Targeting FTO suppresses cancer stem cell maintance and immune evasion

We conducted RNA sequencing with poly A RNA isolated from spleen mononuclear cells of MLL-AF9 (MA9) mouse models after treatment with PBS and FTO inhibitor CS1. Overall design: MLL-AF9 (MA9) primary leukemic mouse bone marrow cells (CD45.2+) were collected and sorted by flow cytometry when the mice developed full-blown AML. The cells were injected into sub-lethally irradiated (320 rads) secondary recipient mice with 0.05x106 donor cells per mouse via tail vein injection. One week after BMT, the mice were randomly divided into CS1 and control groups. The recipient mice were injected with PBS control and 5mg/kg CS1 i.p. every other day for 20 days. Spleens were removed from mice at the end point, and homogenized into a single-cell suspension using RPMI 1640 supplemented with 2% FBS. Red blood cells were lysed using ammonium chloride (07850, STEMCELL Technologies), washed with cold PBS. Cell pellet were then resuspended in 45 µL pre-cold MACS buffer (0.5% BSA and 2mM EDTA in PBS) per 107 total cells. Biotin-labeled CD45.2 (130-101-903, Miltenyi Biotec) were added to the cells and incubated for 10 minutes in the refrigerator (2-8 °C). Cells Cells were subsequently washed with MACS buffer and resuspended in MACS buffer (1 × 108 cells/mL). Streptavidin microbeads were added (MSPB-6003-74, Thermo Fisher Scientific) and the cell mixture was incubated at room temperature for 10 minutes. Cells were washed again and resuspended in 500 µL MACS buffer and then isolated with the MACS seperation Columns (130-042-201, Miltenyi Biotec). After collecting the CD45.2 positive cells, total RNA was extracted using miRNeasy Mini Kit (217004, Qiagen) and polyA RNA were enriched for RNA sequence.