1016 - DEFACTINIB AFFECTS CHRONIC LYMPHOCYTIC LEUKEMIA CELL SURVIVAL WITH ADDITIVE EFFECT IN COMBINATION WITH BTK INHIBITORS

INTRODUCTION
The emergence of BTK inhibitors (BTKi) has improved the outcomes of patients with chronic lymphocytic leukemia (CLL). The pioneering bruton tyrosine kinase (BTK) inhibitor, ibrutinib along with the second-generation inhibitors acalabrutinib and zanubrutinib, have displayed remarkable therapeutic efficacy in patients with CLL. Despite the effectiveness of these agents, resistance and severe side effects can arise, resulting in treatment failure, highlighting the need for alternative treatments. Research on molecules involved in the increased survival and drug resistance of leukemic B cells, may unveil novel therapies for a successful outcome in CLL that can be employed clinically alone or in combination with the currently approved BTKi. Focal adhesion kinase (FAK) is a 125 kDa protein, which upon phosphorylation on tyrosine (Y) 397, is activated and can recruit numerous signalling proteins controlling several cellular processes such as adhesion, migration, apoptosis and proliferation, especially in solid tumors. Consequently, several inhibitors have been developed to target FAK phosphorylation at Y397 or its kinase activity. One such inhibitor is defactinib that has demonstrated safety and efficacy in clinical trials for solid tumors. However, limited efforts are made on investigating the effects of defactinib specifically on CLL. Based on this knowledge, we hypothesized that defactinib may exert a significant effect on the survival of CLL cells, and that its combination with BTKi may improve their cytotoxic effects, thereby enhancing their in vitro activity.
METHODS
B lymphocytes were isolated from eight CLL patients and then were treated with either 5uM of ibrutinib, acalabrutinib, zanubrutinib alone, or in combination with defactinib at a 1:1 ratio. Parallel experiments were performed where these cells were co-cultured with the human bone marrow stromal HS5 cell line, applying the same treatments. After 24 and 48 hours of treatment, apoptosis was evaluated through two methods: PARP cleavage analysis by western blot and Annexin V/Propidium iodide assay by flow cytometry. The expression level of FAK phosphorylation on Y397 representing its kinase activity, was examined using western blot analysis.
RESULTS
We demonstrated that defactinib alone exhibits greater apoptosis-inducing effects compared to BTKi alone. In particular, when defactinib was combined with BTKi, an increase in PARP cleavage was observed using western blot analysis, indicating that defactinib enhances BTKi activity. This finding was further supported by the Annexin V/Propidium iodide assay, where leukemic B cells exhibited a significant reduction in their viability when co-treated with both agents compared to treatment with either agent alone. Interestingly, the phosphorylation of FAK was not affected by BTKi alone. However, samples treated only with defactinib or in combination with BTKi showed a reduction in FAK phosphorylation. This suggests that the enhanced apoptosis observed in the co-treated conditions is specifically due to the selective inhibition of FAK activation. Furthermore, when CLL B cells were cultured with HS5 stromal cells, which are known to enhance CLL cell survival in vivo, co-treatment of defactinib and BTKi prevented the protective effect of HS5 cells and instead resulted in higher levels of apoptosis. This effect may be attributed to the alteration of FAK in the HS5 cell line upon treatment.
CONCLUSIONS
In summary, our findings demonstrate that defactinib, by inhibiting FAK activation, exhibits greater capability as an apoptotic agent compared to BTKi. Moreover, defactinib enhances the cytotoxic effects of BTKi in vitro and counteracts the protective effect of HS5 stromal cells on CLL cell survival. These results highlight FAK as a promising target for developing of novel therapeutic approaches in CLL. Furthermore, the combination of FAK inhibitors with BTKi may enhance their therapeutic efficacy, leading to deeper treatment responses and better outcomes for CLL patients. These findings suggest the potential clinical relevance of targeting FAK and warrant further investigation in clinical settings.