SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells
- Translational Biology and Molecular Medicine Graduate Program, Baylor College of Medicine, United States
- Medical Scientist Training Program, Baylor College of Medicine, United States
- Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, United States
- Department of Molecular and Cellular Biology, Baylor College of Medicine, United States
- Center for Cell and Gene Therapy, United States
- Department of Haematology, Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, United Kingdom
- Integrated Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, United States
- Texas Children’s Hospital Department of Hematology/Oncology, Baylor College of Medicine, United States
- Cancer and Cell Biology Graduate Program, Baylor College of Medicine, United States
- Department of Leukemia, The University of Texas MD Anderson Cancer Center, United States
- Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, United States
- Department of Neurosurgery, Baylor College of Medicine, United States
- Donders Centre for Neuroscience, Radboud University Medical Center, Netherlands
- Department of Education, Innovation and Technology, Advanced Technology Cores, University of Texas, United States
- Department of Genome Medicine, The University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1 with 1 supplement
SOD1 is a synthetic-lethal vulnerability of PPM1D-mutant leukemia cells.
(A) Schematic of whole-genome CRISPR dropout screen. Wild-type (WT) Cas9-expressing OCI-AML2 and two isogenic PPM1D-mutant lines were transduced with the Human Improved Whole Genome Knockout CRISPR library V1 containing 90,709 guide RNAs (gRNAs) targeting 18,010 human genes at low multiplicity of infection (MOI~0.3). Each condition was performed in technical triplicates. Three days post-transduction, cells underwent puromycin selection for 3 days. Cells were harvested at day 10 as the initial timepoint and then harvested every 3 days afterward. sgRNA-sequencing was performed on cells collected on day 28. (B) Top biological processes based on gene ontology analysis of the top 37 genes essential for PPM1D-mutant cell survival. Enrichment and depletion of guides and genes were analyzed using MAGeCK-VISPR by comparing read counts from each PPM1D-mutant cell line replicate with counts from the initial starting population at day 10. (C) Volcano plot of synthetic-lethal hits ranked by fitness score with a negative score indicating genes for which their knockout leads to decreased growth or survival. SOD1 (highlighted) was the top hit from the screen. (D) Left: Schematic of competitive proliferation assays used for validation of CRISPR targets. Right: WT and PPM1D-mutant Cas9-OCI-AML2 and Cas9-OCI-AML3 cells were transduced with lentiviruses containing a single SOD1-gRNA with a blue fluorescent protein (BFP) reporter. Cells were assayed by flow cytometry every 3–4 days and normalized to the BFP percentage at day 3 post-transduction. Two unique gRNAs against SOD1 were used per cell line and each condition was performed in technical duplicates; multiple unpaired t-tests, **p<0.01, ***p<0.001. (E) Left: Cas9-expressing WT and PPM1D-mutant cells were transduced with control or sgSOD1-containing lentiviruses and underwent puromycin (3 µg/mL) selection for 3 days prior to transplantation. Sublethally irradiated (250 cGy) NSG mice were intravenously transplanted with 3×106 cells. Right: Kaplan-Meier survival curve of mice transplanted with WT or PPM1D-mutant (gray) leukemia cells with or without SOD1 deletion. The median survival of mice transplanted with WT, WT/SOD1–/–, PPM1Dmut, and PPM1Dmut/SOD1–/– leukemia cells was 32, 43, 32, and 55 days, respectively; Mantel-Cox test, **p<0.01, ***p<0.001.
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Figure 1—source data 1
CRISPR dropout screen raw data and top 37 gene candidates.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
SOD1 is a synthetic-lethal vulnerability of PPM1D-mutant leukemia cells.
