Fig. 3
From: Targeted inhibition of STAT/TET1 axis as a therapeutic strategy for acute myeloid leukemia
STAT3 and STAT5 are potential direct targets of NSC-370284 in AML. a A top network identified with IPA involving all the 14 genes carrying recurrent mutations in THP-1 NSC-370284-resistant clones. Genes with mutations are labeled in bold font. b Knockdown of STAT3 and/or STAT5 reduces TET1 level. MONOMAC-6, THP-1, and NB4 cells were electroporated with control siRNA (siNC), siSTAT3, siSTAT5 or a combination of siSTAT3 and siSTAT5. TET1 expression level was detected by qPCR 48 h post transfection. c Four genomic sites designed for ChIP-qPCR analysis to identify potential binding sites of STAT3 and STAT5 on TET1 promoter and other regions. d–h MONOMAC-6 cells (d–g), were treated with DMSO control or 500 nM NSC-370284. ChIP-qPCR assay was carried out 48 h after drug treatment. Enrichment of STAT3, STAT5, or IgG at the TET1 promoter region and other regions are shown. NB4 cells (h), were applied as a negative control. i Predicted binding of DNA (upper panel) or NSC-370284 (lower panel) with the DNA-binding domain (DBD) that is conserved between STAT3 and STAT5 proteins, from docking study on PDB ID 1bg1 using MolSoft ICM. j The association between STAT3 and NSC-370284 as determined with NMR chemical shift perturbation (CSP). Complex formation with compound 370284 induced extensive CSPs at the Ile residues of STAT3 at 1:2 of protein:ligand molar ratio (red peaks: free STAT3; green peaks: STAT3-NSC-370284 complex). The CSP occurs at residues adjacent to the DNA-binding site (I464), and residues at or near the DBD. k NSC-370284 suppresses the binding between STAT3 and TET1 CpG island, as determined through EMSA. *P < 0.05; **P < 0.01, two-tailed t-test. Error bar indicates SD of triplicate experiments
