Evaluation of the Ras/B-Raf/SHP-2 Axis in B Cell Malignancies.

Abstract

The Ras/Raf/MEK/MAPK pathway is a major regulator of tumor cell proliferation. Ras mutations have been known in many malignancies for several years, whereas more recent reports have shown that activating mutations of the BRAF gene are present in a large percentage of human malignant melanomas and thyroid carcinomas and in a substantial proportion of colon carcinomas (Nature 2002; 417:949–54). The vast majority of these mutations represent a T1796A change resulting in a V599E substitution within the activation segment of B-Raf. This mutant B-Raf kinase is constitutively active and results in inappropriate stimulation of downstream MAPK/ERKs and tumor cell proliferation. The PTPN11 gene encodes SHP-2 (Src homology 2 domain-containing protein tyrosine Phosphatase), a nonreceptor tyrosine protein tyrosine phosphatase (PTPase) that modulates Ras signaling. Germline mutations in PTPN11 are responsible for Noonan syndrome (NS), whereas somatic mutations have been detected in juvenile myelomonocytic leukemia (JMML). We investigated the presence of mutations in the Ras/B-Raf/SHP-2 axis in 25 lines from B-cell malignancies, including 19 multiple myeloma (MM) cell lines. DNA was isolated and B-Raf (exons 11 and 15), PTPN11 (3 and 13) and K-, N- and H-Ras sequences were amplified by PCR using specific flanking primers. The PCR products were purified and sequenced in automatic sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). Ras gene mutations were found in 11/25 (44%) of the studied cell lines. None of the previously described activating B-Raf mutations, including the most prevalent V599E, were found in our panel. Only one MM cell line demonstrated a C1332A substitution, which corresponds to a D444E change, which is predicted to have a minimal, if any, impact on kinase activity. A single PTPN11 G178C substitution, corresponding to a G60R change (previously described in NS and AML), was detected in another MM line. These data confirm the previously known role of Ras mutations in B-cell malignancies and identify a rare occurrence of a PTPN11 mutation in our panel. On the other hand, activating mutations of the B-Raf kinase are unlikely to play a significant part in the pathogenesis of B cell malignancies, in contrast to their prominent role in several types of solid tumors. In total, our data suggest that the Ras/Raf/MEK/MAPK can be overactive in these malignancies due to activating genetic events upstream of B-Raf, such as the previously described activating Ras mutations.