Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

Abstract

Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.

Figure 1

Expression of BCL2 family genes in the LL-100 panel. (a) Heat map of RNA-seq gene expression data of BCL2 family members. Framed in red are expression levels of MCL1 and BCL2 in ALCL and PEL. (b) Results of qRT-PCR showing expression of BCL2 family members. (c) Western blot analysis showing the protein expression of MCL1, BCL2 and BCLXL in ALCL and PEL cell lines. DLBCL and MM cell lines were included as controls. Note that ALCL and PEL cell lines are consistently MCL1^pos/BCL2^neg.

Figure 2

SiRNA-mediated knockdown of BCL2 family members in ALCL cell lines. Life cell imaging shows effects of knockdown of BCL2 family members on growth (cell counts) and apoptosis (caspase 3/7 activity) 48 h after onset of treatment with siRNA oligos. (a) Knockdown of MCL1 and BCLXL; (b) knockdown of MCL1 and BCL2A1. Note synergistic effects of MCL1 and BCLXL knockdown in cell line L-82 and additive effects of MCL1 and BCL2A1 knockdown in cell line SU-DHL-1. The horizontal line shows the expected additive effects of the combination of siRNA oligos.

Figure 3

Effect of AZD-5991 and ABT-263 on growth and apoptosis of ALCL cell lines. Life cell imaging data showing the effect of the MCL1 inhibitor AZD-5991 (300 nM) and the BCL2/BCLXL inhibitor ABT-263 (300 nM) on growth (48 h) and apoptosis (12 h) of (a) L-82, SU-DHL-1 and (b) SR-786, KARPAS-299. (c) shows effects of AZD-5991 and BEZ-235 on growth and apoptosis of cell lines L-82 and SU-DHL-1. Apoptosis was assessed by caspase 3/7 activity. The horizontal line shows the expected additive effects of the combination of both inhibitors. Note that the only cell line that does not respond to ABT-263 is the BCLXL^low cell line KARPAS-299.

Figure 4

Effect of BEZ-235 on growth and apoptosis of ALCL cell lines. Life cell imaging data showing the effect of the PI3K/mTOR inhibitor BEZ-235 on (a) growth (48 h) and (b) apoptosis (12 h) of ALCL cell lines L-82 and SU-DHL-1. Apoptosis was assessed by caspase 3/7 activity.

Figure 5

Effect of BEZ-235 on PI3K/AKT activity and on expression of BCL2 family members. Western blots (12% PAA gel) showing (a) dose- and (b) time-dependent effects of BEZ-235 on the expression of BCL2 family members and on phosphorylation of AKT, the mTOR target S6 and the BCL2 family member BAD. Dose-dependent effects were assessed after 24 h, time-dependent effects with a concentration of 300 nM BEZ-235. (c) Kinetics of apoptosis induced by BEZ-235 and AZD-5991 as determined by caspase 3/7 activity. Note that the kinetics of caspase 3/7 activity stimulated by BEZ-235 and AZD-5991 do not differ.