RESUMEN
Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic acid-binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6-specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Inmunoterapia Adoptiva , Lectinas/inmunología , Leucemia Mieloide Aguda/terapia , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Humanos , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/inmunología , Linfocitos T/inmunología , Células U937RESUMEN
The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.
Asunto(s)
Citoesqueleto de Actina/fisiología , Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Citoesqueleto de Actina/química , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pironas/farmacología , Quinolinas/farmacología , Estaurosporina/farmacología , Sulfonamidas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/fisiologíaAsunto(s)
Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Mieloma Múltiple/terapia , Receptores Quiméricos de Antígenos/uso terapéutico , Linfocitos T/fisiología , Técnicas de Transferencia de Gen , Humanos , Leucemia Mieloide Aguda/inmunología , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Mieloma Múltiple/inmunología , Viroterapia Oncolítica/efectos adversos , Virus Oncolíticos/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Antígenos de Linfocitos T/uso terapéutico , Receptores Quiméricos de Antígenos/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/antagonistas & inhibidores , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/inmunologíaRESUMEN
FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8+ and CD4+ T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.