(A) Immunoblot validation of PPM1D-mutant Cas9-expressing OCI-AML2 cells generated and used for CRISPR screening. Blots were probed with anti-PPM1D (1:1000) and GAPDH (1:1000). Clones 2102 and 2113 were selected for the dropout screen. (B) Venn diagram of genes that were depleted from the two PPM1D-mutant clones (#2102, 2113) used in the dropout screen, but not depleted in the wild-type (WT) control lines. 37 genes were found to be depleted in both mutant clones. For a full list of genes, see Figure 1—source data 1. (C) Volcano plot of synthetic-lethal hits ranked by fitness score with the Fanconi anemia pathway genes highlighted in blue. (D) Immunoblot validation of SOD1 deletion. WT and PPM1D-mutant Cas9-OCI-AML2 cells were transduced with control (empty vector [EV]) or sgSOD1 lentiviruses. Two sgRNAs targeting SOD1 were tested. Three days post-transduction, the cells underwent puromycin selection (3µg/mL) for 3 days after which they were harvested for western blot. Blots were probed with anti-PPM1D (1:1000), anti-SOD1 (1:500), and anti-vinculin (1:2500). (E) Cas9-OCI-AML2 and Cas9-OCI-AML3 WT or PPM1D-mutant cells were transduced with the empty vector control backbone tagged with a blue fluorescent protein (BFP) reporter. Cells were assayed by flow cytometry between 3 and 24 days post-transduction and normalized to the BFP percentage at day 3. Data shown are mean ± SD (n=2 per condition).
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Figure 1—figure supplement 1—source data 1
Western blot validation of OCI-AML2 PPM1D-mutant clones after CRISPR editing.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Western blot validation of SOD1 deletion in WT and PPM1D-mutant cells.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig1-figsupp1-data2-v1.zip
Figure 2 with 2 supplements
PPM1D-mutant cells are sensitive to SOD1 inhibition and have increased oxidative stress.
(A,B) Dose response curves for cell viability with SOD1-inhibitor (LCS-1) (A) or LCS-1 in combination with 0.25 uM NAC (B) in WT and PPM1D-mutant leukemia cell lines after 24-hours. Mean + SD (n=3) is shown with a non-linear regression curve. All values are normalized to the baseline cell viability with vehicle, as measured by MTT assay. (C) Endogenous cytoplasmic superoxide levels of WT and PPM1D-mutant leukemia cell lines were measured using dihydroethidium (5 uM). The mean fluorescence intensity (MFI) of dihydroethidium was measured by flow cytometry. Mean + SD (n=3) is shown. (D) Lipid peroxidation measured using BODIPY 581/591 staining (2.5 uM) of WT and PPM1D-mutant OCI-AML2 cells. The MFI was measured by flow cytometry. Mean + SD (n=3) is shown. (E-F) Measure of total reactive oxygen species using 2’,7’–dichlorofluorescin diacetate (DCFDA) staining (10 uM) measured by flow cytometry. WT and PPM1D-mutant OCI-AML2 cells were measured at baseline and 24-hrs after SOD1 inhibition (ATN-224 12.5 uM, LCS-1 0.625 uM) (E) or 24-hrs after pharmacologic PPM1D inhibition (GSK2830371, 5 uM) (F); unpaired t-tests were used for statistical analyses, ns=non-significant (p>0.05), **p<0.01, ***p<0.001, ****p<0.0001.
Figure 2—figure supplement 1
PPM1D-mutant cells have increased oxidative stress.
(A) Superoxide dismutase (SOD) activity assays in OCI-AML2 and OCI-AML3 cells at baseline (NT), or treated with high (12.5 µM) or low (6.25 µM) doses of ATN-224 for 16 hr. (B) Left: Representative flow cytometry plots of wild-type (WT) and PPM1D-mutant cells treated with ATN-224 (25 µM for 24 hr) and stained for Annexin V-APC and PI for apoptosis; multiple unpaired t-tests, ns = non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C) Endogenous mitochondrial superoxide levels of WT and PPM1D-mutant leukemia cell lines were measured using MitoSOX Green staining (1 µM). The mean fluorescence intensity (MFI) of MitoSOX Green was measured by flow cytometry. Mean ± SD (n=3) is shown.
Figure 2—figure supplement 2
PPM1D-mutant cells have increased oxidative stress.
