Activation of K-Ras in Arf-Null Mice Is Not Sufficient for Malignant Transformation of Post-Germinal Center B-Cells

Abstract 5016

Activating mutations in Ras (N- and K-) are the most common mutations in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. As previously reported, we re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found the most common mutations to affect Ras oncogenes (N and K-). To test the role of Ras activation in MM pathogenesis directly, we generated mice harboring a mutant K-ras allele, Lox-stop-lox(LSL)- K-ras G12D (K-ras^+/G12D) with Cγ1-Cre knock-in mice (Cγ1^+/Cre), activated specifically in germinal center (GC) cells. K-ras^+/+/Cγ1^+/Cre mice served as negative controls. We found 58% of unstimulated K-ras^+/G12D/Cγ1^+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12), phenotypes that have previously been reported for K-ras^+/G12D mice. Tumor cells demonstrated successful recombination of the K-Ras locus. We showed recombination in splenic germinal center cells (B220+, GL7+ and IgG1+), as well as in post-germinal center cells (B220-, CD138+). No evidence of plasma cell proliferation was seen by serum ELISA, SPEP, flow cytometry or histological examination. We confirmed these results by crossing the K-ras^+/G12D mice to a second strain, AID-Cre, which expresses the recombinase earlier and more specifically in the GC. We found 100% (n=20) of K-ras^+/G12D/AID-Cre-YFP mice possessed visible, small benign papillomas on the ventral neck, as early as 3 weeks of age. To stimulate malignant transformation, vitamin D deficient chow and/or sub-lethal radiation was given to K-ras^+/G12D/AID-Cre-YFP and negative control, K-ras^+/G12D mice. Dual treatment increased the size and number of papillomas, giving a median survival of 26 weeks, compared to 52 weeks with control. However, no evidence of MM was detected by serum ELISA, SPEP, flow cytometry or histological examination. Finally, we generated AID-Cre-K-ras^+/G12D triple transgenic mice in a tumor prone Arf-null background. After 13 wks, we found sarcomas in 66% (n=3) of K-ras^+/G12D/AID-Cre-YFP/Arf^−/− mice. Additionally, 100% (n=3) of these mice had rapid growing benign papillomas and only a minimal increase in the plasma cell compartment, compared to K-ras^+/G12D/AID-Cre/Arf ^+/− control. Together, these data demonstrate that Ras activation is not sufficient to transform primary germinal center or plasma cells, even in an Arf-null context, and suggests that specific myeloma events, yet to be identified, are required for malignant transformation.