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 May 1, 2019; First published February 21, 2019
Citation Information: J Clin Invest. 2019;129(5):1878-1894. https://doi.org/10.1172/JCI120654.
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Categories: Research Article Cell biology Oncology

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

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 3

ARV-825 downregulates the chemokine and adhesion receptors in leukemic cells and disrupts stromal–leukemic cell interactions to inhibit BM microenvironment niche–mediated drug resistance.

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ARV-825 downregulates the chemokine and adhesion receptors in leukemic c...
(A) OCI-AML3 (normal or hypoxia-adapted) cells were cultured with or without a monolayer of NMSCs and treated with ARV-825 (50 nM) or cytarabine (1 μM) under either normoxic or hypoxic conditions for 72 hours, and apoptosis was assessed by annexin V assay using flow cytometry (n = 3). (B) OCI-AML3 cells were treated with ARV-825 (10 nM) for 12 or 24 hours and subjected to CyTOF, and a heatmap was generated using the publicly available Broad Institute Gene Pattern Heatmap viewer. OCI-AML3 cells treated for 24 hours were stained for CXCR4 with or without permeabilization to quantify the changes in intracellular or surface CXCR4 expression using (C) a PE-conjugated antibody for the flow-based assay (n = 3) and (D) a AF594-conjugated secondary antibody for confocal imaging (original magnification, ×40). (E) OCI-AML3 cells were treated with ARV-825 (10 nM) or plerixafor (100 nM) (positive control) for 24 hours and subjected to a migration assay 4 hours after incubation in media containing SDF-1 (100 ng). Surface expression of CXCR4 was measured by flow cytometry, and cell migration was measured by collecting the cells from the lower chamber containing SDF-1 following Beckman Vi-CELL counts (n = 3). Whole-cell lysates obtained from OCI-AML3 cells treated with or without ARV-825 in the presence of SDF-1 were processed for immunoblotting with the indicated antibodies. β-Actin served as a loading control. (F) OCI-AML3 cells were treated with ARV-825 (10 nM) for the indicated durations, and whole-cell lysates were analyzed with the indicated antibodies (β-actin served as a loading control). Numbers indicate normalized intensity. PIM1-overexpressing OCI-AML3 cells were treated with ARV-825 (10 nM) for 24 hours, and then (G) whole-cell lysates were analyzed by immunoblotting with the indicated antibodies (α-tubulin was used as a loading control). (H) Surface expression of CXCR4 was analyzed by flow cytometry followed by a migration assay (n = 3). **P < 0.01 and ***P < 0.001, by standard Student’s t test.