High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells
Bastien Gerby, … , Philippe P. Roux, Trang Hoang
Bastien Gerby, … , Philippe P. Roux, Trang Hoang
Published December 1, 2016; First published October 31, 2016
Citation Information: J Clin Invest. 2016;126(12):4569-4584. https://doi.org/10.1172/JCI86489.
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Research Article Hematology Stem cells

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 2

High-throughput assay for primary pre-LSCs in thymic-like microenvironment.

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High-throughput assay for primary pre-LSCs in thymic-like microenvironme...
(A and B) Generation of an optimal MS5-DL4 stromal cell line for pre-LSC viability. Following DL4 gene transfer, MS5 stromal cells were assessed for DL4 surface expression levels. Three MS5 subclones were purified according to levels of DL4 expression (low, medium, and high) (A). DN3 preleukemic SCLtgLMO1tg thymocytes were cocultured with MS5-CTL or MS5-DL4lo, -DL4med, or -DL4hi stromal cells during 5 days, and the absolute number of DN3 cells was assessed using FACS. Each dot represents an individual well, and dashed lines represent the mean for each condition (B). (C) Purified DN3 WT and preleukemic SCLtgLMO1tg thymocytes were cocultured with MS5-DL4lo stromal cells. DN3-derived thymocytes were immunophenotyped 5 days later using the CD4, CD8, CD25, and CD44 markers. (D) Miniaturized coculture system for HTS-FACS in 384-well plate. The proportion of DN3 (left panel) and DP (right panel) thymocytes was then assessed using HTS-FACS, and assay reproducibility was calculated. Each dot represents an individual well. Solid lines and dashed lines represent the mean and 3-fold the SD, respectively. The Z′ factor is a statistical measure of the robustness of the HTS assay (ideally between 0.5 and 1). Representative of 3 independent experiments. (E and F) DN3 preleukemic SCLtgLMO1tg thymocytes were cocultured with MS5-DL4lo stromal cells for 5 days in the presence or absence (Vehicle) of γ-secretase inhibitor (GSI, 1 μM), and the absolute number of DN3 cells was assessed using HTS-FACS. Representative FACS analysis of DN population is shown (E) together with assay reproducibility (F).