Characterization of a Novel R-RAS Mutation Cloned from Primary AML Cells.

Mammalian RAS genes encode a family of small GTPases, which are involved in signal transduction pathways resulting in cell growth and differentiation. Many human cancers, including hematologic malignancies, have been found to harbor activating H-, K- and N-RAS mutations. We report here the discovery and characterization of a novel, activating R-RAS mutation cloned from primary AML cells. Total RNA was purified from AML blasts, reverse transcribed into cDNA and R-RAS-specific PCR products were sequenced on an ABI310 automated DNA sequencer. A mutation in codon 84 of R-RAS (Thr to Ala, T84A) was detected in a female AML patient with normal karyotype. The R-RAS T84A mutant was cloned into a hygromycin B-resistant pCGN vector and used to stably transfect BA/F3 cells, an IL-3-dependent murine pro B-cell line. The well-described R-RAS Q87L activating mutant was employed as a positive control for R-RAS-induced transformation of BA/F3 cells as well as for activation of signal transduction pathways. The R-RAS T84A mutant protected BA/F3 cells from the apoptotic effects of IL-3 starvation, albeit to a lesser extent than the R-RAS Q87L mutant. Interestingly, these two R-RAS mutants activate distinct signal transduction pathways. For example, the R-RAS Q87L mutant preferentially caused activation of the RAS-to-MAPK signal cascade as evidenced by Western blots of affinity precipitations with the GST-conjugated c-Raf-1 RAS binding domain, which is designed to bind GTP-loaded (activated) RAS proteins. The R-RAS T84A mutant activated the PI-3K pathway as demonstrated by Western blots of affinity precipitations with the GST-coupled RAS binding domain of the p110-alpha subunit of PI-3K. Additionally, the R-RAS T84A mutant induced a stronger phosphorylation of the PI-3K down-stream target Akt upon IL-3 stimulation (0.1 and 10 ng/mL, 10 minutes) of BA/F3 cells cultured overnight without IL-3. In conclusion, a novel R-RAS mutation was found in primary AML blasts and the mutant protein activates anti-apoptotic signaling through the PI-3K/Akt pathway.