VS-5584, a novel and highly selective PI3K/mTOR kinase inhibitor for the treatment of cancer.

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC(50) = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC(50): PI3Kα = 16 nmol/L; PI3Kß = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials.

Conservation defines functional motifs in the squint/nodal-related 1 RNA dorsal localization element.

RNA localization is emerging as a general principle of sub-cellular protein localization and cellular organization. However, the sequence and structural requirements in many RNA localization elements remain poorly understood. Whereas transcription factor-binding sites in DNA can be recognized as short degenerate motifs, and consensus binding sites readily inferred, protein-binding sites in RNA often contain structural features, and can be difficult to infer. We previously showed that zebrafish squint/nodal-related 1 (sqt/ndr1) RNA localizes to the future dorsal side of the embryo. Interestingly, mammalian nodal RNA can also localize to dorsal when injected into zebrafish embryos, suggesting that the sequence motif(s) may be conserved, even though the fish and mammal UTRs cannot be aligned. To define potential sequence and structural features, we obtained ndr1 3'-UTR sequences from approximately 50 fishes that are closely, or distantly, related to zebrafish, for high-resolution phylogenetic footprinting. We identify conserved sequence and structural motifs within the zebrafish/carp family and catfish. We find that two novel motifs, a single-stranded AGCAC motif and a small stem-loop, are required for efficient sqt RNA localization. These findings show that comparative sequencing in the zebrafish/carp family is an efficient approach for identifying weak consensus binding sites for RNA regulatory proteins.

A novel androgen receptor amino terminal region reveals two classes of amino/carboxyl interaction-deficient variants with divergent capacity to activate responsive sites in chromatin.

The androgen receptor (AR) is an important signaling molecule in multiple tissues, yet its mode of action and cell-specific activities remain enigmatic. AR function has been best studied in the prostate, in which it is essential for growth and homeostasis of the normal organ as well as each stage of cancer development. Investigation of mechanisms responsible for continued AR action that evolve during prostate cancer progression or after hormonal management of the disease have been instructive in defining AR signaling pathways. In the current paper, we use sequence similarity and the collocation of somatic mutations in prostate cancer to define residues 501-535 of the AR amino-terminal domain as an important mediator of receptor function. Specifically, the 501-535 region is required for optimal interaction of the amino-terminal domain with both the p160 coactivator, nuclear receptor coactivator-2, and the AR-ligand binding domain in the amino/carboxyl (N/C) interaction. The N/C interaction is decreased by deletion of the 501-535 region but is distinct from deletion of the (23)FQNLF(27) peptide in that it does not affect the capacity of the AR to activate transcription from a chromatin integrated reporter or recruitment of the receptor to androgen-responsive loci in vivo. Collectively, we have been able to outline two classes of N/C-deficient AR variant that are divergent in their capacity to act in a chromatin context, thereby further defining the interplay between N/C interaction and coregulator recruitment via multiple receptor domains. These mechanisms are likely to be key determinants of the cell and promoter specific activities of the AR.

The zebrafish dorsal axis is apparent at the four-cell stage.

A central question in the development of multicellular organisms pertains to the timing and mechanisms of specification of the embryonic axes. In many organisms, specification of the dorsoventral axis requires signalling by proteins of the Transforming growth factor-beta and Wnt families. Here we show that maternal transcripts of the zebrafish Nodal-related morphogen, Squint (Sqt), can localize to two blastomeres at the four-cell stage and predict the dorsal axis. Removal of cells containing sqt transcripts from four-to-eight-cell embryos or injection of antisense morpholino oligonucleotides targeting sqt into oocytes can cause a loss of dorsal structures. Localization of sqt transcripts is independent of maternal Wnt pathway function and requires a highly conserved sequence in the 3' untranslated region. Thus, the dorsoventral axis is apparent by early cleavage stages and may require the maternally encoded morphogen Sqt and its associated factors. Because the 3' untranslated region of the human nodal gene can also localize exogenous sequences to dorsal cells, this mechanism may be evolutionarily conserved.

PC-3 cells with enhanced androgen receptor signaling: a model for clonal selection in prostate cancer.

