Abstract
BackgroundJuvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML.MethodsWe constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34+ cells.ResultsGMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38− cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth.ConclusionsLigand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0256-3) contains supplementary material, which is available to authorized users.
Highlights
Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood
A total of 1.0 × 107 peripheral blood mononuclear cells (PBMCs) from approximately 10 ml of blood from healthy donors was electroporated with a granulocyte-macrophage colony-stimulating factor receptor (GMR) CARcontaining piggyBac transposon and the piggyBac transposase plasmids
The number of MO7e cells observed following co-culture with mock T cells at an identical ratio was 94.0 ± 13.0 % of the GMR chimeric antigen receptor (CAR) T cells inhibit the colony growth of JMML CD34 + cells, but not that of cord blood or bone marrow CD34+ Cells We evaluated the anti-proliferative effects of GMR CAR T cells on JMML CD34+ cells of patients carrying PTPN11 mutations, NRAS mutations, or monosomy 7
Summary
Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML. Juvenile myelomonocytic leukemia (JMML) is a fatal, mixed myeloproliferative, and myelodysplastic disorder that occurs in infancy and early childhood. Patients with JMML have genetic abnormalities in granulocyte-macrophage colonystimulating factor (GM-CSF) signaling pathways, such as inactivation of NF1 or mutations in PTPN11, NRAS, KRAS, and CBL [1, 2]. In our genetic analyses of individual granulocyte-macrophage colonies, these nonRAS pathway gene mutations may represent the second genetic aberration in a proportion of JMML children with PTPN 11 mutations [4]. Their study defined PRC2 loss that switches the methylation/acetylation status of histone H3 lysine 27
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