UCH-L1 Provides An Essential Survival Signal in Malignant B-Cells by Enhancing NF-κB Activation.

Abstract Using an activity-based approach, we previously described the over-expression of the deubiquitinase ubiquitin carboxy terminal hydroxylase 1 (UCH-L1) in several B-cell malignancies including multiple myeloma, Burkitt lymphoma, non-Hodgkin lymphoma and chronic lymphocytic leukemia. UCH-L1 over expression also correlates with a poor prognosis in non-lymphoid tumors including non-small cell lung, esophageal, colorectal and pancreatic cancers. Whether UCH-L1 expression contributes to the malignant phenotype in these conditions, however, has not been established. We therefore sought to determine the role of UCH-L1 in lymphoid malignancies through the use of lentivirusencoded short hairpin RNA (shRNA) targeting UCH-L1. When compared with a control non-silencing shRNA, reduced UCH-L1 levels are associated with a profound block in proliferation and loss of viability (>90%) in the UCH-L1 expressing myeloma cell line KMS11. The introduction of these shRNAs into the UCH-L1 negative myeloma cell line KMS12 does not affect proliferation or viability. The association of cell death with reduced UCH-L1 levels was further reinforced using a doxycycline inducible shRNA construct. KMS11 and KMS12 cells grow equally well following transduction and selection of stable viral integrants. However, upon induction of shRNA production with doxycycline, greater than 90% of KMS11 cells die within 48-hours timed with the depletion of UCH-L1 while KMS12 cells continue to proliferate. These results strongly suggest that UCH-L1 is essential for survival in UCH-L1 expressing myeloma cells. Given the critical role of NF-kB signaling in many lymphoid malignancies, we examined the impact of UCH-L1 depletion on NF-kB activity. Using a secreted luciferase reporter assay, we determined NF-kB activity in control and UCH-L1 depleted KMS11 cells. We observed a 5-fold reduction of NF-kB activity in UCH-L1 knockdown cells compared to control. In order to establish a correlation between loss of UCH-L1 expression and NF-kB down-regulation, cells transduced with an inducible shRNA were treated with doxycycline and monitored daily for UCH-L1 and NF-kB activation. We observed a direct relationship between the level of UCH-L1 and NF-kB activity in these cells with a 5-fold reduction in NF-kB activity by 48-hours accompanied by reduced viability. These effects are entirely reversible with the removal of doxycycline from the growth medium. Doxycycline induction had no effect on the NF-kB activity of the UCH-L1 negative cell line KMS12. Knockdown of UCH-L1 does not affect TNF-alpha-induced NF-kB activity in these cells suggesting that UCH-L1 modulates the non-canonical NF-kB pathway. A relationship between UCH-L1 and NF-kB is further suggested by observing a 3.8-fold increase in baseline NF-kB activity in HeLa cells over-expressing UCH-L1. These data strongly indicate that UCH-L1 enhances NF-kB through the non-canonical pathway. In order to determine whether UCH-L1 over-expression can by itself promote tumorigenesis in vivo, we have engineered a UCH-L1 transgenic mouse. These mice will provide us the opportunity to study the effect of UCH-L1 expression on spontaneous as well as induced (carcinogen or oncogene) tumorigenesis. Further studies are examining the effect of forced UCH-L1 expression on B-cell development. This study will provide a better understanding of the role of UCH-L1 in hematological malignancies and may validate UCH-L1 as an important drug target for B-cell malignancies.