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Title: The complex regulatory mechanisms of the BCL-2 family of proteins in cancer
Author: Greaves, G. J.
ISNI:       0000 0004 7964 1889
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Resistance to apoptosis is a key hallmark of most cancers. The BCL-2 family of proteins are the major arbiters of the cell death program and the interactions between pro- and anti-apoptotic members of this family are believed to shift the fate of a cell towards life or death. The anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1, BCL-w and BCL-2A1) are often overexpressed in cancer, thereby promoting abnormal cell survival. As a result, these proteins are highly attractive targets for novel chemotherapeutic approaches. The recent clinical success of inhibitors against BCL-2 (Navitoclax and Venetoclax) has launched the compounds known as BH3 mimetics into the chemotherapeutic spotlight and has led to the successful development of several highly potent MCL-1 inhibitors. The targeting of MCL-1 in cancer is critical to restoring sensitivity to other chemotherapeutic approaches, as MCL-1 is strongly associated with abnormal cell survival and chemoresistance in many cancers. The aims of this study were to (1) assess the specificity and potency of the novel MCL-1 inhibitor, S63845, as a single agent and in conjunction with other BH3 mimetics, to induce apoptosis in a range of cancer cell lines (2) characterise the dependence on BH3-only members for apoptosis induced by BH3 mimetics and (3) identify novel MCL-1 interacting partners which could explain BH3-only protein-independent apoptosis. Using a panel of cancer cell lines with various dependencies on BCL-2 family members, it was shown that S63845 is an extremely potent MCL-1 inhibitor which can induce extensive apoptosis alone or in combination with other BH3 mimetics. Immunoprecipitation studies of the BCL-2 family during BH3 mimetic-induced apoptosis revealed that the interactions of a pro-survival protein with certain BH3-only proteins might not exclusively dictate sensitivity to certain BH3 mimetics. Moreover, extensive study of cells lacking the 8 key BH3-only proteins showed that BH3 mimetics can induce a BAX-dependent but BH3-only protein-independent apoptosis, going against the accepted dogma of BH3 mimetic action. With the possibility of other BH3-only-like proteins existing to mediate the apoptosis seen in the absence of the 8 major BH3-only proteins, novel interacting partners of MCL-1 were explored by mass spectrometry. DRP-1, a mitochondrial fission regulator, was thus identified as an MCL-1-interacting protein. While knockdown of DRP-1 could abrogate BH3 mimetic-induced apoptosis in H1299 cells, it did not interact with MCL-1 via a BH3 motif and exacerbated apoptosis in the cells lacking the BH3-only proteins. Thus, DRP-1, while confirmed as a regulator of apoptosis, was not proposed to function like a novel BH3-only protein which can activate BAX/BAK in the absence of the BH3-only proteins. Overall this study reveals that S63845 is an important and highly effective MCL-1 inhibitor with strong potential for use in the clinic both as a monotherapy and in combination with other approaches. Moreover, the mechanism underlying BH3 mimetic-induced apoptosis and BAX activation has been revealed to be much more complicated than currently known, as it appears that the BH3-only proteins are dispensable for apoptosis following pro-survival protein neutralisation. Future studies should clarify how BAX can initiate MOMP without the BH3-only proteins, be it through activation of BAX by the outer mitochondrial membrane directly or, perhaps, the existence of novel BH3-only-like proteins which can activate BAX independently of the 8 key BH3-only proteins.
Supervisor: Varadarajan, Shankar ; Cohen, Gerry ; Pettitt, Andy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral