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BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment
Sujan Piya, … , Michael Andreeff, Gautam Borthakur
Sujan Piya, … , Michael Andreeff, Gautam Borthakur
Published February 21, 2019
Citation Information: J Clin Invest. 2019;129(5):1878-1894. https://doi.org/10.1172/JCI120654.
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Research Article Cell biology Oncology Article has an altmetric score of 4

BETP degradation simultaneously targets acute myelogenous leukemic stem cells and the microenvironment

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Abstract

The antileukemic effect of inhibiting bromodomain and extra-terminal domain-containing (BET-containing) proteins (BETPs) such as BRD4 has largely been largely attributed to transcriptional downregulation of cellular anabolic and antiapoptotic processes, but its effect on the bone marrow microenvironment, a sanctuary favoring the persistence of leukemic stem/progenitor cells, is unexplored. Sustained degradation of BETP with the small-molecule BET proteolysis-targeting chimera (PROTAC) ARV-825 resulted in a marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interactions, in acute myeloid leukemia (AML) cells. Abrogation of surface CXCR4 expression impaired SDF-1α–directed migration and was mediated through transcriptional downregulation of PIM1 kinase, which in turn phosphorylates CXCR4 and facilitates its surface localization. Downregulation of CD44, including isoforms CD44v8–10 impaired cystine uptake, lowered intracellular reduced glutathione, and increased oxidative stress. More important, BETP degradation markedly decreased the CD34+CD38–CD90–CD45RA+ leukemic stem cell population and, alone or in combination with cytarabine, prolonged survival in a mouse model of human leukemia that included AML patient-derived xenografts (AML-PDX). Gene expression profiling and single-cell proteomics confirmed a downregulation of the gene signatures associated with “stemness” in AML and Wnt/β-catenin and Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell-intrinsic signaling, stromal interactions, and metabolism in AML.

Authors

Sujan Piya, Hong Mu, Seemana Bhattacharya, Philip L. Lorenzi, R. Eric Davis, Teresa McQueen, Vivian Ruvolo, Natalia Baran, Zhiqiang Wang, Yimin Qian, Craig M. Crews, Marina Konopleva, Jo Ishizawa, M. James You, Hagop Kantarjian, Michael Andreeff, Gautam Borthakur

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Figure 7

ARV-825 downregulates the Wnt/β-catenin pathway, c-Myc transcriptional programs, and other oncogenic pathways and modulates the stromal environment.

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ARV-825 downregulates the Wnt/β-catenin pathway, c-Myc transcriptional p...
(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.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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