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

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.

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.

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.

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.

No relevant conflicts of interest to declare.