A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity
Samir H. Barghout, … , Rima Al-Awar, Aaron D. Schimmer
Samir H. Barghout, … , Rima Al-Awar, Aaron D. Schimmer
Published January 21, 2021
Citation Information: JCI Insight. 2021;6(5):e141518. https://doi.org/10.1172/jci.insight.141518.
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Research Article Oncology Therapeutics

A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity

Abstract

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.

Authors

Samir H. Barghout, Ahmed Aman, Kazem Nouri, Zachary Blatman, Karen Arevalo, Geethu E. Thomas, Neil MacLean, Rose Hurren, Troy Ketela, Mehakpreet Saini, Moustafa Abohawya, Taira Kiyota, Rima Al-Awar, Aaron D. Schimmer

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Figure 4

BEND3 knockout dampens TAK-243 effects and reduces the intracellular transport of TAK-243 into AML cells.

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BEND3 knockout dampens TAK-243 effects and reduces the intracellular tr...
(A and B) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with DMSO or TAK-243 (30 nM) for 24 hours. After treatment, whole cell lysates were prepared, and levels of UBA1, UBA3, UBA6, UBA2, poly-ubiquitylated proteins, activating transcription factor 4 (ATF4), poly (ADP-ribose) polymerase (PARP), cleaved PARP (C. PARP), DNA-damage inducible transcript 3 (CHOP), phospho-JNK (p-JNK), and Ser139 phosphorylated H2AX (γH2AX) were measured by immunoblotting. GAPDH and β-actin were used as loading controls. (C) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with DMSO or increasing concentrations of TAK-243 at 15–120 nM for 1 hour followed by heating the intact cells at 54°C. After heating, whole cell lysates were prepared, and levels of UBA1 and GAPDH were measured by immunoblotting. (D) Control and BEND3-knockout OCI-AML2-Cas9 cells were washed, seeded in equal numbers, and lysed. Luminescence was then measured after adding an ATP-dependent luciferase reagent. Relative luminescence obtained from BEND3-knockout OCI-AML2-Cas9 cells was calculated by normalizing to control cells. Data points represent means ± SEM of 3–5 independent experiments. (E) Control and BEND3-knockout OCI-AML2 cells were treated with increasing concentrations of TAK-243 (300–1200 nM) for 1 hour and washed, and pellets were then extracted with acetonitrile. TAK-243 concentrations were then measured by LC-MS. Data points represent means ± SEM of triplicate data from a representative experiment (n = 2). **P ≤ 0.01; ****P ≤ 0.0001 using 2-way ANOVA and Sidak’s multiple comparisons test.