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Journal of Clinical Oncology, 2005 ASCO Annual Meeting Proceedings.
Vol 23, No 16S (June 1 Supplement), 2005: 7533
© 2005 American Society of Clinical Oncology
Anthrax tumor toxin (LeTx) selectively kills B-RAF mutant melanomas
A. E. Frankel,
R. J. Abi-Habib,
J. O. Urieto,
S. Liu,
S. H. Leppla,
G. F. van de Woude and
N. S. Duesbery
Wake Forest Univ Sch of Medicine, Winston-Salem, NC; National Insts of Health, Bethesda, MD; Van Andel Research Institute, Grand Rapids, MI
7533
The efficacy of anthrax tumor toxin (LeTx) was tested on a panel of 18 human melanoma cell lines using a 3H-Leucine incorporation inhibition assay. 11/18 cell lines were sensitive to LeTx (IC50 < 350 pM, percent cell kill > 75%), while the remaining 7/18 were not sensitive to LeTx (IC50 > 750 pM, Percent cell kill < 60%). 10/11 sensitive melanoma cell lines carried the V599E BRAF. Only 1/7 resistant cell line was BRAF mutation positive (p < 0.0001). 6/7 resistant cell lines carried the Q61R or Q61K N-Ras mutation. Next, LeTx effects on 3H-Thymidine incorporation were measured. In 11/11 sensitive cell lines, the IC50 for 3H-thymidine incorporation was lower than that of 3H-Leucine incorporation indicating that the majority of surviving cells at maximal concentration were in cell cycle arrest. 4/7 cell lines that were not sensitive to LeTx by 3H-Leucine incorporation had low IC50 values in the 3H-Thymidine incorporation inhibition assay (IC50 < 275 pM) and a significantly lower percentage of 3H-thymidine uptake at maximum concentration suggesting significant tumor cell growth arrest rather than tumor cell death. Anthrax toxin receptor (ATR) levels, measured by saturation assay with 125I-PA, varied between 3562 and 32440 receptors/cell with resistant cell lines having the same levels of ATR expression as sensitive cell lines and no correlation of ATR receptor levels with IC50 for LeTx sensitive cell lines. Total and phosphorylated levels of MEK1/2 and ERK1/2 in the cell lines were quantified by western blots and densitometry. LeTx killing correlated with MEK1/2 activation levels. 9/11 sensitive melanoma cell lines and 2/7 resistant melanoma cell lines had a P-MEK1/2 to MEK1/2 ratio > 0.3 (p < 0.001). P-ERK1/2 to ERK1/2 levels did not correlate with melanoma cell line sensitivity to LeTx. The small molecular weight MEK1/2 inhibitor U0126 was tested for effects on the cell lines. 6/6 LeTx resistant cells were resistant to U0126; 5/9 LeTx sensitive cells were sensitive to U0126. In summary, melanoma cell line LeTx sensitivity correlated with B-Raf mutation, P-MEK1/2 levels, and U0126 sensitivity, but not ATR or P-ERK1/2. In future clinical trials of LeTx in metastatic melanoma, these results may provide molecular targets to identify responders.
No significant financial relationships to disclose.
Abstract presentation from the 2005 ASCO Annual Meeting
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