Analysis of PKB/Akt-Signaling and Oncogenic Ras in Multiple Myeloma.

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Abstract 831 Introduction:Dysregulated signaling pathways contribute to cell growth, survival and drug resistance in multiple myeloma (MM). In about 50 percent of primary MM samples the protein kinase B (PKB)/Akt pathway appears to be constitutively active and its blockade strongly impairs survival, thus defining Akt-dependent versus Akt-independent MM. Because illegitimate activation of survival pathways can be a consequence of oncogenic Ras mutations, we analyzed the role of Ras in Akt-dependent primary MM samples. Methods:Primary MM samples from different patients (n=35) were tested for cell death induction with the small molecule Akt-inhibitor Akti 1/2 and the sensitivity was correlated to the mutation status of N- or K-Ras in these samples, which was obtained by RT-PCR. Immunohistochemistry was used to demonstrate the presence of phosphorylated Akt in corresponding patient biopsies. Apoptosis was measured by flow cytometry with Annexin V-APC or Annexin V-FITC and propidium-iodide staining. We further analyzed the consequences of Ras depletion with regard to Akt activation in a Ras wildtype (AMO-1) versus a K-Ras mutated (MM.1s) and an N-Ras mutated (INA-6) myeloma cell line using siRNA knockdown and Western blotting. Isoform specificity of the siRNAs was confirmed through the effects on ectopically expressed HA-tagged Ras proteins. Results:Of the 35 primary MM samples tested 17 (48.6%) showed sensitivity to pharmacologic inhibition of Akt and thus constituted the Akt-dependent myeloma subgroup. Activating mutations of either K-Ras (n=7) or N-Ras (n=7) were found in 14 samples (40%). However, we did not observe any obvious correlation between the mutation status of Ras and sensitivity to the Akt inhibitor, suggesting that oncogenic Ras is not a prerequisite for the constitutive activation of Akt. We extended our analysis to MM cell lines and performed isoform-specific siRNA mediated Ras knockdown. Depletion of K-Ras induced apoptosis selectively in K-Ras mutated MM.1s cells, while N-Ras mutated INA-6 cells were most sensitive to N-Ras knockdown. Contrarily, knockdown of either of these isoforms had no effect on survival in the Ras wildtype cell line AMO-1. Ras knockdown did not lead to a notable decrease in Akt(Ser473) phosphorylation, again indicating that oncogenic Ras signaling may not be mediated through the PKB/Akt-pathway in MM. Conclusion:Whereas MM cell survival might well be promoted by activating Ras mutations, oncogenic Ras does not appear to be instrumental in the constitutive activation of the PKB/Akt pathway in primary MM. Targeting Ras isoforms themselves can, however, lead to apoptosis in Ras mutated MM cell lines. Thus, even though oncogenic mutated Ras is currently not a druggable target, these experiments underscore that it is nonetheless desirable to develop ways to specifically block its function in MM. Disclosures:No relevant conflicts of interest to declare.