Bromodomain Inhibitors Correct Bioenergetic Deficiency Caused by Mitochondrial Disease Complex I Mutations

Abstract

Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.

Keywords: BRD4; OXPHOS; PGC-1α; bromodomain inhibitors; mitochondria; mitochondrial disorders.

Figure 1. Brd4 is identified as a target by both high-throughput chemical and CRISPR genetic screens to enhance OXPHOS proteins and rescue mitochondrial bioenergetic defects

A, Schematic of high-throughput chemical screen. B, Scatterplot of chemicals tested (blue) plotted with first test (x-axis) and repeated test (y-axis) of Cox5a fluorescent values relative to the average PGC-1α positive control (green). DMSO baseline fluorescent values (black) are indicated. Vertical dotted line indicates compounds selected at the 70% cutoff. C, Cox5a fluorescent values from selected compounds tested in secondary screen plotted relative to PGC-1α positive control set to 1.0. Values represent the mean ± s.e.m. n=3. D, Chemical structure of the I-BET 525762A compound with Cox5a fluorescent values below. E, Workflow of genome-wide CRISPR Screen. f, Scatterplot of two replicate CRISPR screens showing enrichment of sgRNAs after galactose treatment. Points highlighted in red represent the different sgRNAs for Brd4. See also Figure S1.

Figure 2. Bromodomain inhibition increases OXPHOS proteins and remodels the mitochondrial proteome

A, I-BET 525762A increases OXPHOS proteins (left). Cycs densitometry of IBET-treated ND1-mutated cybrids is comparable to PGC-1α positive control (right). B, I-BET 525762A increases OXPHOS proteins in a dose-responsive manner. C, Blue Native Page- I-BET 525762A treatment increases OXPHOS complex levels. D, Diverse Brd4 inhibitors increases OXPHOS proteins. Immunoblots shown are representative of greater than 3 independent experiments. E, Proteomic heat map of the hierarchical clustering of all proteins (∼4,800) in duplicate (left). I-BET 525762A-upregulated proteins (> 1.4 fold) are indicated (vertical black bar). Proteomic heat map indicating that I-BET treatment in ND1-mutated cybrids increases core ETC proteins compared to control (middle). Gene Ontology (GO) categories from upregulated proteins enriched after Benjamin-Hochberg correction are indicated to the right. Corresponding −log10 p-values (x axis) and the number of proteins (in parentheses) are listed. See also Figure S2 and Table S1.

Figure 3. Bromodomain inhibition increases mitochondrial bioenergetics and protects against galactose-induced cell death

A-C, Total oxygen consumption (A) as well as CII (B) and CIV (C) activities are increased with I-BET 525762A. D, I-BET 525762A protects against galactose-induced cell death. Microscopy image (left) is representative of at least 3 independent experiments and time course (top right) and cell number quantification (bottom right) are the mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. Horizontal black scale bar = 200 μm. E, Different Brd4 inhibitors rescue ND1-mutated cybrids from galactose-induced cell death. F-G, I-BET 525762A enhances oxygen consumption in ND6-mutated (LHON) cybrids (F) and protects against galactose-induced cell death (G). H, I-BET 525762A rescues galactose-induced cell death in CI knock-down control cells. i, I-BET 525762A protects against galactose-induced cell death in ACAD9 human patient-derived fibroblasts. Microscopy image (right) is representative of at least 3 independent experiments and cell number quantification (left) is the mean ± s.e.m., n=3. Horizontal black scale bar = 200 μm. J, I-BET 525762A protects against galactose-induced cell death in control human cybrids with rotenone-induced CI deficiency. Data represents the mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. All I-BET and Brd4 inhibitor treatments are delivered at final concentration of 0.9 μM. See also Figure S2.

