In vivo studies

Mouse care and experimental procedures were performed under pathogen-free conditions in accordance with approved protocols from the institutional animal care and use committee of City of Hope National Medical Center. For the systemic AML model, 10⁶ luciferase-expressing Molm14 cells were injected into the tail vein of 6-to 8-week old NSG mice (Jackson Laboratory). To determine the leukemic burden, we injected the mice (I.P.) with 3 mg D-Luciferin (Promega) 4 days after tumor injection and imaged them in an IVIS 100 (Caliper Life Sciences). A standard region of interest (ROI), which included the entire mouse, was used to determine the total body bioluminescence. Data were expressed as photons/s/mm². The mice were then distributed into groups bearing equal tumor burdens and treated by gavage with NT1721 or the vehicle control (5% DMSO/30% solutol (Sigma)) as indicated.

To assess changes in gene expression levels in the human cells, we sacrificed mice after 14 days of treatment, extracted the bone marrow cells and isolated human cells using anti-human CD45 microbeads (Miltenyi) according to the manufacturer's protocol. The RNA was then isolated and used for qPCR assays.