(A,B) Dose-response curves for cell viability with SOD1 inhibitor (ATN-224) (A) or ATN-224 in combination with 0.25µM N-acetylcysteine (NAC) (B) in wild-type (WT) and PPM1D-mutant leukemia cell lines after 24hr. Mean ± SD (n=3) is shown along with a non-linear regression curve. All values are normalized to the baseline cell viability with vehicle, as measured by MTT assay. (C) Immunoblot of SOD2 expression in WT and PPM1D-mutant cells at baseline and after SOD1 deletion. WT and PPM1D-mutant Cas9-OCI-AML2 cells were transduced with control (empty vector [EV]) or sgSOD1 lentiviruses. Two sgRNAs targeting SOD1 were tested. Three days post-transduction, the cells underwent puromycin selection (3 µg/mL) for days after which they were harvested for western blot. Blots were probed with anti-PPM1D (1:1000), anti-SOD2 (1:1000), and anti-vinculin (1:2500). (D) Total reactive oxygen species (ROS) of WT and Ppm1d-mutant mouse embryonic fibroblasts (MEFs) measured by 2’7’-dichlorofluorescein diacetate (DCFDA) (10 µM) staining. Mean fluorescence intensity (MFI) was determined by flow cytometry. n=6 biological replicates were used for each genotype. Data shown are the mean of each biological replicate; unpaired t-test. (E) Total ROS of WT GM12878 (gray) and PPM1D-mutant (pink) patient lymphoblastoid cell lines (LCLs) at baseline and after 24 hr of SOD1 inhibition measured by DCFDA (10 µM) staining. MFI was determined by flow cytometry; multiple unpaired t-tests. (F) Dose-response curve of WT and PPM1D-mutant LCLs after ATN-224 treatment. IC50s of WT and PPM1D-mutant LCLs were 48.8 µM and 20.51 µM, respectively, as measured by MTT assay; non-linear regression analysis, ns = non-significant (p>0.05), **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 2—figure supplement 2—source data 1
Western blot of SOD2 expression at baseline and after SOD1 deletion.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig2-figsupp2-data1-v1.zip
Figure 3 with 1 supplement
PPM1D-mutant cells have altered mitochondrial function.
(A) Mitochondrial mass of wild-type (WT) and PPM1D-mutant leukemia cells was determined using MitoTracker Green (100 nM) and the mean fluorescence intensity was analyzed by flow cytometry. Data represents mean ± SD of triplicates. At least three independent experiments were conducted with similar findings; unpaired t-tests. (B) Immunoblot of WT and PPM1D-mutant cell lysates probed with the human OXPHOS antibody cocktail (1:1000) and vinculin (1:2000). (C) Measurement of mitochondrial oxygen consumption rate (OCR) by seahorse assay in WT and PPM1D-mutant OCI-AML2 cells after treatment with oligomycin (1.5 µM), FCCP (0.5 µM), and rot/AA (0.5 µM). Quantification of basal, maximal, and ATP-linked respiration are shown. Data shown are the mean ± SD of technical triplicates. (D) Mitochondrial membrane potential of WT and PPM1D-mutant OCI-AML2 cells was measured using MitoTracker CMXRos (400 nM). The mean fluorescence intensity (MFI) was measured and analyzed by flow cytometry. Data represents mean ± SD of triplicates, unpaired t-test, ns = non-significant (p>0.05), *p<0.05, **p<0.01.
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Figure 3—source data 1
Western blot of mitochondrial proteins in WT and PPM1D-mutant cells.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig3-data1-v1.zip
Figure 3—figure supplement 1
PPM1D-mutant cells have altered mitochondrial function.
(A,B) Measurement of mitochondrial oxygen consumption rate (OCR) by seahorse assay in wild-type (WT) vs. PPM1D-mutant MOLM-13 (A) and OCI-AML3 (B) cells after treatment with oligomycin (1.5 µM), FCCP (0.5 µM), and rot/AA (0.5 µM). Quantification of basal, maximal, and ATP-linked respiration shown. Each cell line was performed in technical triplicates, Student’s t-test. (C) Growth curves of WT and PPM1D-mutant leukemia cell lines at 24, 48, and 72 hr. Cell counts were normalized to day 0. ns = non-significant (p>0.05), *p<0.05, ***p<0.001.
