etics and being selleck chemicals llc heterozygous for BRAFV600E. This differential response to PL 4032 in BRAFV600E mutant melanoma cell lines may be e plained by several mechanisms. It may be that there is preferential MAPK pathway addiction in sensitive cell lines, and cells with lower sensitivity are less dependent on the BRAFV600E oncogenic signaling, relying on the co activation of other signaling pathways including the PI3K Akt pathway. We e plored this possibility with SNP arrays and high throughput oncogene sequencing with a particular inter est in looking at this pathway. The genomic analysis revealed that alterations in PI3K Akt, including deletions of PTEN, amplifications of AKT and activating mutations in AKT were distributed throughout the cell line list with S ndergaard et al.
However, in two cell lines phospho specific Inhibitors,Modulators,Libraries Western blot staining suggested that the resistant cell Inhibitors,Modulators,Libraries line had increased Akt signaling upon PL 4032 e po sure. Another possibility is that PL 4032 resistant BRAFV600E mutants have alternative signaling at the level of Raf, as has been described for cell lines with acquired resistance to a different Raf inhibitor, AZ628, which show increased signaling through C Raf. The increase in pErk in an NRAS Q61L mutant cell line could be e plained by abrogation of negative feedback loops medi ated mainly by dual specificity phosphatases, as reported with Mek inhibitors, and the recent description of increased C Raf signaling when het erodimerizing with inhibited B Raf in BRAF wild type cells.
Therefore, the modulation of feed back loops and alteration of Raf dimerization upon treatment with Raf inhibitors may also have a role in the differential sensitivity to PL 4032 in BRAFV600E mutant cell lines. Finally, differences in e pression of pro and anti apop totic molecules like Bim and Bad may allow some BRAFV600E mutant Inhibitors,Modulators,Libraries cell lines to undergo growth arrest but not die from apoptosis upon e posure to PL 4032. Stud ies are ongoing to further e plore these possibilities. We e plored the Inhibitors,Modulators,Libraries use of PET imaging as a mean to GSK-3 non invasively detect PL 4032 sensitivity. In vitro we found that any of the three PET tracers FDG, FLT and FAC could be used to distinguish between melanomas with a NRAS or a BRAFV600E mutation based on the differential effects of PL 4032 on cell cycle and metabolism. FDG could furthermore be used to distinguish between BRAFV600E mutant melanomas with high or low sensitiv ity to PL 4032.
The PI3K Akt pathway has been widely selleck chem inhibitor regarded as having a role in the regulation of glucose metabolism through mTOR, but recently the LKB1 AMPK pathway has been found to be regulated by onco genic BRAFV600E signaling, which together may e plain the marked and rapid effects of PL 4032 on inhibiting FDG uptake. We e plored this possibility in two cell lines. Our data suggests a minor increase in pAMPK upon PL 4032 e posure, which may be in line with the proposed hypothesis. Conclusions These studies in melanoma cell lines may allow to better interpret the