Figure 4. Loss-of-function of Brd4 enhances mitochondrial bioenergetics while gain-of-function impairs OXPHOS capacity

A, CRISPR ablation of Brd4 enhances OXPHOS proteins. B, Total oxygen consumption rates are increased in sgBrd4 ND1-human cybrids compared to sgNeg control. C-D, Complex II (C) and complex IV (D) activities are increased in sgBrd4 ND1-human cybrids compared to sgNeg controls. E, Representative microscopy image (right) of sgNeg, sgBrd4, and IBET-treated sgNeg ND1-mutant cybrids cultured in galactose for 72 hours. Cell quantification after 72 hours in galactose media is indicated to the left. Horizontal black scale bar = 200μm. F, Competition assay. GFP-negative sgBrd4 is more abundant that GFP-positive sgNeg ND1-mutated cybrids. G, Brd4 overexpression in control human cybrids reduces OXPHOS proteins. H, Total oxygen consumption is reduced with Brd4 overexpression in control human cybrids. I, Control human cybrids are sensitized to galactose-induced cell death with Brd4 overexpression. Immunoblots shown are representative of greater than 3 independent experiments and all other experiments are the mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. All I-BET treatments are delivered at final concentration of 0.9 μM. See also Figure S3.

Figure 5. IBET 525762A treatment displaces Brd4 from OXPHOS promoters and increases transcription in complex I-deficient cells

A, ChIP- I-BET 525762A displaces Brd4 from nuclear-encoded mitochondrial promoters. Negative control promoter region Atp1A3 is unaltered. B, ChIP- PGC-1α occupancy increases upon I-BET 525762A-mediated Brd4 displacement. C, ChIP- Brd4 is displaced upon adPGC-1α over-expression. Data is representative of mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. D-F, I-BET 525762A-mediated displacement of Brd4 (D) and ablation of Brd4 by CRISPR (E) increases expression of OXPHOS genes while over-expression of Brd4 decreases OXPHOS gene expression (F). Data represents mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. G-H, ShPGC-1α KD in ND1-mutated cybrids fail to increase OXPHOS transcripts (G) and partially blunts rescue from galactose-induced cell death (H) when treated with I-BET 525762A. See also Figure S4.

Figure 6. I-BET 525762A treatment rewires and enhances metabolic and energetic states of complex I-deficient cells

A, Metabolomics Heatmap in control and ND1-mutated cybrids in duplicate. I-BET 525762A-treated ND1-mutated human cybrids increases metabolites linked to energy, CII, and CoQ, and redox metabolism. B-C, I-BET 525762A treatment increases dihydroxyacetone phosphate (B) and succinyl CoA (C) metabolite levels. D-F, I-BET 525762A treatment increases ATP (D), NAD⁺ (E) and FAD (F) metabolite levels in ND1-mutated cybrids. Data represents mean ± s.e.m., n=2. G, ¹⁴C Glutamine oxidation is enhanced by I-BET 525762A treatment in ND1-mutated cybrids. H, I-BET-mediated galactose rescue is blunted when glutamine is removed from culture media in ND1-mutated cybrids. I, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) at 25 μM which selectively inhibits Glutaminase (GLS1) to block glutamine to glutamate conversion abolished the positive of effects of I-BET 525762A in ND1-mutated cybrids. J, I-BET 525762A displays minimal rescue upon Sdha knockdown. Immunoblot indicates complete Sdha knockdown at the protein level. Data represent the mean ± s.e.m., n=3. Asterisks denote *p<0.05 or **p<0.01 via Student's t-test. All I-BET treatments are delivered at a final concentration of 0.9 μM. K, Model detailing Brd4 inhibition rescues bioenergetic defects in complex I-deficient cells. Arrows indicate metabolites that were increased (green) or decreased (red) upon I-BET 575762A treatment. αKG, alpha-ketoglutarate, G-3-P, glyceraldehyde-3-phosphate, F-1-6-P, Fructose 1,6 bisphosphate, and CoQ, Coenzyme Q. See also Figure S5.