BCL2 family proteins determine cell fate and comprise pro-apoptotic "initiators" (NOXA, BIM, PUMA), anti-apoptotic "guardians" (BCL2, MCL1, BCLX) and pro-apoptotic effectors (BAX/BAK). Venetoclax, a BCL2 inhibitor, received regulatory approval in therapy of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. However, venetoclax is modestly effective in NHL. MCL1 is a short-lived pro-survival protein that is frequently overexpressed in NHL, leading to increased cell survival and drug resistance. MCL1 transgenic mice develop lymphoma, mostly of the diffuse large B-cell (DLBCL) type. Thus, selective targeting MCL1 represents a promising pharmacologic strategy in NHL. AZD5991 is a small molecule inhibitor highly selective towards MCL1 (Tron et al, 2018) in clinical trials. Here we explored pre-clinical activity of AZ5591 in NHL.
Experiments were conducted in activated B-cell (ABC)-like (OCI-LY3, U-2932) and germinal center (GC)-like (OCI-LY18/19, SUDHL4/6/10, VAL) DLBCL cell lines; parental and ibrutinib-resistant Mino mantle cell lymphoma (MCL) cell lines. Peripheral blood mononuclear cells were isolated from patients with CLL and MCL and co-cultured with BAFF- or CD40L-expressing stroma. AZD5991 was obtained from Activ Biochem Ltd.
MCL1 expression was detected in 10 tested DLBCL cell lines, highly expressed in 7/10. Meanwhile, BCLX was upregulated in eight, and BCL2 in 5/10 lines. Four cell lines expressed all three proteins. To confirm relevance of this data, we conducted analysis of primary DLBCL lymph nodes (n=30). MCL1 and BCLX were expressed in 50% and 80% of GC-like, and 10% and 25% of non-GC tumors, respectively.
Treatment with AZD5991 restricted growth of DLBCL cells in a dose-dependent manner. GC-like cell lines VAL, SU-DHL4 and SU-DHL6 were highly susceptible with IC50~0.2 µM. Interestingly, they expressed relatively low levels of MCL1. Similarly, parental and ibrutinib-resistant Mino MCL cells were susceptible to MCL1 inhibition, with IC50 of 0.1 µM and 0.5 µM, respectively. Meanwhile, SU-DHL10, OCI-LY19 (GC-like), OCI-LY3 and U-2932 (ABC-like) cells were resistant to AZD5591. Consistent with its mechanism of action, immunoprecipitation assays showed that AZD5591 displaced BIM from MCL1 in NHL cells.
We then evaluated MCL1 inhibition in primary neoplastic B-cells. CLL and MCL cells from patients were co-cultured with either CD40L- or BAFF-expressing stroma for 24 h. While CD40L predominantly induced BCLX, BAFF upregulated MCL1 in those cells. Consistent with this, BAFF-stimulated cells were highly sensitive to AZD5991, while CD40L-stimulated cells exhibited resistance.
Since MCL1 acts in balance with pro-apoptotic effectors BAX and BAK at the mitochondrial membrane, we assessed the physiological effects of MCL1 inhibition on the mitochondrial function. Treatment with AZD5991 induced mitochondrial depolarization and led to a reduction in mitochondrial mass as well as increased generation of reactive oxygen species. This was accompanied by a decrease in cellular maximal respiratory capacity in both DLBCL and parental/ibrutinib-resistant MCL cells. Meanwhile, the rate of glycolysis was not significantly impacted. Interestingly, MCL1 inhibition induced mitophagy in sensitive (VAL) but not resistant cells (OCI-LY3).
Next, we evaluated AZD5991 for synthetic lethality in a functional MTS-based screening assay using a panel of 189 small molecule inhibitors that target a variety of distinct signaling pathways activated in cancer (Tyner et al, 2018). AZD5991 demonstrated synergy with other BH3-mimetics. Co-treatment of DLBCL cells with BCL2/X inhibitors AZ4320, venetoclax and navitoclax overcame resistance to AZD5991.
In summary, MCL1 inhibition using selective BH3-mimetic AZD5991 restricts cell proliferation and induces apoptosis in a subset of DLBCL, ibrutinib-resistant MCL cells and primary neoplastic B cells. MCL1 inhibition leads to mitochondrial dysfunction and mitophagy. Resistance to MCL1 inhibition may be overcome by concurrent targeting of alternative anti-apoptotic proteins (BCL2/X) in NHL.
Tyner:Syros: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Aptose: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Agios: Research Funding. Danilov:Abbvie: Consultancy; Celgene: Consultancy; Rigel Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Research Funding; Aptose Biosciences: Research Funding; Astra Zeneca: Consultancy, Research Funding; Verastem Oncology: Consultancy, Research Funding; Takeda Oncology: Research Funding; Gilead Sciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; TG Therapeutics: Consultancy; Nurix: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Karyopharm: Consultancy.
Asterisk with author names denotes non-ASH members.