A growing body of evidence shows that Signal Transducer and Activator of Transcription 5 (STAT5) protein, a key member of the STAT family of signaling proteins, plays a pivotal role in the progression of many human cancers, including acute myeloid leukemia and prostate cancer. Unlike STAT3, where significant medicinal effort has been expended to identify potent direct inhibitors, Stat5 has been poorly investigated as a molecular therapeutic target. Thus, in an effort to identify direct inhibitors of STAT5 protein, we conducted an in vitro screen of a focused library of SH2 domain binding salicylic acid-containing inhibitors (∼150) against STAT5, as well as against STAT3 and STAT1 proteins for SH2 domain selectivity. We herein report the identification of several potent (K(i) < 5 μM) and STAT5 selective (>3-fold specificity for STAT5 cf. STAT1 and STAT3) inhibitors, BP-1-107, BP-1-108, SF-1-087, and SF-1-088. Lead agents, evaluated in K562 and MV-4-11 human leukemia cells, showed potent induction of apoptosis (IC(50)'s ∼ 20 μM) which correlated with potent and selective suppression of STAT5 phosphorylation, as well as inhibition of STAT5 target genes cyclin D1, cyclin D2, C-MYC, and MCL-1. Moreover, lead agent BP-1-108 showed negligible cytotoxic effects in normal bone marrow cells not expressing activated STAT5 protein. Inhibitors identified in this study represent some of the most potent direct small molecule, nonphosphorylated inhibitors of STAT5 to date.

DLK1 is an imprinted gene on chromosome 14. Using informative coding single nucleotide polymorphisms, we found DLK1 expression to be monoallelic in normal bone marrow, whereas it was biallelic in 76% of acute myeloid leukemia (AML) overexpressing DLK1 (61% of all AML). Quantitative methylation analysis of 7 cytosine-phosphate-guanosine-rich areas (3 upstream of or within DLK1, the putative intergenic-differentially methylated region and 3 upstream of or within MEG3) revealed a strong association between biallelic DLK1 expression and hypermethylation of a cytosine-phosphateguanosine-rich region 18 kb upstream of DLK1. Allele-specific methylation analysis of this region revealed the alleles to be differentially methylated in normal bone marrow and monoallelic DLK1 AML, whereas there was increased methylation of both alleles in AML with biallelic expression. Moreover, chromatin immunoprecipitation analysis revealed that CCTCbinding factor binds to this region in monoallelic but not biallelic expression samples. Taken