Abstract 3112: Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH, 2012). Children with BL who relapsed or progressed have chemotherapy resistant disease and can rarely be cured after salvage therapy (Cairo et al.,JCO, 2012). Bruton's tyrosine Kinase (BTK) is a major regulator of normal B-cell development and functions through activation of the BCR, MAPK, NF-κB and AKT signaling pathways (Qiu et al., Oncogene, 2000). Chronic active B-cell receptor signaling through activation of the BTK, can be specifically inhibited by a selective BTK inhibitor, ibrutinib (Davis et al., Nature, 2010). However, the mechanism(s) and function of BTK is still poorly understood in B-cells, especially in BL. Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in a variety of organisms and stem cells for new experimental and therapeutic tools (Sander et al, Nat. Biotech., 2011; Carlson et al., Mol Ther., 2012). OBJECTIVES: We hypothesize that 1) TALEN nuclease technology is suitable for the modification of endogenous BTK gene expression in BL cells, and 2) a TALENs-mediated BTK knockout model will provide a useful tool for understanding the mechanism and function of BTK and the development of therapeutic agents that target BTK in BL. METHODS: BTK TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011; Lee/Cairo et al., ASH, 2012) for BTK gene disruption. BTK TALENs validities in 293 and Raji cells were confirmed by surveyor assay and sequencing analysis. Western blotting was performed and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional BTK TALENs (BTK-T1 and BTK-T2) targeting endogenous BTK gene were constructed and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293 cells. We confirmed that entire BTK coding sequences were deleted by PCR genotyping and sequencing analysis in stable BTK-knockout Raji cell clones (BTK+/-). BTK-T12-25 and BTK-T12-29 showed a significant decrease of BTK protein expression (50±1.1% reduction, p<0.0005; 27±0.7% reduction, p<0.0005, respectively) compared to empty vector-transfected Raji cells as mock control. There was a significant decrease in Erk phosphorylation in BTK knockout Raji cells compared to mock control (30±1.6%, p<0.001). CONCLUSIONS: These preliminary data suggest that BTK-TALENs are useful tools for the disruption of endogenous BTK gene expression. This TALEN mediated BTK knockout model will be used for studying BTK function in BL and the efficacy of BTK inhibitors as potential therapeutic agents in BL. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3112. doi:10.1158/1538-7445.AM2014-3112