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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Categories: 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 5

2-ME2 inhibits glucocorticoid-resistant T-ALL cells and targets MYC protein synthesis.

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2-ME2 inhibits glucocorticoid-resistant T-ALL cells and targets MYC prot...
(A) Dose-response curves of DEXA (left panel) and 2-ME2 (right panel) of 4 human T-ALL cell lines (JURKAT, KOPTK1, P12-ICHIKAWA, and HPB-ALL) treated for 48 hours (mean ± SEM, n = 3, *P ≤ 0.05). Cell viability was assessed by FACS (percent control). (B) Levels of SCL and c-MYC protein expression were determined by Western blot in the 3 indicated T-ALL cell lines after total protein extraction. (C–E) JURKAT, KOPTK1, and P12-ICHIKAWA cells were treated for 16 hours with the indicated doses of 2-ME2. Nuclear protein extracts were subjected to immunoblotting with the indicated antibodies. α-Actin was used as a loading control. (F) SCL and c-MYC half-lives were determined in JURKAT (SCL+c-MYC+) and KOPTK1 (c-MYC+) T-ALL cells by cycloheximide chase and Western blotting according to Supplemental Figure 9D. Representative of 2 independent experiments. (G) JURKAT, KOPTK1, and P12-ICHIKAWA cells were treated for 16 hours with the indicated doses of 2-ME2. Total protein extracts were subjected to immunoblotting with the indicated antibodies. α-Tubulin was used as a loading control. Representative of 2 independent experiments. (H) JURKAT cells were treated with 2-ME2 (3 μM) or not (Vehicle) during 3 hours in the presence of 35S-methionine. SCL (top panel) and c-MYC (bottom panel) immunoprecipitation was then performed on labeled proteins. Immunoglobulins were used as control of immunoprecipitation (IP CTL). Radiolabeling was revealed using a PhosphorImager. (I) T-ALL blasts from a thymoma of SCLtgLMO1tg mice were treated for 16 hours with the indicated doses of 2-ME2. The human SCL transgene and endogenous c-MYC protein expression levels were then determined by immunoblotting. α-Actin was used as a loading control. Representative of 2 independent experiments.