Preclinical Anti-Lymphoma Activity of the BCL-2 BH4 Domain Antagonist Bda-366 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell non-Hodgkin lymphoma. MCL is genetically characterized by t(11;14) translocation, which leads to cyclin D1 overexpression. Despite the introduction of high-dose chemotherapy followed by autologous stem cell transplantation and novel agents including the Bruton's tyrosine kinase inhibitor ibrutinib, the proteasome inhibitor bortezomib and the immunomodulator lenalidomide, MCL still remains an incurable disease. BCL-2 is often amplified and/or overexpressed in MCL; high-level copy number gains of the BCL2 locus at 18q21.3 are most frequent. The novel BCL-2BH4 domain antagonist BDA-366 converts anti-apoptotic BCL-2 into the pro-apoptotic molecule (Han et al., Cancer Cell 2015). Considering the promising clinical activity of the BH3 mimetics venetoclax (Davids et al., JCO 2017), we investigated anti-lymphoma effects of BDA-366 in MCL. A total of eight MCL cell lines (Z-138, JVM-2, Granta-519, MINO, JeKo-1, REC-1, MAVER-1 and NCEB-1) were used. Z-138, Granta-519, MINO and NCEB-1 have high-level DNA amplification of BCL-2 . BDA-366 induced dose- and time-dependent phosphatidylserine externalization in MCL cells, with 72-h ED₅₀ (effective concentration inducing 50% cell killing as measured by annexin V positivity) values ranging from 9.3 to 240 nM. 72-h IC₅₀ values (concentration at which cell growth is inhibited by 50%) were also determined, which ranged from 5 to 30 nM. BDA-366 showed smaller differences in sensitivity than venetoclax, raising the possibility that BDA-366 may exhibit broader anti-lymphoma activity against heterogeneous MCL backgrounds. Basal levels of BCL-2 (determined by flow cytometry) did not significantly affect MCL cell susceptibility (ED₅₀) to BDA-366 ( r = -0.08, P = 0.85). The presence of high-level DNA amplification of BCL-2 was not associated with BDA-366 sensitivity either. BDA-366 did not increase total BCL-2 levels but it actively promoted the exposure of pro-apoptotic BH3 domain of BCL-2, supporting its proposed mode of action. BDA-366 did not significantly affect cell cycle distribution; S and G2/M phase cells appeared to be susceptible to BDA-366-induced apoptosis. BDA-366 induced loss of mitochondrial membrane potential, caspase-3 cleavage and BAX activation, indicating mitochondrial apoptosis. p53 knockdown (>85% knockdown efficiencies, as determined by Western blotting) desensitized p53 wild-type cell lines Z-138 and JVM-2 to BDA-366 (ED₅₀ values of 29 nM and 10 nM in Z-138; 303 nM and 108 nM in JVM-2), implying the interference of p53 with BCL-2 signaling. BDA-366 synergized with venetoclax to induce apoptosis in venetoclax-sensitive Z-138 (averaged combination index (aCI) = 0.25) and MINO cells (aCI = 0.14), indicating that the combination may abrogate BCL-2-mediated survival signaling in MCL. The combination effects of BDA-366 with doxorubicin or AraC were additive (aCI ~ 1.0). BDA-366 induced apoptosis in primary leukemia cells from patients with B-cell chronic lymphoproliferative disorders. BDA-366 appeared to take longer time to fully induce apoptosis than ABT-199, perhaps reflecting the distinct mode of action of BDA-366 that changes BCL-2 conformation. From a clinical viewpoint, the slower pharmacodynamics in inhibiting BCL-2 and inducing MCL cell death may reduce the risk of tumor lysis syndromes in patients receiving BDA-366. Collectively, BCL-2 BH4 inhibition may offer a novel therapeutic strategy for MCL.