Genomic Stability: A Novel Function of NF-Kb in Lymphomas.

Abstract 3240

Poster Board III-177

Lymphomas are heterogeneous diseases comprising multiple clinical and biological subgroups. Several studies have shown that genomic instability and constitutive activation of the NF-kB pathway are key features for lymphoma development (Shen M,Hematologica 2007). However, it remains unclear whether changes in activation of the canonical and non-canonical NF-kB pathways (rel-A/p50 and rel-B/p52, respectively) reflect a response to genomic instability and therefore promotes lymphomagenesis. To answer this question, we first demonstrated in two lymphoma cell lines (Daudi and OCI-Ly3) that DNA damage induced by Doxorubicin (2 mcg/mL) resulted in nuclear localization of rel-A and Rel-B. Then, to determine the role of each NF-kB pathway in DNA repair and centrosome duplication we compared the number of cells positive for phospo-H2aX (pH2aX) and centrosomes numbers (measured by gamma-tubulin) in p105-siRNA (canonical) and P100-siRNA (non-canonical) with luciferase-siRNA (control) expressing cells. Our results showed that the expression of p105 and p100 siRNAs increase the number of pH2aX (+) cells compared to control. Subsequently, a time course measuring pH2aX (+) cells was performed after treating p105, P100 and luciferase siRNA OCI-ly3 expressing cell lines with Doxorubicin (2 mcg/mL). In cells expressing luciferase siRNA, pH2aX (+) cells peak (60%) at 60 minutes (min) and return to normal at 120 min, in p105 siRNA –cells pH2aX peak at 90 min (90% + cells) and then decrease similarly to luciferase siRNA cells. P100 siRNA cells demonstrate a continuous increased in pH2aX (+) cells up to 80%. In addition, p100 siRNA expression was associated with centrosome amplification (>2 centrosomes in 20-30% of the cells vs. < 8% in p105 or Luciferase siRNA expressing cells). Also, the expression of NF-kB siRNAs delayed doxorubicin-induced phosphorylation of p53 (serine 15 – target of ATM) and CHK2. To evaluate whether the genomic instability caused by both NF-kB siRNAs affects tumor development, we performed xenograft experiments. Our results demonstrated that NF-kB siRNAs not only slow down tumor initiation but prevented tumor development (p105 siRNA= 8 days delayed and 22% were tumor free and p100 siRNA= 13.5 days delayed and 55% were tumor free compared to luciferase siRNA). To investigate these findings in primary tissues we measured the number of phospo-H2aX (+) cells and the levels of rel-A and rel-B nuclear localization in 40 primary lymphoma tumor samples. Our results demonstrated that phospo-H2aX levels inversely correlated with rel-B nuclear localization (r=-0.58, p<0.0001). To identify possible explanations for these results, gene expression analysis was performed in cells expressing NF-kB siRNAs. Our results demonstrated that p105 siRNA regulated genes involved in DNA repair (PPP2R5C, ING5, SYF2, SYF2, XRCC6, etc) and p100 siRNA regulated genes involved in both DNA repair and centrosome duplication (GADD45 alpha, cyclin G, REDD1, PCBP4, etc) consistent with our results above. Quantitative PCR for some these genes during a doxorubicin-time course confirmed GADD45 alpha, cyclin G, PCBP4 and SFRS6 to be induced. We explored further the role of GADD45 alpha in lymphomas and found that knock down of this protein increase doxorubicin sensitivity by 50-fold. Overall this study demonstrated that activation of each NF-kB pathway is essential for maintaining genomic stability and therefore promoting tumor resistance to chemotherapy in lymphomas. In addition, we identified that GADD45 alpha is important target of the non-canonical NF-kB pathway for mediating genomic stability. These findings provide the rationale for designing novel agents aiming at targeting key genes involved in genomic stability.