ETV1 Is a Survival Gene That Is Expressed in a Subset of Multiple Myeloma

Abstract 2884

While several genes have been shown to participate in primary Ig translocations in about 50% of cases of multiple myeloma, it is still remains unclear how activation of these genes ultimately leads to a transformed phenotype. In nearly 15% of myeloma cases, t(4;14) results in the over-expression of MMSET and FGFR3. However the FGFR3 translocation is lost in about 25% of these patients. To identify genes associated with these changes we initially compared the gene expression profiles (GEP) of t(4;14) patients that expressed FGFR3 with those that did not. Using both supervised and unsupervised hierarchal clustering we were unable to identify significant differences associated with FGFR3 expression. Therefore we pooled these samples and compared them to GEP from normal plasma cells and MGUS patients to identify genes that were up-regulated in t(4;14) myelomas. As validation of this approach the two highest up-regulated genes were FGFR3 and MMSET (WHSC). Interestingly an additional gene that was significantly up-regulated (33 fold) was the ETS family transcription factor Ets variant gene 1 (ETV1; also called ER81). Further analysis of GEP data of MM patient samples revealed that ETV1 is primarily expressed in the genomically defined CD1, CD2 and MS subgroups in newly diagnosed patients (Arkansas dataset GSE4204). These data were confirmed in relapsed/refractory patients where expression was greatest in the 11q13 and 4p16 subsets (Tc classification, Mulligan dataset GSE9782). ETV1 is a member of the ETS family transcription factors and has been shown to be involved in recurrent translocations in prostate cancer and is also up-regulated in GIST. However no data exists on the role of ETV1 in MM. We investigated the expression of ETV1 in human myeloma cell lines (HMCL) and determined the effect of silencing on cell viability, proliferation, and drug responses.

To determine whether ETV1 is expressed in MM cell lines, 10 HMCL were subjected to real-time PCR and western blot analysis. All ten cell lines expressed ETV1 with MM.1s expressing the lowest levels. Consistent with the GEP data from the patient samples, the t(4;14) and t(11;13) lines expressed 5.5–13-fold higher levels of ETV1 than MM.1s. Surprisingly the highest expresser was not from one of these translocation groups, as RPMI8226 displayed a 74-fold increase compared with MM.1s. Determination of the potential reason for this level of over-expression is currently underway. Protein levels of ETV1 also correlated with mRNA level for all MM cell lines. To functionally characterize the role of ETV1 in myeloma, we utilized shRNA-mediated knockdown in the KMS11 line. ETV1 knockdown had no effect on cell proliferation, but did result in increased apoptosis compared to the vector control KMS11 cells, suggesting a biological role for ETV1 in myeloma cell survival. Consistent with this possibility, we observed increased sensitivity to the Bcl-2 inhibitor ABT-737 and the HDAC inhibitors SAHA and Romidepsin in ETV1-shRNA bearing KMS11 when compared to vector control. These results provide the first line of evidence that ETV1 plays a significant role in a subset of MM and suggest that therapies targeting this gene could have an impact in a subset of MM patients.