Mechanism of caspase-3 activation and Mcl-1 inhibition in the regulation of apoptosis Open Access
Ponder, Katelyn (Fall 2018)
Abstract
Intrinsic apoptosis is regulated by the Bcl-2 family of proteins. Upon an apoptotic stimuli proapoptotic proteins, Bak and Bax will form a multimer on the outer membrane of the mitochondria releasing cytochrome c and activate the caspase cascade. Understanding this mechanism and how the proteins involved are regulated is important for advances in cancer therapies. We focused our attention on the mechanism by which caspase-3 becomes activated at the end of the apoptotic pathway and on how the anti-apoptotic, Mcl-1 could be targeted in the treatment of Multiple Myeloma.
Caspase-3 is found in the cytoplasm as an inactive zymogen dimer. Upon cleavage of the interdomain linker by an initiator caspase and subsequent removal of the N-terminal prodomain, caspase-3 is active. It is unclear if removal of the prodomain is necessary for caspase-3 activity as previous studies were done in cell free systems and have conflicting results. Using a more physiologically relevant model we found the prodomain negatively regulates the activation and activity of caspase-3. Surprisingly a single point mutation at D9 rendered caspase-3 inactive. We also found that for the complete removal of the 28 amino acid prodomain, an initial cleavage event must occur at D9.
Multiple myeloma (MM) is the second most common hematologic malignancy that arises from bone marrow plasma cells. MM cells are dependent on the anti-apoptotic protein, Mcl-1 for survival. Carfilzomib, a proteasome inhibitor, is FDA approved for single-agent use in relapsed/refractory MM. We combined carfilzomib with TG02, a multi-kinase inhibitor, and found that the combination not only had at least additive effects in MM cells lines but also in patient samples. This combination targets Mcl-1 via two different mechanisms as carfilzomib causes an increase in NOXA, an inhibitor or Mcl-1, and TG02 caused a decrease in Mcl-1 protein. Based on our preclinical studies a phase I trial was initiated to test this combination for multiple myeloma.
Table of Contents
I. INTRODUCTION 1
A. Programmed Cell Death: Apoptosis 1
i. Extrinsic Apoptosis 1
ii. Intrinsic Apoptosis 2
B. Cysteine-Aspartic Acid Proteases 4
i. Activation and Structure of Apoptotic Caspases 4
C. Bcl-2 Family of Proteins 8
D. Multiple Myeloma 11
i. Protease Inhibitors 12
ii. TG02 13
E. Statement of Problem 15
II. THE PRODOMAIN OF CASPASE-3 REGULATES ITS OWN REMOVAL AND CASPASE ACTIVATION 17
A. Abstract 18
B. Introduction 19
C. Materials and Methods 21
D. Results 24
E. Discussion 29
F. Acknowledgements 32
III. DUAL INHIBITION OF MCL-1 BY THE COMBINATION OF CARFILZOMIB AND TG02 IN MULTIPLE MYELOMA 45
A. Abstract 46
B. Introduction 47
C. Materials and Methods 50
D. Results 53
E. Discussion 57
F. Acknowledgements, Disclosures and Contributions 60
IV. UNPUBLISHED DATA 68
A. Introduction 68
B. Results/Discussion 68
V. DISCUSSION 73
A. Implications of Caspase-3 studies 73
i. Caveats and opportunities for further study 76
ii. Implications for future research 77
B. Implications from MCl-1 studies in multiple myeloma 79
i. Caveats and opportunities for further study 79
ii. Implications for future research 80
VI. REFERENCES 83
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