The Microenvironmental Stromal Cells Abrogate NF-κb Inhibitor Induced Apoptosis in Chronic Lymphocytic Leumkemia

Abstract Introduction: In recent years, the emergence of kinase inhibitors has drastically altered treatment strategies and improved outcomes in CLL patients, but lack of cure and resistance to therapy still remain serious issues. The transcription factor NF-κB influences several cellular functions such as proliferation, apoptosis and inflammation and is known as a key factor contributing to CLL development and progression. NF-κB is constitutively active in CLL and the NF-κB subunit RELA has been proposed as a prognostic marker in CLL with high RELA DNA-binding activity being predictive of short time to first treatment and overall survival. Therefore, NF-κB has gained attention as a promising therapeutic target. NF-kB inhibition induces apoptosis in CLL cells in vitro. However, whether this effect pertains in vivoremains unclear. Since the microenvironment is crucial for CLL cell viability circumventing apoptosis, we tested whether NF-κB inhibition modulates CLL viability in the presence of the microenvironment. Methods: The specific NF-κB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone (2-5 µg/ml) or combined with fludarabine (10 µM), rhBAFF (50 ng/ml), rhAPRIL (500 ng/ml), rhSDF-1a (100 ng/ml) or CD40 ligand (1 µg/ml) on primary CLL cells cultured alone (monoculture) or on bone marrow stromal cells (BMSC) (co-culture with a ratio of 20 CLL cells per stromal cell) for 48-144 h. Viability and apoptosis were measured by flow cytometry using AnnexinV/PI stainings. Protein expression was analyzed by western blot using standard protocols. NF-κB DNA-binding activity after DHMEQ treatment (5 µg/ml) for 6 h was measured by ELISA for all subunits using 1 µg of protein lysate for the NF-κB1 subunit and 10 µg protein lysate for the subunits RELA, NF-κB2, RELB and c-REL. RELA gene knockdown was performed by siRNA transfection (2 µM targeting and non-targeting siRNA). Results: NF-κB inhibition using DHMEQ led to apoptosis in monocultured CLL cells (viability 74% vs. 24%, n=17, p Conclusion: NF-κB inhibition in primary CLL cells shows great discrepancy between in vitro and in vivo scenarios. While DHMEQ treatment leads to apoptosis in mono-cultured cells by BAX upregulation and increased PARP cleavage, CLL cell viability is not affected in the presence of microenvironment, suggesting that the NF-κB pathway can be bypassed in vivo. Soluble ligands, especially BAFF, appear to be involved in mediating this protective effect. However, the combination of NF-κB inhibition with standard chemotherapy might represent a promising approach and warrants further clinical assessment. Disclosures No relevant conflicts of interest to declare.