Abstract: Classical Hodgkin Lymphoma (cHL) is a malignancy of mature (post germinal center) B-lymphocytes that afflicts almost 20,000 individuals annually in North America and Europe alone. While the majority of patients diagnosed with HL are cured with multi-agent chemotherapy, 15 % of patients are refractory to conventional chemotherapy and almost half of patients with high-risk disease relapse. Only 2 agents have been approved in the last 30 years for relapsed HL and novel therapies are needed. Bruton’s Tyrosine Kinase (BTK) is a member of the TEC family and plays a central role in B-cell signaling, activation, proliferation and differentiation. Several studies have shown that BTK is upregulated in a proportion of Reed-Sternberg cells and has been associated with PI3K, mTOR, FAS and NF-κB signaling. Furthermore, pathways upregulated in cHL such as CD40 ligand binding, elevated chemokines such as IL-6 and activation of toll like receptor (TLR) pathways have been associated with BTK upregulation. Thus, BTK inhibition may have therapeutic potential in cHL.
Methods: The BTK inhibitor Ibrutinib at 0-15 micro molar doses was incubated for 72 hrs. with a panel of HL cell lines (KM-H2, L1236, L-428). Cell viability was evaluated using trypan blue. Apoptosis was analyzed using annexin V FITC and histone DNA ELISA. Protein expression was analyzed using western blotting with RNA post ibrutinib treatment subjected to RT PCR.
Results: Ibrutinib suppressed viability of these HL cell lines in a dose dependent manner (IC50s ~5-7.5 µM in KM-H2, L1236 and L-428). The drug induced statistically significant apoptosis in the responsive cell lines in a dose dependent manner (>50% apoptosis at IC50 doses). Molecular analysis using western blotting and RT-PCR revealed significant induction of apoptotic markers and down-regulation of pro-survival markers. Further characterization of the effect of Ibrutinib on PI3K, mTOR, FAS and NF-kB signaling is ongoing. Ibrutinib is now being evaluated in ICR SCID mice with sub-cutaneous xenografts of KM-H2 and L1236. A pre-clinical efficacy trial using maximum tolerated dose (MTD) given by gavage is ongoing.
Conclusion : These are the first studies showing in vitro and ex vivo effects of BTK inhibitors in HL and may provide support for further clinical investigation.