A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity
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
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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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 2

BEND3 knockout confers resistance to TAK-243 in AML cells.

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BEND3 knockout confers resistance to TAK-243 in AML cells. (A) OCI-AML2...
(A) OCI-AML2 cells overexpressing Cas9 were stably transduced with gRNAs targeting LacZ (control) or BEND3. After transduction, whole cell lysates were prepared, and levels of BEND3 and β-actin serving as a loading control were measured by immunoblotting. (B) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with increasing concentrations of TAK-243 for 72 hours. Cell growth and viability was measured by the MTS assay. Inset: IC50 values (nM) are shown. Data points represent means ± SEM of 3 independent experiments. (C) WT OCI-AML2 cells were stably transduced with a single-plasmid system encoding spCas9 and gRNAs targeting LacZ (control) or BEND3. After transduction, whole cell lysates were prepared and levels of BEND3, spCas9, and GAPDH serving as a loading control were measured by immunoblotting. (D) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with increasing concentrations of TAK-243 for 72 hours. Cell growth and viability was measured by the MTS assay. Inset: IC50 values (nM) are shown. Data points represent means ± SEM of 3 independent experiments. (E) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with 2 concentrations of TAK-243 for 96 hours. Cell viability was measured by annexin V/PI staining and flow cytometry. Data points represent means ± SEM of 3–4 independent experiments. (F) Control and BEND3-knockout OCI-AML2-Cas9 cells were seeded with or without TAK-243 (30 nM), and trypan blue–negative cells were counted every 2–3 days. Data points represent means ± SEM of 2–3 counts. (G) Control and BEND3-knockout OCI-AML2-Cas9 cells were treated with TAK-243 (30 nM) and then plated into colony-forming assays. After 7 days of incubation, colonies of at least 50 cells were counted. The y axis shows the number of colonies as a percentage of the DMSO-treated controls taking into account plating efficiency as detailed in the Methods section. ***P ≤ 0.001; ****P ≤ 0.0001 using 2-way ANOVA and Sidak’s multiple comparisons test.