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 7

2-ME2 reduces human T-ALL development in xenograft models.

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2-ME2 reduces human T-ALL development in xenograft models. (A) Immunophe...
(A) Immunophenotype of human T-ALL samples (nos. 14H025 and 14H148) at diagnosis and after transplantation in NSG mice. The expression of human CD34/CD7 and CD4/CD8 markers of peripheral blood cells from the patients (left panel) and of engrafted human CD45+ T-ALL blasts in the BM of NSG mice 6 weeks after transplantation (right panel) is shown. (B) Purified engrafted human CD45+CD7+ T-ALL blasts from patients 14H025 and 14H148 were cocultured on MS5-DL4 stromal cells with a dose-response of 2-ME2 and DEXA for 4 days (n = 3, *P ≤ 0.05). The percentages of cell viability are shown for both patients. (C) Blasts from both patients were treated with the indicated doses of 2-ME2 for 16 hours. SCL and c-MYC protein expression levels were determined by immunoblotting. (D) Blasts were treated with 1 μM of 2-ME2 during 16 hours, and mRNA expression levels of the indicated genes were assessed by quantitative RT-PCR and were normalized to β-actin mRNA (n = 3, **P ≤ 0.01, ***P ≤ 0.001). (E) Experimental procedure to study the effect of 2-ME2 on human T-ALL in vivo (left panel). Engrafted T-ALL blasts of both patients were transplanted into secondary NSG mice (n = 10), and the proportion of CD45+CD7+ T-ALL blasts in BM aspirations was assessed 3 weeks after transplantation (right panel). (F) Engrafted secondary mice were randomly selected for treatment or not (Vehicle, n = 5) with 2-ME2 (40 mg/kg/d, n = 5) using osmotic pumps during 7 days. Human T-ALL reconstitution (number of CD45+CD7+ blasts) was monitored by FACS in the BM and the spleen (*P ≤ 0.05, ***P ≤ 0.001).