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 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

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 3

Small-molecule screening identifies 2-ME2 as an inhibitor of pre-LSCs.

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Small-molecule screening identifies 2-ME2 as an inhibitor of pre-LSCs. (...
(A) Experimental procedure for chemical screening of pre-LSCs. (B) The inhibition of pre-LSC viability was calculated for each compound (GSI set as 100% and DMSO as 0%; average pre-LSC viability: 0.4%). The top 5% (n = 96) of hits were selected. (C) Clustering of 44 selected compounds by structural similarities using the ChemMine tool (http://chemmine.ucr.edu/). The multidimensional scaling algorithm maps a compound described by its chemical structure into a 3D space and where neighbor compounds exhibit similar molecular structure. The axes (D1, D2, and D3) refer to the dimensions generated by the multidimensional scaling. Black arrow identifies 2-ME2 and gray arrow DEXA. MTDs, microtubule-targeting drugs. (D) 2-ME2 is the only MTD that does not affect the viability of human HSCs. Human CD34+ peripheral blood was collected from cord blood cells and treated with the indicated MTDs at 1 μM during 1 week as described previously (38). Numbers (n = 2) represent the fractions of viable cells compared with vehicle control. (E) 2-ME2 structure (top left panel) and dose-response on purified pre-LSCs and DN3 WT thymocytes. The percentages of cell viability are shown (right panel, n = 6, *P ≤ 0.05). IC50 was calculated (bottom left panel). Representative of 2 independent experiments. (F) MS5-DL4 stromal cells alone were treated in the same conditions. DL4 expression was assessed by FACS (left panel, n = 2), and IC50 for cell viability was calculated (right panel, n = 2). (G) Treated and untreated viable cells were then transplanted by limiting dilution assay into recipient mice according to Supplemental Figure 5A. Thymic engraftment was analyzed 6 weeks after transplantation, and pre-LSC frequencies were calculated (left panel) by Poisson statistics (CIs in parentheses; ***P ≤ 0.001). Right panel: Thymic reconstitution by control or 2-ME2–treated cells.