Activated K-RAS Down-Regulated Small GTPases and Cell Motility of Myeloid Sarcoma-Inducing Leukemia Cells Through Erk Signaling Cascade

Abstract 2459

The molecular mechanism of myeloid sarcoma (MS) formation in the patients with acute myeloid leukemia (AML) is not clear. By analyzing the genetic mutations, we found that AML patients with combined MLL/AF10 and activated N-/K-RAS mutation had a high occurrence of association with MS. We had previously performed a retroviral transduction/transplantation assay and demonstrated that the activated KRAS mutation could not immortalize murine bone marrow (BM) cells, whereas MLL/AF10 alone induced myeloproliferative disease-like myeloid leukemia (MPD) in the transplanted mice. Combination ofMLL/AF10 and activated K-RAS mutation induced MPD in a shorter latency, in addition, multiple MS were detected in the abdominal-, subcutaneous-, or intramuscular fat of the transplanted mice. To understand the role of activated KRAS mutation in cooperation with MLL/AF10 in the MS formation, we performed an in vivo imaging analysis to compare cell migration between cells carrying single and combined mutations. Our results showed that the cells carrying combined mutations were slowly migrating to the omental and gonadal adipose tissues of the transplanted mice after intra-peritoneal injection. On the contrary, the cells carrying only MLL/AF10 were rapidly condensed and entered into blood stream. An in vitro transwell migration assay confirmed that the cells carrying combined mutations exhibited a reduced cell motility in response to SDF-1 treatment. Analysis of the Cxcr4 gene expression did not show significant difference between cells carrying single and combined mutations, suggesting that the slow migration was not attributed to lacking receptor for SDF-1. Further determination of the cell migration-related small GTPases revealed that the amount of total and GTP–bound Rac, Cdc42 and Rho was reduced in the cells carrying combined mutations. Moreover, treatment of SDF-1 further down-regulated protein expression and activity of these small GTPases. To understand which signaling pathway(s) linked the KRAS mutation to the down-regulated small GTPases, we analyzed the amount of total and phosphorylated signaling molecules and found that Erk, but not Jnk1 and p38 Mapk, had a similar protein expression and activation pattern as those of the GTPases, suggesting that the reduction in cell motility of the MS-inducing leukemia cells might be mediated via KRAS-Mek-Erk axis.