic progression

doi: 10.1038/s43018-022-00486-8

Figure Lengend Snippet: a, Relative RPS6KA1 expression across 804 pan-cancer cell lines from the CCLE (Source data). Box plots represent minimum to maximum ranges with the median and 25th and 75th percentiles. b, Gene dependency score for RPS6KA1 CRISPR knockout across 804 pan-cancer cell lines from DepMap (Source data). Box plots represent minimum to maximum ranges with the median and 25th and 75th percentiles. KO, knockout. c, Kaplan–Meier event-free survival curve of patients stratified by RPS6KA1 expression from the TCGA LAML cohort (n = 173). d, Kaplan–Meier overall survival curve of patients from c. e, HEL and UKE-1 cells were treated with increasing concentrations of BI-D1870 for 72 h, and viability was normalized to the control treatment. n = 6 independently treated cell cultures at each dose. Mean and s.d. are presented. f, Immunoblot of HEL and UKE-1 cells treated with BI-D1870 for 24 h. The immunoblot is representative of at least three experiments. g, Cell cycle assay of HEL cells treated with 5 μM BI-D1870 (BI-D) or control for 12 h and 24 h. Cells were treated in triplicate (n = 3 independent experiments). Data are presented as mean values ± s.d. h, Annexin V apoptosis assay of HEL cells treated with 5 μM BI-D1870 for 48 h and 72 h (n = 3 independent experiments). Data are presented as mean values ± s.d. i, GSEA showing top altered pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling and metabolism gene set from RNA-seq of HEL cells treated with 10 μM BI-D1870 or control for 24 h. n = 2 independently treated cell cultures. ECM, extracellular matrix; NK, natural killer. j, Top 50 DEGs from the KEGG PI3K–AKT signaling pathway of RNA-seq from HEL cells treated with 10 μM BI-D1870 for 24 h versus control treatment. k, Dot plots of lin−CD34+ cells from one unique NBM donor and PB of two patients with MF by CyTOF. Samples were treated with 5 μM BI-D1870 for 4 h, 20 ng ml−1 TPO for 1 h or the combination. Signals were normalized to the control treatment of each individual sample and reported as a 90th-percentile arcsinh ratio. l, Colony assays of lin−CD34+ cells from one unique NBM donor, two patients with MF and one patient with sAML. Cells were grown for 12 d in 1.5 μM BI-D1870 or an RPMI control and plated in duplicate. m, Pearson correlation of DUSP6 and RPS6KA1 expression from lin−CD34+ cells from MF (n = 14) and sAML (n = 6) patient samples. Expression values represent RMA from the microarray. n, Immunoblot of cells treated with 1 μM BCI for 24 h. The immunoblot is representative of at least three experiments. o, Immunoblot after knockdown with two shDUSP6 species or a non-targeting pLKO control. The immunoblot is representative of at least three experiments.

Article Snippet: Gene expression of DUSP family genes across AML cell lines and RPS6KA1 expression (public 21Q1; https://depmap.org/portal/download/ ) were accessed from the CCLE by the Broad Institute.

Techniques: Expressing, CRISPR, Knock-Out, Control, Western Blot, Cell Cycle Assay, Apoptosis Assay, RNA Sequencing, Microarray, Knockdown