EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha-ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5 flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5 deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning ؊634 to ؊531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (؊540) motif, AP-1 sites (؊533, ؊79), a NF-B (؊600) consensus sequence, and a GT box (؊52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activated protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent the activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/ets and AP-1 sites and is MEK1-independent.The 92-kDa type IV matrix metalloproteinase (92-kDa gelatinase B also known as MMP-9) plays a major role in cell migration in both physiological and pathological processes (1-3) by facilitating the destruction of the type IV collagencontaining basement membrane which separates the epithelial and stromal compartments (4). The 92-kDa type IV collagenase is secreted as a proenzyme (5) and subsequently activated by multiple enzymes, including cathepsin G, trypsin, stromelysin 1 (6), and 72-kDa gelatinase A (7) by the removal of 73 amino acids from the amino terminus of the protease. The active enzyme, which is capable of digesting native type I, III, IV, and V collagens at nondenaturing temperatures (4, 6), consists of five domains: the aminoterminal and zinc-binding domains shared by all members of the metalloproteinase family, a collagen-binding fibronectinlike domain, a carboxyl-terminal hemopexin-like domain, and a unique 54-amino acid ...

Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2-and VEGF-mediated phosphorylation of ERK1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases ( angiogenesis ͉ cancer ͉ redox ͉ tetrathiomolybdate ͉ ATN-224

The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)-and extracellular signal-regulated kinase-(ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we ®rst determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning 7144 to 773 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides 791 to 768 and containing a consensus AP-1 motif at 779. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinasede®cient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-de®cient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinasede®cient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK-and ERK-dependent signaling pathways.

Long tracts of CCG trinucleotide or CCGNN pentanucleotide repeats in DNA have previously been shown to resist assembly into nucleosomes. This may provide a molecular explanation for the nature of certain rare, folate-sensitive fragile sites in human chromosomes that contain expanded CCG triplet tracts. Further, it is known that methylation of CpG dinucleotides at or near these fragile sites enhances the fragile phenotype. Here DNAs containing 76 tandem CCG triplets or 48 CCGNN pentanucleotide repeats were methylated with SssI methylase at three different levels of methylation. Using competitive nucleosome reconstitution/gel shift assays, the ability of these DNAs and a mixed sequence DNA from the pUC19 plasmid were compared in their ability to assemble into nucleosomes. DNA methylation had no significant effect on nucleosome formation over the pUC 19 fragment. However, the highly methylated DNAs containing 76 CCG triplets or 48 CCGNN pentanucleotide repeats were 2.0 +/- 0. 2-fold and 2.1 +/- 0.3-fold less efficient in nucleosome assembly than the respective unmethylated forms, and 4.4 +/- 0.4-fold and 12. 6 +/- 1.6-fold less efficient than a pUC19 fragment of similar length.