Myeloma Patient-Derived MCL1 Point Mutations Can Influence MCL1-Inhibitor Function Restricted; Files Only

Chen, Brandon (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/zk51vh94g?locale=en
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Abstract

Multiple myeloma is a malignancy of long-lived plasma cells that is rarely curable. Despite recent drug development, additional approaches are required. We decided to investigate molecular vulnerabilities of the BCL2 family. Using the MMRF CoMMpass study, we determined nonsynonymous mutations frequency in the BCL2 family. Analysis from 982 patients revealed that mutations in the BCL2 family are relatively rare. Interestingly, MCL1 was mutated in 10 baseline samples (1.02%) and the frequency of the mutations in these samples was high (median 0.391, range 0.066-0.531). Therefore, we further investigated the functional consequences of the MCL1 mutation.

Of the 10 mutations detected, we focused on the 4 mutations that lie near the functional BH1 (V249L and L267V) and within the BH3 (N223S, and R214Q) domains. Wild-type (WT) MCL1 and the four mutant MCL1 constructs were introduced into murine B-ALL cell line that has endogenous murine MCL1 flanked with LoxP sites and confirmed expression by western blot analysis. Human MCL1 can replace murine MCL1 in this cell model, therefore we are determining if the myeloma-derived mutants of MCL1 can complement the loss of mouse Mcl1 and will report on these findings. We treated cells with MCL1 inhibitor S63845 and measured apoptosis (Annexin V/PI) at 24 hours. Cells with empty vector were highly resistant to death (less than 20% at 1000 nM) while cells expressing the human WT MCL1 were susceptible to the MCL1 inhibitor at 300 and 1000 nM. The V249L, N223S and R214Q mutations mimicked the sensitivity of the WT MCL1. However, L267V mutation resulted in a dose curve similar to the empty vector control suggesting this mutation was either resulted in the loss of function or impaired drug binding. Since drug binding stabilizes MCL1 by competing for E3 ligase, we determined the effect of S63845 on MCL1 protein levels. We found S63845 increased human MCL1 protein expression in all mutants, ruling out lack of drug binding. We next co-immunoprecipitated MCL1 and found that BIM release correlated with S63845 sensitivity. In addition to not releasing BIM, L267V did not effectively release NOXA and BAK after S63845 treatment. Taken together, the L267V mutation blocks the ability of the drug to displace pro-apoptotic proteins required to induce cell death.

We tested another MCL-1 inhibitor in clinical trials, AZD5991. Interestingly, all four mutations resulted in diminished killing activity when compared to cells expressing WT MCL1, suggesting that these mutations may also influence drug function. Together these data suggest that MCL1 mutations may not necessarily influence MCL1 function, yet they could alter responses to an emerging class of inhibitors where 3 drugs are currently in clinical trials.

Table of Contents

Introduction…p1-10

Results…p11-28

Discussion…p29-32

Material and methods…p33-36

References…p37-39

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