Molecular Cell Biology An ERG Enhancer–Based Reporter Identifies Leukemia Cells with Elevated Leukemogenic Potential Driven by ERG-USP9X Feed-Forward Regulation Nasma Aqaqe 1 , Muhammad Yassin 1 , Abed Alkader Yassin 1 , Nour Ershaid 1 , Chen Katz-Even 1 , Adi Zipin-Roitman 1 , Eitan Kugler 3,4,5 , Eric R. Lechman 2 , Olga I. Gan 2 , Amanda Mitchell 2 , John E. Dick 2 , Shai Izraeli 3,4,5 , and Michael Milyavsky 1 Abstract Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERGþ85 enhancer– based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFP High ) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFP Neg counterparts. The tagBFP High fraction was capable of regen- erating the original cellular heterogeneity and demonstrat- ed increased invasive ability. Moreover, the tagBFP High fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERGþ85–positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibi- tion of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional hetero- geneity and suggest that targeting ERG via USP9X inhibi- tion may be a potential treatment strategy in patients with leukemia. Significance: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells. Introduction Acute leukemia is a highly aggressive group of blood malig- nancies that originate from hematopoietic stem cells (HSC). Accumulation of blast cells in the bone marrow due to deregu- lation of molecular pathways controlling self-renewal and differ- entiation of immature blood cells is the main feature of leuke- mia (1, 2). Well-recognized and prognostic genetic heterogeneity, as well as functional variability, exists among the subsets of leukemia cells obtained from the same patient (3, 4). Functional heterogeneity model posits that a fraction of acute leukemia cells displays sufficient regenerative capacity to propagate the disease, withstand chemotherapy, and cause leukemia relapse. Functional resemblance of these leukemic cells to normal hematopoietic stem cells (HSC) contributed to their nomination as leukemia stem cells (LSC; refs. 5, 6). Although recent studies have shown that phenotypic and genetic heterogeneity within tumors constitutes a major source of therapeutic resistance (7), efficient tools to identify and pull out functional stem cell from the heterogeneous cell population are still lacking. Experimentally, the presence of functional human LSCs can be proved by their capacity to engraft immunodeficient mice and induce leukemia growth in their hematopoietic organs (8, 9). Up to date, enrichment for LSCs has been reached by focusing on: cell surface markers (10), metabolism (11, 12), cell-cycle quiescence (13), and miRNA bioactivity (14). These studies and others demonstrated extraordinary inter- and even intra-sample (3) heterogeneity for LSC activity that was influ- enced by disease stage and type of therapy (10, 15, 16). Further- more, prior studies emphasized the need to devise approaches that enable identification and isolation of viable human LSCs based on stemness state of the single cell to pinpoint molecular regulators that maintain LSC properties. Here, we capitalized on our recent findings that a þ85 enhancer of ERG transcription factor (TF) can be used as a 1 Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 2 Princess Margaret Cancer Centre, University Health Network and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 3 Department of Pediatric Hemato-Oncology, Schneider Children Med- ical Center Petah-Tikva, Israel. 4 The Gene Development and Environment Pediatric Research Institute, Pediatric Hemato-Oncology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel. 5 Department of Molecular Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). N. Aqaqe and M. Yassin contributed equally to this article. Corresponding Author: Michael Milyavsky, Tel Aviv University, Sackler Faculty of Medicine, Tel Aviv 6997801, Israel. Phone: 9725-4591-5351; E-mail: mmilyavsky@post.tau.ac.il Cancer Res 2019;79:3862–76 doi: 10.1158/0008-5472.CAN-18-3215 Ó2019 American Association for Cancer Research. Cancer Research Cancer Res; 79(15) August 1, 2019 3862 Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/15/3862/2784655/3862.pdf by guest on 19 June 2022