CBFA2T3-GLIS2 model of pediatric acute megakaryoblastic leukemia identifies FOLR1 as a CAR T cell target
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
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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Research Article Oncology

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 6

Preclinical efficacy of FOLR1 CAR T cells against C/G AML cells.

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Preclinical efficacy of FOLR1 CAR T cells against C/G AML cells. (A) Cyt...
(A) Cytolytic activity of CD8+ T cells unmodified or transduced with FOLR1 CAR following 6 hours of coculture with C/G-CB (cells taken after >9 weeks in EC coculture), WSU-AML, Kasumi-1 FOLR1+, and Kasumi-1 parental cells. Data presented are mean leukemia specific lysis ± SD from 3 technical replicates at indicated effector/target (E:T) ratios. Data are representative of 3 donors. (B) Concentration of secreted IL-2, IFN-γ, and TNF-α in the supernatant following 24 hours of T cell/AML coculture at 1:1 E:T ratio as measured by ELISA. Data are representative of 2 donors and are presented as mean ± SD from 3 technical replicates. Where concentrations of cytokines were too low to discern, the number above the x axis indicates the average concentration. Statistical significance was determined by unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.005, ***P < 0.0005. Data are representative of 2 donors. (C) Representative flow cytometric analysis of cell proliferation of cell proliferation dye–labeled (CellTrace-labeled) unmodified and FOLR1 CAR T cells after 4-day coculture with target cells at a 1:1 E:T ratio. CAR T cells divided rapidly and diluted their CellTrace fluorescence after 4-hour coincubation with FOLR1+ AML cells. Data are representative of 2 donors. (D) Bioluminescence imaging of C/G-CB, WSU-AML, Kasumi-1 FOLR1+, and Kasumi-1 leukemias in mice treated with unmodified or FOLR1 CAR T cells at 5 × 106 T cells per mouse. n = 5 mice/group. Radiance scale indicates an increase in leukemia from blue to red; X indicates death. (E) Kaplan-Meier survival curves of xenografts treated with unmodified or FOLR1 CAR T cells. n = 5 per group. Statistical differences in survival were evaluated using Mantel-Cox log-rank test. Note: 2 C/G-CB–bearing mice treated with CAR T cells died without leukemia and T cells present in bone marrow, spleen, and liver tissues and in peripheral blood as determined by flow cytometric analysis. n = 5 mice/group.