Siglec6 CAR T cells suppressed progression of AML via inhibiting Siglec6 and SHP2 induced Src and ERK signaling activation.

To explore the specific molecular mechanisms of Siglec6 CAR-T therapy in acute myeloid leukemia (AML). AML samples were selected from the GEO database for bioinformatics analysis. Siglec6 was knocked down and overexpressed in MOLM-13 cells through lentiviral infection, and then injected into NOD/SCID mice via the tail vein to detect the distribution of AML in mice using in vivo imaging. MOLM-13 cells were divided into six groups: oe-NC, oe-Siglec6, oe-Siglec6 + PHPS1, oe-Siglec6 + Dasatinib, oe-Siglec6 + Dasatinib + Lovastatin, and oe-Siglec6 + Dasatinib + Lovastatin + IL-3 neutralizing antibody. The expression levels of related proteins were detected by Western blot and immunofluorescence, and the cell invasion and proliferation abilities were tested by Transwell and CCK8 assays. Finally, MOLM-13 cells were co-cultured with CD19-CAR-T and Siglec6-CAR-T cells, and the apoptosis level of MOLM-13 cells after co-culture was detected by flow cytometry. In vivo imaging found that after overexpression of Siglec6 in MOLM-13 cells, AML cells were more widely distributed in mice; Western blot and immunofluorescence detected the protein levels in AML cells and found that compared with the oe-NC group, the expression levels of Siglec6, p-SHP2, IL-3, and p-ERK1/2 proteins were increased in the oe-Siglec6 group; cell invasion, migration, and proliferation abilities were enhanced, and these abilities were reversed after treatment with SHP2 inhibitors, Src inhibitors, SHP2 agonists, and IL-3 neutralizing antibodies. Finally, both in vitro and in vivo, it was found that compared with CD19 CAR-T, the apoptosis level of AML cells treated with Siglec6 CAR-T was increased, and their distribution in mice was reduced. Siglec6 CAR-T reduces the proliferation, invasion, and migration abilities of AML cells by acting on the SHP2/Src/ERK/IL-3 axis.