Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell, and proliferation genes and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.
The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.
Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase a-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase S. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These rindings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.The replication of chromosomal DNA in mammalian cells is not yet understood. To gain better insight into this process, we have been studying the replication of the genome of the papovavirus simian virus 40 (SV40) (1, 2). SV40 DNA replication takes place in the nucleus of permissive primate cells. Except for the participation of a single virus-encoded protein, the SV40 large tumor (T) antigen, viral DNA replication is dependent upon the resident cellular replication machinery. For this reason the fundamental mechanisms of SV40 DNA replication are probably quite similar to those of chromosomal DNA replication. Thus, the detailed biochemical analysis of SV40 DNA replication represents an attractive approach to the identification and functional characterization of cellular DNA replication proteins.We previously established a crude in vitro system capable of supporting the complete replication of plasmid DNA molecules that contain the SV40 origin of DNA replication (3). This system consists of a soluble extract from permissive cells supplemented with purified SV40 T antigen. DNA replication in the system exhibits most of the characteristics of SV40 DNA replication in vivo (3-7). In particular, DNA synthesis begins in the neighborhood of the SV40 origin and proceeds bidirectionally via replication intermediates identical to those observed in vivo. We and others have made use of the cell-free SV40 DNA replication system to explore the enzymatic mechanisms of viral DNA replication and to identify the proteins involved (1, 2). These studies have provided evidence for the involvement ofa number of cellular proteins in SV40 DNA replication in vitro: DNA polymerase a-primase complex (8, 9), topoisomerases I and II (10),
The ability of simian virus 40 (SV40) large T antigen to Cellular kinases and phosphatases regulate the initiation of simian virus 40 (SV40) DNA replication. Unphosphorylated large T antigen is unable to unwind the viral origin of replication until phosphorylated on threonine 124 by a cyclindependent kinase (24). T antigen purified from mammalian cells is heavily phosphorylated on a number of additional serines and threonines (reviewed in reference 11). Phosphorylation on serines 120 and 123 by a nuclear form of casein kinase I (CKI) blocks T antigen's origin-unwinding activity, apparently by preventing functional interactions between Tantigen hexamers bound to the minimal origin of replication (5,6,21,44). This phosphorylation-mediated inhibition of T-antigen activity can be reversed in vitro by treatment of T antigen with alkaline phosphatase (13,28) or with the isolated catalytic subunit of protein phosphatase 2A (PP2AJ) (42,43).Since the phosphoryl groups on serine 123 (and probably serine 120) of T antigen have a high turnover rate in vivo (31) and T-antigen mutants with alanines instead of serines at these positions are inviable (34), these phosphorylation-dephosphorylation cycles are likely to be physiologically relevant. Two lines of evidence suggest that PP2A is the cellular phosphatase that dephosphorylates serines 120 and 123. First, PP2A was purified from HeLa extracts as a factor required for efficient in vitro SV40 DNA replication (42, 43). Second, addition of okadaic acid but not inhibitor 2 inhibited in vitro SV40 DNA replication (21). Since okadaic acid inhibits both PP2A and protein phosphatase 1, while inhibitor 2 inhibits protein phosphatase 1 but not PP2A, this suggests that PP2A is the sole cellular phosphatase which activates SV40 DNA replication. intracellular serine/threonine protein phosphatases (9). All members of the family appear to share a -36-kDa catalytic C subunit. This PP2Ac polypeptide can be dissociated from PP2A regulatory subunits in vitro but does not appear to exist as a monomer in vivo. Rather, the PP2A holoenzymes isolated to date contain both a catalytic subunit and a -65-kDa regulatory A subunit. These A and C subunits are each encoded by a pair of highly homologous genes (17). In addition, several heterotrimeric PP2A holoenzymes have been purified in a number of laboratories (reviewed in references 29 and 36); these enzymes contain a third B subunit. At least three distinct B subunits of 72, 55, and 54 kDa have been purified to date from skeletal and cardiac muscle and erythrocytes. cDNAs for the 55-and 72-kDa forms (and a 130-kDa splice variant) have been cloned (16,22). Interestingly, small t antigen of SV40 and small and middle T antigens of polyomavirus can also bind to the A-C complex in place of endogenous B subunits, leading to alterations in phosphatase activity (references 4 and 38 and references therein). Disruption of the gene encoding the 55-kDa B subunit homolog in Drosophila melanogaster leads to lethal defects in mitosis and wing development (23,40), ...
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