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 1

C/G-CB cells induce leukemia, recapitulating primary disease.

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C/G-CB cells induce leukemia, recapitulating primary disease. (A) Diagra...
(A) Diagram of experimental design. (B) Kaplan-Meier survival curves of NSG-SGM3 mice transplanted with GFP-CB control and C/G-CB cells. Statistical differences in survival were evaluated using the Mantel-Cox log-rank test. n = 4 mice per group. (C) Representative histology of H&E stain of femur taken from a mouse transplanted with C/G-CB cells (top) and cells from a C/G-positive patient sample (bottom) after development of leukemia. Original magnification, ×2.5 (left), ×40 (middle), and ×63 (right). See Supplemental Figure 1 for all H&E stains from C/G-CB–transplanted mice. n = 4 mice for C/G-CB cells and n = 2 mice for PDX. (D) Expression of the RAM immunophenotype in C/G-CB cells harvested from the bone marrow (BM) of a representative mouse at necropsy compared to a primary patient sample and PDX marrow xenograft cells. In all 3 samples, malignant cells were gated based on human CD45 expression and SSC. n = 4 mice for C/G-CB cells, n = 2 mice for PDX derived from BM cells of patient B. (E) Left and middle: Representative immunohistochemistry showing high expression of ERG (×10 magnification) and CD56 (×5 magnification) in the femur of a representative mouse transplanted with C/G-CB cells. Right: Small aggregates of blasts with high CD56 expression detected in a BM biopsy of a chemotherapy-refractory C/G-fusion-positive patient, consistent with residual, adherent, patchy disease distribution (×100 magnification). n = 4 mice. (F) Kaplan-Meier plot showing survival in primary (1°, n = 4 mice), secondary (2°, n = 7 mice), and tertiary (3°, n = 5 mice) transplantations of C/G-CB cells. (G) Engraftment of C/G-CB cells in the BM at time of symptomatic leukemia, shown as percentage human CD45+ cells. Images on the right are H&E stain of femurs taken from mice indicated by “a” and “b.” See Supplemental Figure 1 for all H&E stains from C/G-CB–transplanted mice. (H) Quantification of CD56+ cells among human CD45+ cells isolated from the BM at necropsy following development of symptomatic leukemia. (I) Expression of AMKL markers, CD41 and CD42, in C/G-CB and PDX cells harvested from the BM at necropsy. C/G-CB cells were gated on human CD45+ cells. PDX cells were gated on human CD45+CD56+ cells. (J) Quantification of CD41/CD42 subsets described in I. Bars indicate mean ± SEM. (G–J) 1°, n = 4 mice per group; 2°, n = 7 mice; and 3°, n = 5 mice.