Figure 4 with 2 supplements
PPM1D-mutant cells have a reduced oxidative stress response.
(A) RNA-sequencing (RNA-seq) gene set enrichment analysis (GSEA) of PPM1D-mutant cells compared to wild-type (WT) Cas9-OCI-AML2 cells. Significantly up- and downregulated pathways are indicated by the blue and red bars, respectively. Normalized enrichment scores (NES) are shown with false discovery rate (FDR) < 0.25. (B) Reverse-phase protein array (RPPA) profiling of WT and PPM1D-mutant OCI-AML2 cells. Proteins from the ‘Response to Oxidative Stress’ pathway have been selected for the heatmap. Each column represents a technical replicate. See Figure 4—source data 2 for the raw data. (C) Total- and small-molecule antioxidant capacity of WT and PPM1D-mutant cells performed in technical duplicates. (D) Intracellular glutathione (GSH) levels measured by flow cytometry using the Intracellular GSH Detection Assay Kit (Abcam). Left: Representative flow cytometry plot demonstrating the gating for GSH-high and GSH-low populations. Right: Quantification of the percentage of GSH-high cells for each cell line. Mean ± SEM (n=3) are shown. (E) Immunoblot of WT and PPM1D-mutant OCI-AML2 after transduction with the empty vector (EV) control and after SOD1 deletion (left) or after treatment with SOD1 inhibitors for 16 hr (right, ATN-224 12.5 µM, lung cancer screen-1 [LCS-1] 1.25 µM). Lysates were probed with an anti-oxidative stress defense cocktail (1:250), SOD2 (1:1000), and vinculin (1:2000). SMA = smooth muscle actin. Student’s t-tests were used for statistical analysis; **p<0.01, *p<0.05.
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Figure 4—source data 1
RNA-seq gene expression analysis of WT and PPM1D-mutant cells after transduction with empty vector [EV] or sgSOD1 lentiviruses.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig4-data1-v1.xlsx
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Figure 4—source data 2
Reverse phase protein array (RPPA) analysis of WT and PPM1D-mutant cells at baseline and after SOD1-deletion.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig4-data2-v1.xlsx
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Figure 4—source data 3
Reverse phase protein array (RPPA) over-representation analysis pathways.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig4-data3-v1.xlsx
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Figure 4—source data 4
Western blot analysis of oxidative stress defense proteins after genetic deletion and pharmacologic inhibition of SOD1.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig4-data4-v1.zip
Figure 4—figure supplement 1
PPM1D-mutant cells have reduced oxidative stress response.
(A) Schematic of the experimental setup for the bulk RNA-sequencing and reverse-phase protein array. Wild-type (WT) and PPM1D-mutant Cas9 OCI-AML2 cells were transduced with either empty vector (EV)-blue fluorescent protein (BFP) or SOD1-sgRNA-BFP. Cells were passaged for 10 days and then sorted for BFP expression for downstream analysis. (B, D) Gene set enrichment analysis (GSEA) enrichment plots for PPM1D-mutant cells compared to WT after transduction with EV (B) or after SOD1-knockout (D) for the ‘Regulation of Response to Oxidative Stress’ (GO:1902882) and ‘Response to Oxidative Stress’ (GO:0006979). Normalized enrichment scores (NES) are shown with false discovery rate (FDR) < 0.25. (C) GSEA of RNA-sequencing of SOD1-deleted cells compared to EV control in WT and PPM1D-mutant cells. Blue and red bars indicate significantly up- and downregulated pathways, respectively. NES are indicated. All pathways filtered for FDR < 0.25. See Figure 4—source data 1 for raw data.
Figure 4—figure supplement 2
PPM1D-mutant cells have reduced oxidative stress response.
