Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway.

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.

Inhibition of the signal transducer and activator of transcription-3 (STAT3) signaling pathway by 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid esters.

The JAK-STAT3 pathway regulates genes that are important in cell proliferation and thus is a promising target for cancer therapy. A high-throughput screening (HTS) campaign using an Apo-ONE Homogenous Caspase 3/7 assay in U266 cells identified 4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxylic acid ethyl ester 4 as a potential STAT3 pathway inhibitor. Optimization of this HTS hit led to the identification of the 7-cyano analogue 8, which inhibited STAT3-Y705 phosphorylation with an EC 50 of 170 nM. Compound 8 also inhibited cytokine induced JAK activation but did not inhibit BCR-ABL activated STAT5 phosphorylation in K562 cells.

Exploiting genotypic differences to identify genes important for EAE development.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human autoimmune disease multiple sclerosis (MS) and is primarily driven by T helper type 1 (Th1) cells. Interleukin (IL)-12 and interferon (IFN)-gamma are important cytokines involved in the differentiation and amplification of Th1 cells, however mice deficient in either IFN-gamma or IL-12 still develop EAE. We have used microarray analysis of EAE-affected CNS tissues in wild-type, IFN-gamma -/- and IL-12 -/- animals to identify genes critical for development of EAE. Over 500 genes were regulated in at least one genotype and over 94 genes were regulated in all three. Of those, 17 were also upregulated in spleen during the disease. We show that a majority of the genes regulated in EAE are also regulated in diseased regions of human MS tissues. The genes in the pool of 94 are more likely to be found regulated in MS patients than the genes regulated in only one or two of the mouse strains suggesting that analyzing gene expression under these multiple genetic conditions may lead to better identification of the genes critical for disease development.