Targeting the Pro-Survival BCL2 Proteins with BH3 Mimetic Compounds for Treating Multiple Myeloma
Authors: Gong Jia-Nan , Khong Tiffany , Segal David , Yao Yuan , van Delft Mark F , Spencer Andrew , Roberts Andrew W , Huang David CS 
Published: October 2019

Abstract
Multiple myeloma is a clonal B-cell malignancy characterized by complex genetic aberrations. Despite recent advances in our understanding of genetic basis of this disease and the introduction of novel therapies, the outcome for many patients remains poor. For example, 20% of newly diagnosed patients with stage 3 myeloma have disease marked by high lactate dehydrogenase levels or unfavorable cytogenetic abnormalities (e.g. 17p deletion, t(4;14)). Half of these patients die within 2 years of diagnosis in spite of advanced therapies.
A promising class of novel agents to treat patients with other B-cell malignancies is the BH3 mimetic compounds, which targets the pro-survival BCL2 proteins. For example, the majority of patients with CLL (chronic lymphocytic leukaemia) respond to venetoclax (ABT-199), a BCL2-selective inhibitor. Myeloma cell lines with t(11;14) translocations are also reported to be sensitive to BCL2 inhibition and venetoclax is in clinic trails for patients with relapsed or refractory multiple myeloma. We are eager to know whether the other pro-survival BCL2 proteins, namely BCLXL, BCLW, MCL1 and BCL2A1 (BFL1) play a role in maintaining the survival of myeloma cells.
To determine this, we screened a large panel of immortalized (25) and low-passage (7) myeloma cell lines to killing by BH3 mimetics with varying specificities. We found that a fraction of the myeloma cell lines were rapidly killed when BCL2 (25%, 8/32) or BCLXL (25%, 8/32) were targeted. Interestingly, we also identified a distinct t(4;14) subgroup, as well as the previous recognized t(11;14) subgroup, to be highly sensitive to BCL2 inhibition. Unlike CLL, in which the majority patients respond to venetoclax, BCL2 inhibition is only likely to account for some cases. We then asked which other pro-survival BCL2 protein keeps the other myeloma cell lines alive. A prime candidate is MCL1 since normal plasma cells rely on it and previous studies had implicated MCL1 in myeloma.
By targeting the expression of the pro-survival BCL2 proteins using CRISPR/Cas9 genome editing technology, we found that a significant fraction (74%, 14/19) of our cell line panel died rapidly in the absence of MCL1. The importance of MCL1 was confirmed using an orthogonal approach: we found that expression of a MCL1-selective peptidyl ligand BIM2A rapidly killed a sizeable fraction of both immortalized (17/25) and low-passage (5/7) myeloma cell lines. This activity strongly correlated with the genetic targeting of MCL1 by CRISPR/Cas9. Importantly, even cell lines that harbor the poor prognostic t(4;14) chromosomal translocation were readily killed by BIM2A and the cell lines were killed regardless of their TP53 status. Moreover, targeting MCL1 also constrained the growth of myeloma in vivo.
Since the inhibition of MCL1, BCLXL or BCL2 can on their own can kill almost all the myeloma cell lines screened, we next identified the molecular mechanisms by which these BH3 mimetics act. Firstly, we tested likely candidates for their role in driving the killing induced by a specific BH3 mimetic, for example whether deleting the BH3-only protein BIM impact upon killing by venetoclax. Secondly, we undertook genome-wide recessive CRISPR/Cas9 screens for genes that are critical for the action of the BH3 mimetics. We have identified a number of hits from such screens and are in the process of validating these. By identifying the unique susceptibility of the myeloma cell lines to the BH3 mimetics and elucidating how they act to kill, our studies are critical for the clinical testing of BH3 mimetics that target BCL2 or its close pro-survival relatives in patients with multiple myeloma.

Disclosures
Gong: The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:The Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.