Briefly, multiple-drug-resistant clones were established and transcriptome sequencing was conducted to find mutations in each clone; the assumption was that the crucial components of the signaling of the drug target(s) would have a high chance to carry mutations in drug-resistant clones26. is one of the most common and fatal forms of hematopoietic malignancies1C4. Despite the improved risk stratifications and treatment-adapted strategies, with standard chemotherapies, still only 35C40% of more youthful (aged 60) and 5C15% of older (aged60) patients with AML can survive over 5 years4,5. Many AML subtypes, such as the family, was first identified as a fusion partner of the gene associated with t(10;11)(q22;q23) in AML12,13. In contrast to the repression and tumor-suppressor role of TET2 observed in hematopoietic malignancies14C17, we recently showed that was significantly upregulated in expression shows only very minor effects on normal development including hematopoiesis21, TET1 is an attractive therapeutic target for AML. In the present study, through a series of in vitro drug testing and in vivo preclinical animal model studies, we identified chemical compounds NSC-370284 and UC-514321 (a more JTK12 effective analog of NSC-370284) as potent inhibitors that significantly and selectively suppress the viability of AML cells with high level of expression (i.e., transcription and TET1 signaling, leading to potent anti-leukemic effects. Results NSC-311068 and 370284 inhibit in AML18,19. In fact, high expression of was found not only in AML, but also in various tumors including uterine malignancy, glioma, etc., and especially, in testicular germ cell malignancies (Supplementary Fig.?1). This indicates potential oncogenic role of in many cancers where expression level is relatively high. In order to identify chemical compounds that may target TET1 signaling, we searched the drug-sensitivity/gene expression database of a total of 20,602 chemical compounds in the NCI-60 collection of malignancy cell samples22. We found the expression levels of endogenous showed a significant positive correlation with the responsiveness of malignancy cells across the NCI-60 panel to 953 compounds (values and Mepenzolate Bromide tested their effects on cell viability of a is highly expressed not only in expression also significantly inhibited t(8;21) fusion gene-induced colony-forming/replating capacity of mouse bone marrow (BM) progenitor cells (Supplementary Fig.?3). Our results showed that NSC-311068 (6-(1-Pyrrolidinyl(3,4,5-trimethoxyphenyl)methyl)-1,3-benzodioxol-5-ol; C21H25NO6) and NSC-370284 (Pyrimidine, 4-[(2,4-dinitrophenyl)thio]-; C10H6N4O4S) exhibited the most significant effects in inhibiting cell viability of all four expression (Fig.?1a, b). In the NCI-60 collection, cell lines with relatively higher expression levels showed more obvious positive correlation between expression level and activity of both NSC-311068 and NSC-370284, compared to that across the entire NCI-60 panel, whereas cell lines with relatively lower expression levels exhibited no obvious positive correlation (NSC-311068) or even unfavorable correlation (NSC-370284) (Supplementary Table?2c, d). In expression (Fig.?1c), as well as the global 5hmC level (Fig.?1d). In order to rule out the possibility of non-specific toxicity, we reduced the dose of NSC-311068 and NSC-370284 to 25?nM, and tested gene expression and cell viability 24?h after treatment. The low dose, short-term treatments again resulted in a significant downregulation of transcription, accompanied with a Mepenzolate Bromide very minor decrease in the viability of MONOMAC-6, THP-1, and KOCL-48 cells (Fig.?1e, f). Thus, it is unlikely that this inhibitory effects of NSC-311068 and NSC-370284 on expression were due to nonspecific toxicity. Open in a separate windows Fig. 1 NSC-311068 and NSC-370284 suppress the viability of AML cells with high level. a, b expression by NSC-311068 and NSC-370284 in AML cell lines. Cells were treated with DMSO, Mepenzolate Bromide or 300?nM NSC-311068 or NSC-370284. expression levels were detected by qPCR 48?h post treatment. d NSC-311068 and NSC-370284 (both at 300?nM) repressed global 5hmC level in THP-1 (left panels) and MONOMAC-6 (right panels) cells. e, f MONOMAC-6, THP-1, and KOCL-48 cells were treated with DMSO, or 25?nM NSC-311068 or NSC-370284. expression levels (e), and cell viability (f), were detected 24?h post treatment. *AML model. NSC-311068 and especially 370284 treatments significantly inhibited (AML mice. Upon the onset of leukemia, the recipient mice were treated with DMSO (control; values were determined by log-rank Mepenzolate Bromide test. b WrightCGiemsa staining of mouse peripheral blood (PB) and BM, or hematoxylin and eosin (H&E) staining of mouse spleen and liver of the treated or control leukemic mice. c, d gene expression levels (c), or Tet1 protein level (d), in BM blast cells of the treated or control leukemic mice. *values were determined by log-rank test NSC-370284 targets STAT3/5 and suppresses expression To decipher Mepenzolate Bromide the molecular mechanism by which NSC-370284 represses expression, we adapted the strategy developed by Kapoor and colleagues26 to identify direct target protein(s) of NSC-370284. Briefly, multiple-drug-resistant clones were established and transcriptome sequencing was conducted to find mutations in.