Discovery of an Atropisomeric PI3Kß Selective Inhibitor through Optimization of the Hinge Binding Motif.

A series of PI3Kß selective inhibitors derived from a novel 4-(1H-benzo[d]imidazol-1-yl)quinoline chemotype has been rationally designed. Crucial to achieving the desired selectivity over the other class I PI3K isoforms, including the challenging δ-isoform, was the identification of a subset of substituted pyridine hinge binders. This work led to the discovery of (P)-14, a highly selective and orally bioavailable PI3Kß inhibitor displaying an excellent pharmacokinetic profile in addition to great cellular potency in various PTEN-deficient tumor cell lines. Results from a dog toxicology study revealing structure-related, off-target ocular toxicity are also briefly discussed.

A comparison of prostate cancer cell transcriptomes in 2D monoculture vs 3D xenografts identify consistent gene expression alterations associated with tumor microenvironments.

BACKGROUND: Prostate cancer (PC) research has relied heavily on patient-derived cell lines, which may be used for in vitro (two-dimensional [2D]) studies or cultivated as three-dimensional (3D) xenografts in mice. These approaches are likely to have differential impacts on cell phenotypes, with implications for experimental outcomes. Therefore, defining and comparing the transcriptional signatures associated with 2D and 3D approaches may be useful for designing experiments and interpreting research results. METHODS: In this study, LNCaP, VCaP, and 22Rv1 human PC cells were either cultivated in monolayers or as xenografts in NOD SCID mice, and their gene transcription profiles were quantitated and compared using microarray and real-time polymerase chain reaction techniques. Immunohistochemistry was used to evaluate protein expression in cancer cell xenografts. RESULTS: Comparisons of gene expression profiles of tumor cells grown in 2D vs 3D environments identified gene sets featuring similar expression patterns in all three cancer cell lines and unique transcriptional signatures associated with 3D vs 2D growth. Pathways related to cell-cell interactions, differentiation, and the extracellular matrix were enriched in 3D conditions. Immunohistochemical analyses confirmed that gene upregulation in xenografts occurred in implanted cancer cells and not in mouse stromal cells. Cultivating cells in vitro in the presence of mouse, rather than bovine serum failed to elicit the gene transcription profile observed in xenografts, further supporting the hypothesis that this profile reflects 3D growth and enhanced microenvironmental interactions, rather than exposure to species-specific serum factors. CONCLUSIONS: Overall, these findings define the expression profiles observed in PC cells cultivated in 2D monolayers and in 3D xenografts, highlighting differentially regulated pathways in each setting and providing information for interpreting research results in model systems.

DMRT1 protects male gonadal cells from retinoid-dependent sexual transdifferentiation.

Mammalian sex determination initiates in the fetal gonad with specification of bipotential precursor cells into male Sertoli cells or female granulosa cells. This choice was long presumed to be irreversible, but genetic analysis in the mouse recently revealed that sexual fates must be maintained throughout life. Somatic cells in the testis or ovary, even in adults, can be induced to transdifferentiate to their opposite-sex equivalents by loss of a single transcription factor, DMRT1 in the testis or FOXL2 in the ovary. Here, we investigate what mechanism DMRT1 prevents from triggering transdifferentiation. We find that DMRT1 blocks testicular retinoic acid (RA) signaling from activating genes normally involved in female sex determination and ovarian development and show that inappropriate activation of these genes can drive sexual transdifferentiation. By preventing activation of potential feminizing genes, DMRT1 allows Sertoli cells to participate in RA signaling, which is essential for reproduction, without being sexually reprogrammed.

DMRT1 prevents female reprogramming in the postnatal mammalian testis.

Sex in mammals is determined in the fetal gonad by the presence or absence of the Y chromosome gene Sry, which controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells. This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which Sry activates Sox9 and a female network involving WNT/ß-catenin signalling. In females the primary sex-determining decision is not final: loss of the FOXL2 transcription factor in adult granulosa cells can reprogram granulosa cells into Sertoli cells. Here we show that sexual fate is also surprisingly labile in the testis: loss of the DMRT1 transcription factor in mouse Sertoli cells, even in adults, activates Foxl2 and reprograms Sertoli cells into granulosa cells. In this environment, theca cells form, oestrogen is produced and germ cells appear feminized. Thus Dmrt1 is essential to maintain mammalian testis determination, and competing regulatory networks maintain gonadal sex long after the fetal choice between male and female. Dmrt1 and Foxl2 are conserved throughout vertebrates and Dmrt1-related sexual regulators are conserved throughout metazoans. Antagonism between Dmrt1 and Foxl2 for control of gonadal sex may therefore extend beyond mammals. Reprogramming due to loss of Dmrt1 also may help explain the aetiology of human syndromes linked to DMRT1, including disorders of sexual differentiation and testicular cancer.