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 2

ECs enhance the proliferative potential and promote leukemic progression of C/G-CB cells.

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ECs enhance the proliferative potential and promote leukemic progression...
(A) Diagram of experimental design. This experiment was performed twice using 2 separate CB units. See Supplemental Figure 4 for the repeat experiment. (B) Growth kinetics of C/G-CB and GFP-CB cells in EC coculture or MC. (C) C/G-CB cells expanded in EC coculture for 9 weeks were reseeded in EC coculture either directly (direct contact) or in EC Transwells (indirect contact) or placed in liquid culture containing SFEM II (with SCF, FLT3L, and TPO). After 7 days, the number of GFP+ cells was quantified by flow cytometry. Data in B and C presented as mean ± SD from 3 technical replicates. Statistical significance was determined by 1-way ANOVA. ****P < 0.00005. (D) At 6 and 12 weeks, a fraction of each culture was transferred to MegaCult cultures. Colonies derived from megakaryocytic (Mk) progenitors were scored and enumerated. Data were normalized to the 500 input cells at the start of the EC coculture or MC culture. A representative colony stained with anti–human CD41 and an alkaline phosphatase detection system is shown. Data shown are the average of 3 technical replicates. Error bars denote SD. (E) Equivalent number of C/G-CB and GFP-CB cells in EC coculture or MC were transplanted into NSG-SGM3 mice at indicated time points (5 × 106/mouse at week 3, and 1 × 107/mouse at weeks 6, 9, and 12). Due to insufficient expansion, GFP-CB cells were not transplanted after 3 weeks in either condition, similarly for C/G-CB cells after 6 weeks in MC culture. Median survival and Kaplan-Meier survival curve are shown. C/G-CB (n = 3 mice/group), GFP-CB (n = 2 mice/group). (F) Expression of the RAM immunophenotype in C/G-CB cells after 6 weeks in EC coculture or MC. Data are pooled from 3 technical replicates. (G) Quantification of CD56+ cells among live CD45+ cells over weeks in culture. (H and I) Expression of CD41 and CD42 (H) and quantification of CD41/CD42 subsets (I) at indicated time points in EC coculture or MC. Data in H and I presented as mean ± SD from 3 technical replicates. (J) Morphological evaluation of the C/G-CB cells cultured with ECs or in MC for 9 weeks showed features of megakaryocytic differentiation, including open chromatin, prominent nucleoli, and abundant focal, basophilic, and vacuolated cytoplasm with cytoplasmic blebbing. Results shown are representative of 3 technical replicates. Scale bars: 100 μm (D) and 20 μm (J).