BACKGROUND: Two sublines of the human prostate cancer cell line, PC-3, which is widely used as a model of prostate cancer progression, have been reported: PC-3(AR-) that do not express androgen receptor (AR), and PC-3AR+ that have measurable AR RNA but little protein. METHODS: We assayed the geneotype, karyotype, AR expression, and physical characteristics of the two PC-3 sublines, and compared their ability to elicit a transactivation response from ectopic AR in the presence and absence of specific AR coregulators. RESULTS: PC-3(AR-) and PC-3AR+ cells are genotypically and karyotypically similar, but exhibit salient differences in their morphology, growth rate, and expression of AR RNA. Whereas endogenous AR expression in PC-3AR+ cells does not result in sufficient protein to confer androgen responsiveness in culture, ectopic AR consistently elicited a much greater transactivation response in PC-3AR+ than in PC-3(AR-) cells, without altered sensitivity to activation by native ligand or AR coregulators including GRIP1, BRCA1, and Zac1. Moreover, phenotypic differences of AR variants implicated in prostate cancer susceptibility and progression were only observed in PC-3AR+ cells. Higher levels of known AR coregulator proteins detected in PC-3AR+ compared with PC-3(AR-) cells likely contribute to these differences. CONCLUSIONS: These studies provide new evidence that the androgen-signaling axis can be sensitized in prostate cancer cells, and have important implications for the analysis and interpretation of AR structure and function in in vitro cell systems.

Structural and functional consequences of glutamine tract variation in the androgen receptor.

The androgen receptor (AR) gene contains a polymorphic trinucleotide repeat region, (CAG)(n), in its N-terminal transactivation domain (NTD) that encodes a polyglutamine (polyQ) tract in the receptor protein. Whereas the length of the CAG repeat ranges from 6 to 39 in healthy individuals, the variations in repeat length both within and outside the normal range are associated with disease, including impaired spermatogenesis and Kennedy's disease, and with the risk of developing breast and prostate cancer. Whereas it has been proposed that the inverse relationship between polyQ tract length within the normal range and AR transactivation potential may be responsible for altered risk of disease, the molecular mechanisms underlying polyQ length modulation of AR function have not been elucidated. In this study, we provide detailed characterization of a somatic AR gene mutation detected in a human prostate tumor that results in interruption of the polyQ tract by two non-consecutive leucine residues (AR-polyQ2L). Compared with wtAR, AR-polyQ2L exhibits disrupted inter-domain communication (N/C interaction) and a lower protein level, but paradoxically has markedly increased transactivation activity. Molecular modeling and the response to cofactors indicate that the increased activity of AR-polyQ2L results from the presentation of a more stable platform for the recruitment of accessory proteins than wild-type AR. Analysis of the relationship between polyQ tract length and AR function revealed a critical size (Q16-Q29) for maintenance of N/C interaction. That between 91 and 99% of AR alleles in different racial-ethnic groups encode a polyQ tract in the range of Q16-Q29 suggests that N/C interaction has been preserved as an essential component of androgen-induced AR signaling.

Genomic organization of human myotonic dystrophy kinase-related Cdc42-binding kinase alpha reveals multiple alternative splicing and functional diversity.

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha) is a Cdc42/Rac interactive binding-containing serine/threonine kinase with multiple functional domains. Its roles in the regulation of peripheral actin reorganization in HeLa cells and NGF-induced neurite outgrowth in PC12 cells have been documented. Here we report the characterization of the genomic structure and alternative splicing of the human counterpart. Human MRCKalpha gene is located on chromosome 1q42.1, spanning a genomic region of 250-300 kb and is composed of 41 exons. Four exons in the internal variable region and six in the 3' end were found to undergo extensive alternative splicing, giving rise to 96 possible transcripts of different combinations. The region of the internal splice site that defines a variable region in between two functional domains of opposite regulatory effects on MRCKalpha catalytic activity, and the 3' end splice site that generates variants with differential GTPase binding activity suggest a role for these alternative splicing events in MRCKalpha regulation.

p80 ROKalpha binding protein is a novel splice variant of CRMP-1 which associates with CRMP-2 and modulates RhoA-induced neuronal morphology.

Using antibody against the Rho binding domain of ROKalpha, two neuronal phosphoproteins of 62 and 80 kDa were co-immunoprecipitated from brain extracts. Peptide analysis revealed their identity as collapsin response mediator proteins (CRMPs); p62 was CRMP-2 whereas p80 was a novel splice form of CRMP-1 with an extended N-terminus. p80 CRMP-1 was able to complex with CRMP-2, suggesting that p80 CRMP-1 and CRMP-2 form oligomers. CRMP-2 was the major substrate of ROK. p80 CRMP-1 interacted with the kinase domain of ROKalpha, resulting in inhibition of the catalytic activity towards other substrates. Over-expression of p80 CRMP-1 and CRMP-2 together counteracted the effects of RhoA on neurite retraction, an effect enhanced by mutation of the ROK phosphorylation site in CRMP-2. p80 CRMP-1 and CRMP-2 may be modulators of RhoA-dependent signaling, through interaction with and regulation of ROKalpha.