(A) Volcano plot of the differentially expressed proteins from the reverse-phase protein array (RPPA) in PPM1D-mutant OCI-AML2 cells compared to wild-type (WT). Red and blue dots indicate significantly up- and downregulated proteins, respectively, with a cutoff false discovery rate (FDR) < 0.2 and linear fold change > |1.2|. (B) RPPA profiling of WT and PPM1D-mutant cells after SOD1 deletion. Proteins from the ‘Response to Oxidative Stress’ pathway have been selected for the heatmap. Each column represents a technical replicate. See Figure 4—source data 2 for the raw data.
Figure 5 with 1 supplement
PPM1D mutations increase genomic instability and impair non-homologous end-joining.
(A) Left: Representative images of comet assays of mouse embryonic fibroblasts (MEFs). Two biological replicates were assessed for each genotype. Right: Quantification of n≥150 comets per experimental group with the Comet IV software; two-way ANOVA. (B) Mean fluorescent intensity (MFI) of 8-oxo-2′-deoxyguanosine (8-oxo-dG) lesions within wild-type (WT) and PPM1D-mutant OCI-AML2 cells as measured by flow cytometry; Student’s t-test. (C) Left: Representative images of metaphase spreads of WT and Ppm1d-mutant mouse primary B-cells treated with low (0.5 µM) or high (5 µM) doses of cisplatin. Right: n≥50 metaphase cells were quantified in each experimental condition for chromosomal aberrations (white arrows). n=2 biological replicates used for each genotype. Student’s t-test was used for statistical analysis. (D–E) Left: Schematic of the homologous recombination (D) or non-homologous end-joining (E) U2OS DNA damage repair cassettes. Right: Quantification of GFP% analyzed by flow cytometry 48 hr after induction of DNA damage by I-SceI transduction; Student’s t-test. (F) Comet assay quantification of WT and PPM1D-mutant Cas9-OCI-AML2 cells 6 days after lentiviral transduction with the empty vector (EV) control, or sgSOD1 to induce SOD1 deletion. Quantification and analyses of tail moments were performed using the Comet IV software. n≥150 comets were scored per experimental group; two-way ANOVA. Data are mean ± SD (n=3), ns = non-significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 5—source data 1
Comet assay assessing baseline levels of DNA damage in WT and Ppm1d-mutant mouse embryonic fibroblasts.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig5-data1-v1.zip
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Figure 5—source data 2
Metaphase spread of WT and Ppm1d-mutant mouse primary B-cells after treatment with cisplatin.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig5-data2-v1.zip
Figure 5—figure supplement 1
PPM1D-mutations increase genomic instability and impairs non-homologous end-joining repair.
(A) Left: Sanger sequencing traces of the parental U2OS cell line harboring a c.1372 C>T mutation in PPM1D and the CRISPR-edited U2OS cell line with mutation corrected to wild-type (WT) PPM1D. Right: Immunoblot validation of these clones are shown. Lysates were probed with anti-PPM1D (1:1000) and anti-GAPDH (1:1000). (B,C) Left: Representative images of Rad51 and 53BP1 immunofluorescence microscopy. Mouse embryonic fibroblasts were treated with 10 Gy irradiation, harvested 1 hr post-irradiation and stained for the indicated markers. Right: Quantification of the number of foci per cell is shown. Analysis was performed using CellProfiler. n>100 cells for each condition; Student’s t-test. (D) Comet assay quantification of mouse embryonic fibroblasts at baseline and after 1 hr post-irradiation (10 Gy). Quantification and analyses of tail moments were performed using the Comet IV software. n≥150 comets were scored per experimental group; two-way ANOVA, ns = non-significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 5—figure supplement 1—source data 1
Western blot analysis of CRISPR-edited U2OS clones validating the correction of the endogenous PPM1D mutations to the wild type form.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Immunofluorescence microscopy of WT and Ppm1d-mutant mouse embryonic fibroblasts stained with Rad51.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Immunofluorescence microscopy of WT and Ppm1d-mutant mouse embryonic fibroblasts stained with 53BP1.
- https://cdn.elifesciences.org/articles/91611/elife-91611-fig5-figsupp1-data3-v1.zip
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SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells
eLife 12:RP91611.
https://doi.org/10.7554/eLife.91611.3
