Determining the Effects of Myeloma-Derived Amino Acid Substitutions in the Functional Domain of Mcl-1 on Stress-Induced Apoptosis Open Access

Abstract

Multiple Myeloma is a cancer of plasma cells. In 2016, there were more than 30,000 estimated new cases of multiple myeloma. Myeloma cells retain many characteristics of plasma cells, and one of which is their dependence on Mcl-1, an anti-apoptotic protein in the Bcl-2 family of apoptotic regulators. Our study investigates whether amino acid substitutions in the functional region of Mcl-1--L267V, V249L, N223S, R214Q--observed in some myeloma patients affect Mcl-1's anti-apoptotic ability. We generated FL5.12 cells that stably overexpress Wild Type(WT) Mcl-1 or mutant Mcl-1. By western blot analysis, real time, quantitative polymerase chain reaction, and proteasome inhibition, we found that the L267V mutation destabilizes the Mcl-1 protein. We then subjected FL5.12 stable cell lines to IL-3 withdrawal, treatment with chemotherapy agents (Vincristine, Etoposide, and Cisplatin), and transient overexpression of Bim. We found that overexpression of WT Mcl-1 protects FL5.12 cells from IL-3 withdrawal only transiently and does not protect against Vincristine, Etoposide or Cisplatin-induced apoptosis. We also observed that overexpression of WT Mcl-1 can protect the FL5.12 stable cell lines against transient Bim overexpression-induced apoptosis. In contrast, overexpression of Mcl-1 V249L does not protect to the extent of WT Mcl-1 overexpression; overexpression of Mcl-1 L267V cannot protect the cells at all, which is consistent with our findings that the L267V mutation destabilizes the protein. Follow up studies need to be done on why Mcl-1 can only protect FL5.12 cells from IL-3 withdrawal transiently, why it can only protect against apoptosis induced from specific methods, and the mechanism through which V249L decreases Mcl-1's anti-apoptotic ability. Our findings will provide insights into why patients with such mutations fare better, which can inform treatment.

Table of Contents

Table of Contents

Introduction................................................................................................ 1

Figure 1.................................................................................................... 6

Figure 2.................................................................................................... 7

Materials and Methods................................................................................ 8

Results....................................................................................................... 14

Figure 3.................................................................................................. 21

Figure 4.................................................................................................. 22

Figure 5.................................................................................................. 25

Figure 6.................................................................................................. 26

Figure 7.................................................................................................. 27

Figure 8.................................................................................................. 28

Figure 9.................................................................................................. 29

Figure 10................................................................................................ 30

Figure 11................................................................................................ 31

Figure 12................................................................................................ 32

Figure 13................................................................................................ 35

Figure 14................................................................................................ 36

Figure 15................................................................................................ 38

Discussion.................................................................................................. 39

Figure 16................................................................................................ 42

References................................................................................................. 43

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Health Sciences, Oncology Biology, Cell
Keyword	Apoptosis Myeloid Cell Leukemia 1 Mcl-1 Bcl-2 Family Multiple Myeloma
Committee Chair / Thesis Advisor	Boise, Lawrence, Emory University
Committee Members	Antia, Rustom, Emory University Eisen, Arri, Emory University

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