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

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

doi:10.1172/jci.insight.141518

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.

Highlights

TAK-243 is a first-in-class inhibitor of the ubiquitin-like modifier activating enzyme 1 (UBA1) that catalyzes the first step of the ubiquitin conjugation cascade [1,2,3]
We previously reported that acute myeloid leukemia (AML) cell lines and primary patient samples are more dependent on the activity of UBA1 compared with normal hematopoietic cells and are more vulnerable to UBA1 inhibition [7]
To identify genes that influence the cytotoxicity of TAK-243, we performed a genome-wide CRISPR/Cas9 knockout screen in AML cells

Summary

Introduction

TAK-243 ( known as MLN7243) is a first-in-class inhibitor of the ubiquitin-like modifier activating enzyme 1 (UBA1) that catalyzes the first step of the ubiquitin conjugation cascade [1,2,3] Through this cascade, protein substrates are tagged with mono- or poly-ubiquitin to induce their proteasomal degradation or to modulate their functions [4, 5]. Protein substrates are tagged with mono- or poly-ubiquitin to induce their proteasomal degradation or to modulate their functions [4, 5] This process is executed through multistep enzymatic reactions whereby ubiquitin is initially activated by the ubiquitin-activating enzyme (E1) in an ATP-dependent manner. While UBA1 is the major ubiquitin E1 in the cell, there are over 30 ubiquitin E2s and hundreds of ubiquitin E3s that mediate the ubiquitylation of substrates in a highly coordinated and specific manner [6]

Methods

Results

Conclusion