CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target
Quy Le, … , Keith R. Loeb, Soheil Meshinchi
Quy Le, … , Keith R. Loeb, Soheil Meshinchi
Published September 22, 2022
Citation Information: J Clin Invest. 2022;132(22):e157101. https://doi.org/10.1172/JCI157101.
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CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target

Abstract

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

Authors

Quy Le, Brandon Hadland, Jenny L. Smith, Amanda Leonti, Benjamin J. Huang, Rhonda Ries, Tiffany A. Hylkema, Sommer Castro, Thao T. Tang, Cyd N. McKay, LaKeisha Perkins, Laura Pardo, Jay Sarthy, Amy K. Beckman, Robin Williams, Rhonda Idemmili, Scott Furlan, Takashi Ishida, Lindsey Call, Shivani Srivastava, Anisha M. Loeb, Filippo Milano, Suzan Imren, Shelli M. Morris, Fiona Pakiam, Jim M. Olson, Michael R. Loken, Lisa Brodersen, Stanley R. Riddell, Katherine Tarlock, Irwin D. Bernstein, Keith R. Loeb, Soheil Meshinchi

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Figure 5

FOLR1 CAR constructs and reactivity of short, intermediate, and long FOLR1 CAR T cells.

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FOLR1 CAR constructs and reactivity of short, intermediate, and long FOL...
(A) Schematic diagram of second-generation FOLR1 CAR constructs with different IgG4 spacer lengths. SP, GM-CSFR signal peptide; scFv, single-chain variable fragment; TM, transmembrane domain; CD, costimulatory domain; SD, stimulatory domain; tCD19, transduced marker truncated CD19. (B) Expression of FOLR1 in C/G-CB, M07e, WSU-AML, Kasumi-1 FOLR1+, and Kasumi-1 parental cells. Blue = stained with PE-labeled anti-FOLR1; gray = isotype control. (C) Cytolytic activity of CD8+ T cells unmodified or transduced with short, intermediate, or long FOLR1 CAR construct against C/G-CB (cells taken >9 weeks in EC coculture), M07e, WSU-AML, Kasumi-1 FOLR1+, and Kasumi-1 parental cells in a 6-hour assay. Shown is the mean percentage specific lysis ± SD from 3 technical replicates at indicated effector/target (E:T) ratios. Data are representative of 3 donors. (D) Concentration of secreted IL-2, IFN-γ, and TNF-α in the supernatant following 24 hours of CD8+ T cell/AML coculture at 1:1 E:T ratio. Mean ± SD from 3 technical replicates is shown. Data are representative of 3 donors. (E) Representative flow plots showing expression of NFAT, NF-κB, and AP-1 in Jurkat J76 TPR reporter cells transduced with FOLR1 CAR constructs cultured alone (top) or coincubated with Kasumi-1 FOLR1+ target cells for 24 hours at 1:1 E:T ratio (bottom). Kasumi-1 FOLR1+ cells were labeled with CellTrace Violet cell proliferation dye to differentiate from Jurkat cells. Transduced Jurkat cells were gated based on tCD19 expression. Number in top right corner indicates the percentage of positive cells. Analysis was performed on day 4 after transduction. (F) Quantification of percentage of NFAT+, NF-κB+, and AP-1+ cells in E. This experiment was repeated once.