An ERG Enhancer–Based Reporter Identifies Leukemia Cells with Elevated Leukemogenic Potential Driven by ERG-USP9X Feed-Forward Regulation

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 enhancerbased 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 regenerating the original cellular heterogeneity and demonstrated 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 inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition 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.

Trib1 promotes acute myeloid leukemia progression by modulating the transcriptional programs of Hoxa9

“…53,54 Previous studies reported that ERG is a downstream target of HOXA9 and that overexpression of ERG is an important biomarker in predicting poor prognoses for patients with normal-karyotype AML 16,40 ; this is in agreement with our findings. Other studies showed that a heptad of transcription factors (FLI1, ERG, GATA2, RUNX1, SCL/TAL1, LYL1, and LMO2) recognize the ERG enhancer 85 kb downstream of a translation initiation site (185 enhancer) 32,55,56 that is well conserved between humans and mice and is a target of Trib1 modification, as revealed by our study. Core transcription factor binding on the 185 enhancer indicates the presence of a core regulatory circuitry (CRC) that is important for the superenhancer activity as well as hematopoietic differentiation and transformation.…”

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells

“…USP9X is a multifunctional posttranslational modulator, which is involved in the regulation of various biological processes in multiple cell types, including proliferation and growth of HEK293T cells (Li et al, 2018), survival of B‐cell precursor acute lymphoblastic leukemia (Schwartzman et al, 2017), apoptosis of spermatogenic cells (Kishi et al, 2017), autophagy of Huh7 cells (Jia et al, 2020), stemness of ES cells (Macrae & Ramalho‐Santos, 2021), heterogeneity and invasive ability (Aqaqe et al, 2019), and radio‐resistance of lung cancer cells (Jie et al, 2021). In mouse ovary, Usp9x is enriched in the GCs of primordial and primary follicles, which indicates that Usp9x is related to mammalian folliculogenesis (Sato et al, 2004).…”

Disruption of a GATA2, TAL1, ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells

“…A second approach would be to focus on transcriptional regulators of these TFs. USP9X, a deubiquitinase that regulates ERG stability 94 and is positively regulated by ERG in a feed forward loop is one such candidate 64 . A third approach would be to focus on specific enhancers such as GATA2-117, which is inaccessible in normal HSCs but open in the transitional progenitor states characteristic of AML, enabling preferential cytotoxicity in leukemic cells.…”

“…Unlike GATA2 -117, the ERG +85 enhancer was open in all HSPC subsets and across AML subtypes ( Figure S1A). This enhancer has been linked to AML prognosis 43 and has been used to identify LSCs within bulk AML populations 64,65 . Enhancers are replete with sequence motifs enabling binding of distinct TF families, either directly to DNA or indirectly via protein scaffolding, as observed for LMO2 66,67 and RUNX1 42,44 .…”