BAY 43-9006 is a powerful inhibitor of the RET kinase. Its potential as a therapeutic tool for RET-positive thyroid tumors, including those expressing V804 mutations merits study.

We have increased the lysine content in the seeds of canola and soybean plants by circumventing the normal feedback regulation of two enzymes of the biosynthetic pathway, aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes encoded by the Corynebacterium dapA gene and a mutant E. coli lysC gene, respectively, were linked to a chloroplast transit peptide and expressed from a seed-specific promoter in transgenic canola and soybean seeds. Expression of Corynebacterium DHDPS resulted in more than a 100-fold increase in the accumulation of free lysine in the seeds of canola; total seed lysine content approximately doubled. Expression of Corynebacterium DHDPS plus lysine-insensitive E. coli AK in soybean transformants similarly caused several hundred-fold increases in free lysine and increased total sed lysine content by as much as 5-fold. Accumulation of alpha-amino adipic acid (AA) in canola and saccharopine in soybean, which are intermediates in lysine catabolism, was also observed.

We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC 50 p100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino-acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC 50 : 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

Purpose: Targeting of KITand platelet-derived growth factor receptor (PDGFR) tyrosine kinases by imatinib is an effective anticancer strategy. However, mutations of the gatekeeper residue (T670 in KITand T681in PDGFRh) render the two kinases resistant to imatinib. The aim of this study was to evaluate whether sorafenib , a multitargeted ATP-competitive inhibitor of KITand PDGFR, was active against imatinib-resistant KITand PDGFRh kinases. Experimental Design: We used in vitro kinase assays and immunoblot with phosphospecific antibodies to determine the activity of sorafenib on KIT and PDGFRh kinases. We also exploited reporter luciferase assays to measure the effects of sorafenib on KIT and PDGFRh downstream signaling events. The activity of sorafenib on interleukin-3^independent proliferation of Ba/F3 cells expressing oncogenic KITor its imatinib-resistant T670I mutant was also tested. Results: Sorafenib efficiently inhibited gatekeeper mutants of KIT and PDGFRh (IC 50 for KIT T670I, 60 nmol/L; IC 50 for PDGFRh T681I, 110 nmol/L). Instead, it was less active against activation loop mutants of the two receptors (IC 50 for KIT D816V, 3.8 Amol/L; IC 50 for PDGFRh D850V, 1.17 Amol/L) that are also imatinib-resistant. Sorafenib blocked receptor autophosphorylation and signaling of KIT and PDGFRh gatekeeper mutants in intact cells as well as activation of AP1-responsive and cyclin D1 gene promoters, respectively. Finally, the compound inhibited KITdependent proliferation of Ba/F3 cells expressing the oncogenic KIT mutant carrying the T670I mutation. Conclusions: Sorafenib might be a promising anticancer agent for patients carrying KIT and PDGFRh gatekeeper mutations.The KIT and platelet-derived growth factor receptors (PDGFR) are members of the type III subclass of receptor tyrosine kinases. KIT is the receptor for stem cell factors (SCF), whereas PDGFRa and PDGFRh are the receptors for platelet-derived growth factors (PDGF; ref. 1). The structure of these receptors includes an extracellular domain with five immunoglobulinlike motifs, a single membrane-spanning domain, and a cytoplasmic tyrosine kinase domain. The kinase domain is split by a kinase insert sequence into an ATP-binding region and a phosphotransferase region (1).KIT, PDGFRa, and PDGFRh are frequently activated in neoplastic diseases. More than 30 gain-of-function mutations in KIT, either single amino acid changes or small deletions/ insertions, have been identified in such highly malignant human neoplastic diseases as gastrointestinal stromal tumors (GIST) and mastocytosis. GISTs are the most common type of sarcoma arising in the digestive tract and are generally distinguished from other abdominal sarcomas by the expression of KIT. Approximately 80% of these tumors show activating mutations in KIT (2). GIST mutations cluster in the KIT juxtamembrane region, whereas most mutations associated with mastocytosis target a specific aspartate residue (D816) in the kinase activation loop (3). Fusion of PDGFRa with different genes has been found in ...

MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.

Background: AXL is an established therapeutic target in various cancers. HSP90 chaperoning is critical in maintaining the stability of several oncogenic kinases. Results: HSP90 blockade by 17-AAG induced cytosolic mature AXL degradation via ubiquitin/proteasome pathway and impeded its membrane localization. Conclusion: AXL depends on HSP90 for its stability and membrane translocation. Significance: Targeting HSP90 would avail a strategy to counteract AXL.