Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC 50 of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cellbased assay measuring phospho-FAK with an IC 50 of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100Â selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC 50 of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATPcompetitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

Aberrant regulation of Notch signaling has been implicated in tumorigenesis. Proteolytic release of the Notch intracellular domain (NICD) by γ-secretase plays a key role in Notch-dependent nuclear signaling. γ-Secretase is an attractive pharmaceutical target for therapeutic intervention in cancer. We describe the potent antitumor effects of PF-03084014, a small molecule that is a reversible, noncompetitive, and selective γ-secretase inhibitor. The ability of PF-03084014 to inhibit γ-secretase activity was shown by the reduction of endogenous NICD levels and by the downregulation of Notch target genes Hes-1 and cMyc in the T-cell acute lymphoblastic leukemia (T-ALL) cell line HPB-ALL. PF-03084014 caused cell growth inhibition of several T-ALL cell lines via cell cycle arrest and induction of apoptosis. PF-03084014 treatment also resulted in robust NICD reduction in HBP-ALL xenograft models. Broad antitumor efficacy at well-tolerated dose levels was observed in six Notch-dependent models. Additional mechanism-of-action studies showed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the antitumor activity of PF-03084014 may be mediated by its direct effects on tumor cell growth or survival. Further studies on PF-03084014-induced gastrointestinal toxicity identified an intermittent dosing schedule that displayed reduced body weight loss and sustained antitumor efficacy. We also showed that glucocorticoids abrogated PF-03084014-induced gastrointestinal toxicity and delayed administration of glucocorticoids did not compromise its protection effect. Collectively, the results show that inhibition of Notch signaling by PF-03084014 while minimizing gastrointestinal toxicity presents a promising approach for development of therapies for Notch receptor-dependent cancers. This compound is being investigated for the treatment of T-ALL and advanced solid tumors in phase I clinical trials.

The Aurora family of highly related serine/threonine kinases plays a key role in the regulation of mitosis. Aurora1 and Aurora2 play important but distinct roles in the G 2 and M phases of the cell cycle and are essential for proper chromosome segregation and cell division. Overexpression and amplification of Aurora2 have been reported in different tumor types, including breast, colon, pancreatic, ovarian, and gastric cancer. PF-03814735 is a novel, potent, orally bioavailable, reversible inhibitor of both Aurora1 and Aurora2 kinases that is currently in phase I clinical trials for the treatment of advanced solid tumors. In intact cells, the inhibitory activity of PF-03814735 on the Aurora1 and Aurora2 kinases reduces levels of phospho-Aurora1, phosphohistone H3, and phospho-Aurora2. PF-03814735 produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. Although PF-03814735 produces significant inhibition of several other protein kinases, the predominant biochemical effects in cellular assays are consistent with inhibition of Aurora kinases. Once-daily oral administration of PF-03814735 to mice bearing human xenograft tumors produces a reduction in phosphohistone H3 in tumors at doses that are tolerable and that result in significant inhibition of tumor growth. The combination of PF-03814735 and docetaxel in xenograft mouse tumor models shows additive tumor growth inhibition. These results support the clinical evaluation of PF-03814735 in cancer patients.Mol Cancer Ther; 9(4); 883-94. ©2010 AACR.

Post-translational modification of oncogenic p21ras proteins with farnesyl, a lipid intermediate in cholesterol biosynthesis, is required for p21ras membrane association and for the ability of p21ras to transform cultured cells. We have tested the ability of lovastatin, a specific inhibitor of cholesterol biosynthesis, to inhibit the growth of ras oncogene-transformed cells in vivo. Balb/c mouse 3T3 cells, transfected with H-ras oncogene from human EJ bladder carcinoma, were highly tumorigenic in nude mice. Immunoprecipitation studies with transformed EJ cells showed that lovastatin (1-100 microM) inhibited p21ras membrane association in a concentration-dependent manner and that a 10 microM concentration reduced the amount of p21ras bound to the membrane by 50%. Lovastatin also inhibited EJ cell growth in a concentration range that closely paralleled that required for inhibition of p21ras membrane association. Treatment of nude mice bearing subcutaneous (s.c.) EJ tumors with lovastatin (50 mg/kg) significantly inhibited the abilities of these tumors to grow as early as four days and, by day 12, the lovastatin treated group of animals had tumors with an average size that was 3-fold smaller than those in the saline treated group. Western blotting studies showed that lovastatin (50 mg/kg) was also able to inhibit p21ras membrane association in EJ tumors implanted s.c. in nude mice. These results demonstrate that lovastatin, an inhibitor of cholesterol biosynthesis, inhibited in vivo tumor growth of H-ras oncogene transformed cells. The results also suggest that inhibition of p21ras membrane association, an essential step in ras oncogene neoplastic transformation, is one mechanism by which lovastatin may express its antitumor activity.

Bleomycin (BLM) hydrolase catalyzes the inactivation of the antitumor drug BLM and is believed to protect normal and malignant cells from BLM toxicity. The normal physiological function of BLM hydrolase is not known. We now provide evidence for its membership in the cysteine proteinase family. BLM hydrolase was purified to homogeneity from rabbit lungs, and a partial amino acid sequence was determined from a tryptic digest peptide. On the basis of this sequence a 36-mer oligonucleotide was synthesized. The 36-mer oligonucleotide probe hybridized to a single mRNA species of 2.5 kb from several species and was used to isolate an 832-bp cDNA insert from a lambda gt11 rabbit liver cDNA library. This insert encoded the tryptic digest peptide previously identified in rabbit lung BLM hydrolase by amino acid sequencing. Analysis of the predicted amino acid sequence coded by the 832-bp BLM hydrolase cDNA fragment indicated no significant homology with any currently known proteins except for a 15 amino acid portion, which displayed remarkable homology with the active site of cysteine proteinases. Within this active-site region, 10 of the amino acid residues of papain and 9 of aleurain, cathepsin H, and cathepsin L were identical with those of rabbit liver BLM hydrolase. The catalytic cysteine of thiol proteinases was also conserved in BLM hydrolase, and cysteine proteinase specific inhibitors, such as E-64, were found to be potent inhibitors of BLM hydrolase activity. Furthermore, bleomycin hydrolase exhibited cathepsin H like enzymatic activity. Bleomycin hydrolase had, however, no significant cathepsin B or L activities.(ABSTRACT TRUNCATED AT 250 WORDS)