title: DRP-1 is required for BH3 mimetic-mediated mitochondrial fragmentation and apoptosis.
abstract: The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs, such as Navitoclax and Venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend on MCL-1 for survival, which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far, while demonstrating early promise in vitro, fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects, we developed a rapid differential scanning fluorimetry (DSF)-based assay, and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested, only A-1210477 induced rapid, concentration-dependent apoptosis, which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-XL, A-1210477 exhibited marked synergy with A-1331852, a BCL-XLspecific inhibitor, to induce cell death. Despite this selectivity and potency, A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression, suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However, A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1, and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands, and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis.
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.
authors.forename: Mateus
authors.initials: M
authors.lastname: Milani
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Byrne
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.
authors.forename: Georgia
authors.initials: G
authors.lastname: Greaves
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Butterworth
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.|Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, Ashton Street, Liverpool L69 3GE, UK.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Cohen
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Eyers
authors.affiliation: Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool L69 3GE, UK.|Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, Ashton Street, Liverpool L69 3GE, UK.
authors.forename: Shankar
authors.initials: S
authors.lastname: Varadarajan
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects*; D052160:Benzothiazoles / Q000008:administration & dosage / Q000737:chemistry; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage / Q000737:chemistry; D045744:Cell Line, Tumor; D064550:Death-Associated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D019337:Hematologic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D006801:Humans; D007211:Indoles / Q000008:administration & dosage / Q000737:chemistry; D007546:Isoquinolines / Q000008:administration & dosage / Q000737:chemistry; D008928:Mitochondria / Q000187:drug effects / Q000473:pathology; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics*; D010446:Peptide Fragments / Q000008:administration & dosage / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000008:administration & dosage / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics*; D013449:Sulfonamides / Q000008:administration & dosage / Q000737:chemistry
chemical_list: C000611392:A-1210477; C000603580:A-1331852; D000970:Antineoplastic Agents; C474007:Bax protein (53-86); D052160:Benzothiazoles; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007211:Indoles; D007546:Isoquinolines; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D064550:Death-Associated Protein Kinases; C579720:venetoclax