High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells
Bastien Gerby, Diogo F.T. Veiga, Jana Krosl, Sami Nourreddine, Julianne Ouellette, André Haman, Geneviève Lavoie, Iman Fares, Mathieu Tremblay, Véronique Litalien, Elizabeth Ottoni, Milena Kosic, Dominique Geoffrion, Joël Ryan, Paul S. Maddox, Jalila Chagraoui, Anne Marinier, Josée Hébert, Guy Sauvageau, Benjamin H. Kwok, Philippe P. Roux, Trang Hoang
Bastien Gerby, Diogo F.T. Veiga, Jana Krosl, Sami Nourreddine, Julianne Ouellette, André Haman, Geneviève Lavoie, Iman Fares, Mathieu Tremblay, Véronique Litalien, Elizabeth Ottoni, Milena Kosic, Dominique Geoffrion, Joël Ryan, Paul S. Maddox, Jalila Chagraoui, Anne Marinier, Josée Hébert, Guy Sauvageau, Benjamin H. Kwok, Philippe P. Roux, Trang Hoang
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Research Article Hematology

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

Abstract

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.

Authors

Bastien Gerby, Diogo F.T. Veiga, Jana Krosl, Sami Nourreddine, Julianne Ouellette, André Haman, Geneviève Lavoie, Iman Fares, Mathieu Tremblay, Véronique Litalien, Elizabeth Ottoni, Milena Kosic, Dominique Geoffrion, Joël Ryan, Paul S. Maddox, Jalila Chagraoui, Anne Marinier, Josée Hébert, Guy Sauvageau, Benjamin H. Kwok, Philippe P. Roux, Trang Hoang

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Figure 4

2-ME2 inhibits the accumulation of SCL protein.

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2-ME2 inhibits the accumulation of SCL protein. (A) Pre-LSCs from SCLtgL...
(A) Pre-LSCs from SCLtgLMO1tg thymocytes were treated with 2-ME2 (1 μΜ) or DEXA (5 nM) for 16 hours, and mRNA levels of SCL and SCL target genes Lyl1 and Hhex were determined by quantitative RT-PCR and normalized to β-actin (mean ± SD, n = 3, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (B) 2-ME2 inhibits the activity of the SCL transcriptional complex. The Gypa promoter (Gypa-Luc) was used as a reporter. Transfected cells were then treated or not (Vehicle) with 2-ME2 (1 μM) or DEXA (2 nM) for 24 hours. Results are expressed as fold activation by SCL over the complex without SCL. Data represent the mean ± SD (n = 3, ***P ≤ 0.001). (C) Immunofluorescence of human SCL oncoprotein by flow cytometry in pre-LSCs from SCLtgLMO1tg thymocytes treated with the indicated doses of 2-ME2 or not (Vehicle) for 16 hours. DN3 WT thymocytes stained with antibody against SCL and cells stained with the secondary antibody only were used as negative controls. (D and E) Real-time imaging of HeLa cells expressing SCL-GFP or H2B-RFP in the presence or absence of 2-ME2 (1 μM). Kinetic of GFP (top panels) and RFP (bottom panels) expression in a representative treated or untreated cell is shown (D), and the averages of mean fluorescence intensities (MFIs) for each time point were calculated (E). MFIs were normalized to the initial intensity of the corresponding cell, which was set as 100%. (F) JURKAT cells were treated with 2-ME2 (1 μM) for the indicated times, and nuclear extracts were subjected to immunoblotting. Pol II, RNA polymerase II. (G and H) SCL and histone H3 intracellular immunofluorescence staining in JURKAT cells treated or not with 2-ME2 (1 μM) for 8 hours. The normalized corrected total cell fluorescence (CTCF) per nucleus of SCL (n = 27–98, ***P ≤ 0.001) and H3 (n = 42–60) was calculated (G). A representative example of SCL (left panel) and H3 (right panel) expression is shown (H). All data shown are representative of 2 independent experiments.