Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent type of B-cell non-Hodgkin lymphomas (B-NHL) in the Western hemisphere. While BCL2 gene deregulation was repeatedly associated with poor prognosis, the role of MCL1 in the biology of DLBCL remains largely unknown. ABT199 is a highly-selective inhibitor of BCL2 protein currently evaluated in clinical trials. Homoharringtonine (HHT) is a plant alkaloid and as a semisynthetic compound (omacetaxine) it was approved for the treatment of relapsed chronic myelogenous leukemia (CML). Anti-tumor activity of HHT includes downregulation of the anti-apoptotic protein MCL1.
The aim of the project was to evaluate the preclinical anti-lymphoma efficacy of BCL2 and MCL1-targeting agents ABT199 and HHT in DLBCL.
Immunophenotype of primary DLBCL samples was determined by immunohistochemistry (IHC) using the Hans algorithm. Sensitivity of DLBCL cell lines to ABT199 and HHT was determined by Annexin V-based apoptotic assay and WST8-based cell proliferation assay. DLBCL clones with downregulation of selected anti-apoptotic proteins were derived using pLKO1-based lentiviral particles containing shRNAs against BCL2, BCL-XL and MCL1. For upregulation, BCL2, BCL-XL and MCL1 were cloned in the lentiviral expression vector pCDHNeo and the prepared lentiviral particles were used for the transduction of DLBCL cell lines.
We analyzed molecular mechanisms of cytotoxic activity of HHT in 7 DLBCL cell lines, and confirmed decreased expression of MCL1 protein in all cases. By semi-quantitative protein expression analysis (western blot or IHC) we demonstrated that BCL-XL and MCL1 were detectable in all DLBCL cell lines (n=18) and primary samples (n=114, GCB=51, ABC=63), while BCL2 was not detectable in 6 out of 18 DLBCL cell lines and 32 out of 114 primary DLBCL samples. 8 out of 12 BCL2-positive DLBCL cell lines were sensitive to 1 microM ABT199 (i.e. did not survive 1 microM ABT199 by standard proliferation assay). In contrary, 6 out of 6 BCL2-negative DLBCL cell lines were resistant to 1 microM ABT199. 11 out of 12 BCL2-positive DLBCL cell lines were sensitive to 30 nM HHT (considered a steady-state plasma level in CML patients treated with HHT). 5 out of 6 BCL2-negative DLBCL cell lines were sensitive to 30 nM HHT. Significant drug synergism between ≤1 microM ABT199 and ≤ 30 nM HHT was observed in 8 out of 12 BCL2-positive, but only in 1 out of 6 BCL2-negative DLBCL cell lines.
We demonstrated that high expression of BCL2 positively correlated with sensitivity to ABT-199, irrespective of expression levels of BCL-XL and MCL1. Expression levels of BCL2 and BCL-XL negatively correlated with sensitivity to HHT. Expression level of MCL1 did not correlate with sensitivity to HHT.
Both targeted downregulation and transgenic overexpression of BCL-XL in selected DLBCL cell lines confirmed that the expression of BCL-XL negatively correlates with sensitivity to HHT (but not to ABT199). While increase in sensitivity to HHT was observed in 3 out of 3 DLBCL cell lines with targeted knock-down of BCL2, increase in sensitivity to ABT199 was observed only in 1 out of these 3 DLBCL cell lines. Targeted knockdown of MCL1 was associated with increased sensitivity to HHT in 1 out of 2 DLBCL cell lines, but with no change of sensitivity to ABT199.
HHT is a promising anti-DLBCL agent in both BCL2-positive and BCL2-negative cases. ABT199, as a single-agent or in combination with HHT, effectively eliminates BCL2-positive DLBCL cells. Based on the observed data two biological categories of DLBCL might be assumed: BCL2-dependent (ABT199-sensitive, HHT-sensitive) and MCL1-dependent (ABT199-resistant, HHT-sensitive) DLBCL.
Grant support: IGA-MZ: NT13201-4/2012, GACR14-19590S, UNCE 204021, SVV-2013-266509, PRVOUK P24/LF1/3, GA-UK 1270214
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.