Mechanism of caspase-3 activation and Mcl-1 inhibition in the regulation of apoptosis Public

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

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular
Mot-clé	Caspase-3 Multiple Myeloma Apoptosis
Committee Chair / Thesis Advisor	Boise, Lawrence, Emory University
Committee Members	Deng, Xingmeng, Emory University Dunham, Christine, Emory University Marcus, Adam, Emory University Kang, Sumin, Emory University

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