Chronic lymphocytic leukemia (CLL) relies on chronic B-cell receptor (BCR) signaling, and as such is effectively treated with Bruton's tyrosine kinase (BTK) inhibitors. However patients who relapse on BTK inhibitors such as ibrutinib have an extremely poor prognosis without additional intervention. Therefore, identifying and characterizing risk factors that predict relapse to BTK inhibitors is important. A series of 308 CLL patients have been enrolled on various ibrutinib clinical trials at the Ohio State University. Seven of these patients were identified to carry the translocation t(14;19), which leads to over-expression of B-cell leukemia 3 (BCL3), Of these 7, 6 (85%) have relapsed on ibrutinib while only 27% of patients (83/308) overall have progressed. BCL3 is known to regulate NF-κB transcription and influence B-cell function, and we found that overall BCL3 expression is increased in CLL compared to normal B-cells, therefore hypothesized that BCL3 may provide a competitive advantage to promote ibrutinib resistance. In order to test the role of BCL3 in ibrutinib resistance using an in vitro system, we overexpressed BCL3 or an empty vector (EV) control in ibrutinib responsive B-cell lines (BCL3 was at least 2-fold over-expressed relative to endogenous BCL3). Cells were treated with vehicle or 1uM ibrutinib for 1 hour followed by a washout, and proliferation and viability were evaluated at various time points. Ibrutinib inhibited cell proliferation and induced apoptosis, however BCL3 expression did not abrogate these effects. However, while examining BCR signaling proteins we found that BCL3 expression enhanced phosphorylation of BTK, suggesting that BCL3 stabilizes activated BTK to circumvent ibrutinib treatment. We next engrafted these EV or BCL3 over-expressing cells into the left or right flank, respectively, of immune-compromised (NSG) mice. Mice were sacrificed when either tumor reached a volume of 2000cm3, and the tumors were isolated and weighed. Tumors which developed from the BCL3 over-expressing cells at sacrifice were larger than those which developed from the EV cells by an average of 1332g. We also evaluated a disseminated tumor model where HBL-1 EV or BCL3 over-expressing cells were injected via the tail vein. Disease was monitored by weekly peripheral blood flow cytometry for human CD19. We saw that 6 of 11 mice intravenously engrafted with HBL-1 BCL3 present with peripheral disease when the mice meet removal criteria, whereas only 1 of 11 mice engrafted with HBL-1 EV cells developed peripheral disease. We conclude that the presence of t(14;19) indicates a higher risk of relapse in patients undergoing ibrutinib therapy, and these patients should be closely monitored for evidence of progression. Our studies also suggest that BCL3 promotes more aggressive disease in a lymphoma xenograft model, and may be involved in tumor migration in vivo.

Citation Format: Timothy L. Chen, Bonnie K. Harrington, Larry Beaver, Amy S. Ruppert, Nyla A. Heerema, Xiaoli Zhang, Hatice Gulcin Ozer, Amy J. Johnson, Jennifer A. Woyach, Erin Hertlein, John C. Byrd. BCL3 over-expression contributes an in vivo growth advantage in a B-cell lymphoma xenograft model and is a risk factor for ibrutinib relapse in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2017-3014