Journal: The Journal of Clinical Investigation
Article Title: BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment
doi: 10.1172/JCI120654
Figure Lengend Snippet: (A) OCI-AML3 and primary AML cells were treated with ARV-825 (10 nM) for 24 hours. GEP analysis was performed on the isolated RNA (n = 3). (A) GSEA was performed on Illumina GEP data and revealed high enrichment (normalized enrichment score [NES] >3) for several gene sets representing downregulation of Myc target genes and Wnt/B catenin pathways, as well as oncogenic pathways and gene sets for cell cycle, hypoxia, metabolism, and Notch). Validation of Wnt/β-catenin pathway downregulation was done by qPCR analysis of the pathway targets AXIN-2 and FRA-1 at 12 and 24 hours (n = 3 independent samples). Statistical significance was calculated using Bonferroni’s method, and adjusted P values were determined. NMSCs were treated with ARV-825 (25 nM) for 24 hours. (B) Representative GSEA found high enrichment for several gene sets representing Myc target genes and oncogenic pathways, as well as cell-cycle, metabolic, hypoxia, oxidative phosphorylation, and Notch gene sets, and GEP of MSCs showed a significant reduction in surface adhesion and SDF-1 expression. w.r.t., with respect to. (C) MSCs treated with DMSO or ARV-825 (25 nM for 24 h) cocultured with OCI-AML3 cells treated with DMSO or ARV-825 (10 nM) for 24 hours and subjected to either determination of ROS using an ENZ-51011 kit (bottom) or whole-cell lysates from AML cells were subjected to immunoblotting with specific antibodies. Tubulin was used as a loading control. (D) GSE76009 data were curated, and GSEA revealed high enrichment (NES >3) of LSC fractions for several gene sets representing hematopoietic stems cells or LSCs (data not shown) and for this gene set of a subgroup of genes regulated by Myc. Illumina GEP data on primary AML cells treated with ARV-825 for 24 hours showed a significant reduction in the number of genes of the 17-gene signature associated with stemness in AML. (E) BM cells were collected from vehicle- and ARV-825–treated mice with AML-PDX and subjected to CyTOF, and data were analyzed using SPADE 3.0. The tree was generated according to the expression of CD34, CD38, CD90, and CD45RA (top). Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34+CD38–CD90–CD45RA+) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom). UP, upregulated; DN, downregulated.
Article Snippet: Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34 + CD38 – CD90 – CD45RA + ) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom).
Techniques: Isolation, Biomarker Discovery, Phospho-proteomics, Expressing, Western Blot, Control, Generated, Activity Assay, Inhibition
![(A) OCI-AML3 and primary AML cells were treated with ARV-825 (10 nM) for 24 hours. GEP analysis was performed on the isolated RNA (n = 3). (A) GSEA was performed on Illumina GEP data and revealed high enrichment (normalized enrichment score [NES] >3) for several gene sets representing downregulation of Myc target genes and Wnt/B catenin pathways, as well as oncogenic pathways and gene sets for cell cycle, hypoxia, metabolism, and Notch). Validation of Wnt/β-catenin pathway downregulation was done by qPCR analysis of the pathway targets AXIN-2 and FRA-1 at 12 and 24 hours (n = 3 independent samples). Statistical significance was calculated using Bonferroni’s method, and adjusted P values were determined. NMSCs were treated with ARV-825 (25 nM) for 24 hours. (B) Representative GSEA found high enrichment for several gene sets representing Myc target genes and oncogenic pathways, as well as cell-cycle, metabolic, hypoxia, oxidative phosphorylation, and Notch gene sets, and GEP of MSCs showed a significant reduction in surface adhesion and SDF-1 expression. w.r.t., with respect to. (C) MSCs treated with DMSO or ARV-825 (25 nM for 24 h) cocultured with OCI-AML3 cells treated with DMSO or ARV-825 (10 nM) for 24 hours and subjected to either determination of ROS using an ENZ-51011 kit (bottom) or whole-cell lysates from AML cells were subjected to immunoblotting with specific antibodies. Tubulin was used as a loading control. (D) GSE76009 data were curated, and GSEA revealed high enrichment (NES >3) of LSC fractions for several gene sets representing hematopoietic stems cells or LSCs (data not shown) and for this gene set of a subgroup of genes regulated by Myc. Illumina GEP data on primary AML cells treated with ARV-825 for 24 hours showed a significant reduction in the number of genes of the 17-gene signature associated with stemness in AML. (E) BM cells were collected from vehicle- and ARV-825–treated mice with AML-PDX and subjected to CyTOF, and data were analyzed using SPADE 3.0. The tree was generated according to the expression of CD34, CD38, CD90, and CD45RA (top). Myc activity, Wnt/β-catenin/Notch, cell-cycle/apoptosis, and PI3K/AKT/mTOR pathways, and target inhibition–, and tumor microenvironment–related protein expression levels in BM cells from vehicle- and ARV-825–treated mice were determined and quantified in LSCs (CD34+CD38–CD90–CD45RA+) as cluster 1 and depicted as a heatmap generated with GraphPad Prism 7 on the basis of the percentiles of intensities with respect to the vehicle (bottom). UP, upregulated; DN, downregulated.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6356/pmc06486356/pmc06486356__jci-129-120654-g053.jpg)