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

Ponder, Katelyn (Fall 2018)

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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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