Impact of TGN-1062 (TGN) on leukemic stem cells and leukemogenesis through inhibition of RNA methylation and mitochondrial metabolism. (A) Effects of TGN (1 μM) on proliferation and apoptosis of LSC-enriched AML blasts. CD34⁺CD38^– cells were isolated from primary MNCs (n = 4) or AML blasts (n = 5). Left, levels of cell proliferation. Right, levels of apoptosis. (B) Effects of TGN on colony forming of LSC-enriched AML blasts. CD34⁺CD38^– AML blasts or MNCs (1 × 10⁵ cells per 1.5 mL, n = 3) were treated with dimethyl sulfoxide (DMSO) control VEH or indicated dose of TGN for 24 hours before plating on methylcellulose. After 14 days, colonies were imagined under light microscope and counted. Data are presented as mean ± standard error, with triplicate determination. Number of colonies are presented as bar graph. Asterisk indicates a statistically significant difference based on unpaired t test analysis. (C-D) Primary CD34⁺CD38^– AML blasts were treated with DMSO control (VEH) or dose-dependent TGN for 24 hours. (C) Effects of TGN on METTL protein regulated m6A RNA methylation. Left, levels of global m6A RNA methylation by triple quadrupole mass spectrometry assay. Right, METTL and BCL-2 protein expression by immunoblotting. N = 2 biological replicates. (D) Effects of TGN on METTL protein regulated m6A BCL-2 mRNA and BCL-2 mRNA decay. Left, levels of m6A BCL-2 mRNA determined by m6A RNA immunoprecipitation assay. Right, levels of BCL-2 mRNA over time following treatment with Act D. N = 2 biological replicates. (E-H) Primary CD34⁺CD38^– AML blasts were treated with VEH or TGN (1 μM) for 24 hours. The effects of TGN on levels of FAO as measured by ³H-palmitate levels (E, left), OXPHOS as indicated by OCR levels (E, right), and ROS (F) are shown. The effects of TGN on mitochondria length (G) and mitochondria membrane potential (MMP) (H) are also presented. (G) Left, TEM was performed to image mitochondria with enlarged images. Scale bar, 1 μm. Right, quantification of mitochondria length. Asterisk indicates significantly different, based on unpaired t test analysis. (H) The treated cells were stained with JC1 probes. The decrease of MMP levels is indicated by the ratio of lower ratio of red (polymer) to green (monomer) JC-1 fluorescence. (I) Effects of TGN on the expression of BCL-2 protein and signaling. Primary CD34⁺CD38^– AML blasts were treated with VEH or TGN (1 μM) for 24 hours. Immunoblotting of indicated antibodies are shown. (J) Effects of TGN on BCL-2/HMGB1 interaction. Primary CD34⁺CD38^– AML blasts were treated with VEH or TGN (1 μM) for 12 hours. Cell lysate was immunoprecipitated with anti-BCL-2 and immunoblotted with anti-HMGB1 antibodies. Input loading controls are shown. (K) Effects of TGN on BCL-2–regulated HMGB1 function. Primary CD34⁺CD38^– AML blasts were treated with VEH or TGN (1 μM) for 24 hours. Left, ubiquitination assay. Cell lysate was immunoprecipitated with anti-HMGB1 and immunoblotted with anti-ubiquitin antibodies. Middle, ChIP assay. Cell lysate was immunoprecipitated with anti-HMGB1 antibody and the binding levels of HSPB1 promoter were measured by quantitative polymerase chain reaction. Right, levels of HSPB1 mRNA. N = 2 biological replicates. (L) Effects of TGN on NRF2 ubiquitination and the expression of NRF2 downstream proteins. Left, cell lysate was immunoprecipitated with anti-NRF2 and immunoblotted with anti-ubiquitin antibodies. Middle, expression of HO-1, NQO-1, and DRP1 by immunoblotting. Right, IM-TEM images of DRP1 expression in mitochondria with enlarged images. Scale bar, 1 μm. (M) Effects of TGN on leukemic cell growth in vivo. Molm-13 cells (0.5 × 10⁶ per mouse) were subcutaneously injected into nude mice. After 7 days of injection, mice were treated with VEH or TGN (75 mg/kg, oral gavage, 21 days). Leukemia growth was determined by tumor size and weight 21 days after the start of treatment. n = 3 mice per group. Left, images of tumor (top) and tumor weight (bottom). Middle, levels of PARP cleavage, PCNA, and DNA fragmentation. Right, phalloidin staining of slide sections from tumors isolated from VEH or TGN-treated mice. N = 2 biological replicates. (N) Effects of TGN on leukemic cell growth in vivo using FLT3-WT AML PDX model. hCD45⁺ BM FLT3-WT AML cells (1 × 10⁶ cells per mouse) were transplanted into NSG mice to generate a cohort of AML bearing PDX mice, which were randomly divided into 2 groups and treated with VEH (n = 10) or TGN (75 mg/kg, oral gavage, n = 10) for 21 days. On day 21, 10⁶ BM MNCs cells from each treatment group were harvested for secondary transplant. Left, FAO and OXPHOS levels in hCD45⁺ BM cells isolated from primary treated mice (each, n = 3). Right, Kaplan–Meier survival curve of primary treated and secondary transplanted leukemic mice. Primary transplant, VEH (purple line, n = 10, median survival [MS] 29 days) or TGN (blue line, n = 10, MS 47 days). Secondary transplant, VEH (purple line, n = 10, MS 40 days) or TGN (blue line, n = 10, MS 50 days). Act D, actinomycin D; ChIP, chromatin immunoprecipitation; CON, control; DAPI, 4′,6-diamidino-2-phenylindole; DRP1, dynamin-related protein 1; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; HO-1, heme oxygenase 1; IP, immunoprecipitation; IB, immunoblotting; IgG, immunoglobulin G; IM-TEM, immuno-TEM; NQO-1, NAD(P)H quinone dehydrogenase 1; ns, nonsignificant; OCR, oxygen consumption rate; PARP, poly (ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; TEM, transmission electron microscope.