The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non-small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF-VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF-VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer. SIGNIFICANCE: These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF-VDAC complex and displays anticancer activity in multiple tumor models.See related commentary by Rao, p. 6074.
Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Membrane Proteins/metabolism , Mitochondria/pathology , Mitochondrial Dynamics/drug effects , Mitochondrial Proteins/metabolism , Animals , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Humans , Lung Neoplasms/drug therapy , Male , Membrane Proteins/antagonists & inhibitors , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/pathology , Mitochondrial Proteins/antagonists & inhibitors , Permeability/drug effects , Protein Multimerization/drug effects , Voltage-Dependent Anion Channel 1/antagonists & inhibitors , Voltage-Dependent Anion Channel 1/metabolism , Xenograft Model Antitumor Assays
The human CST (Ctc1, Stn1 and Ten1) complex binds the telomeric overhang and regulates telomere length by promoting C-strand replication and inhibiting telomerase-dependent G-strand synthesis. Structural and biochemical studies on the human Stn1 and Ten1 complex revealed its mechanism of assembly and nucleic acid binding. However, little is known about the structural organization of the multi-domain Ctc1 protein and how each of these domains contribute to telomere length regulation. Here, we report the structure of a central domain of human Ctc1. The structure reveals a canonical OB-fold with the two identified disease mutations (R840W and V871M) contributing to the fold of the protein. In vitro assays suggest that although this domain is not contributing directly to Ctc1's substrate binding properties, it affects full-length Ctc1 localization to telomeres and Stn1-Ten1 binding. Moreover, functional assays show that deletion of the entire OB-fold domain leads to significant increase in telomere length, frequency of internal single G-strands and fragile telomeres. Our findings demonstrate that a previously unknown OB-fold domain contributes to efficient Ctc1 telomere localization and chromosome end maintenance.
Bone Marrow/metabolism , Protein Folding , Telomere Homeostasis , Telomere-Binding Proteins/chemistry , Telomere/metabolism , Amino Acid Sequence , Bone Marrow/pathology , Crystallography, X-Ray , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding , Protein Domains , Sequence Homology, Amino Acid , Syndrome , Telomere/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism
