PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells

Abstract

Truncating mutations in the terminal exon of protein phosphatase Mg2⁺/Mn2⁺ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.

Graphical abstract

Figure 1.

Truncating PPM1D mutations lead to the selective outgrowth of PPM1D-mutant hematopoietic cells during chemotherapy treatment in vitro and in vivo. (A) The location of the absolute number of somatic frameshift and nonsense mutations in PPM1D identified in the blood cells of a cohort of 28 418 persons is shown. (B) The prevalence of PPM1D mutations in 28 418 people unselected for malignancy was compared with 401 lymphoma patients who had received chemotherapy and were undergoing stem cell collection for autologous transplantation (Auto-SCT).^, Only PPM1D mutations with a variant allele frequency (VAF) > 0.05 are included. Error bars represent the 95% confidence intervals in each age bin. (C) Whole-cell lysates from Molm13 isogenic control cells (control) and PPM1D frameshift (fs) mutant (mut) single-cell clones probed with anti-PPM1D and anti-Actin. (D-E) Viability assays in Molm13 PPM1D-mutant or control single-cell clones that were treated with increasing concentrations of cytarabine (D) or cisplatin (E) for 72 hours. Experiments were performed in triplicate and data are shown as the mean ± standard deviation (SD). Nonlinear logistic regression analyses and a sum of squares F test was performed to compare inhibitory responses. (F-G) Flow cytometric readout of competition experiment with 5% Molm13 PPM1D-mutant cells (tdTomato⁺) and 95% Molm13 control cells (tdTomato⁻), exposed to 150 nM cytarabine (F) and vehicle or 1 μM cisplatin (G) and vehicle for 24 days. Experiments were performed with biological triplicates and data are shown as the means ± SD. (H-J) Flow cytometric analysis of the peripheral blood of chimeric mice transduced with a gRNA-targeting PPM1D (BFP⁺) or a control locus (tRFP⁺). Mice were exposed to 3 rounds of cytarabine or vehicle treatment. Data represent the mean ± standard error of the mean (SEM) of 18 mice in the vehicle and 18 mice in the treatment arm, and are shown for the leukocyte (H), lymphocyte (I), and monocyte (J) cell compartments. Peripheral expansion of Ppm1d-mutant cells was analyzed using Wilcoxon rank sum tests.

Figure 2.

Truncating PPM1D mutations lead to decreased degradation of PPM1D. (A) Log2-fold enrichment of gRNAs (black dots) in Molm13 cells exposed to cytarabine treatment versus vehicle treatment of 12 days. The experiment was performed with biological triplicates, and the red line represents the locally weighted scatterplot smoothing (LOESS) of 0.1. gRNAs with a z score ≥ 3 are shown in green. Overlaid are the absolute number of somatic PPM1D frameshift and nonsense mutations (black bars) identified in the blood cells of 28 418 individuals as described in Figure 1A. (B) Whole-cell lysates from Molm13 cells overexpressing full -length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) were collected at different time points following cycloheximide (50 μg/mL) treatment. Blots were probed with anti-PPM1D and anti-COXIV. (C) Whole-cell lysates before and after 4 hours of 10 μM MG132 treatment from Molm13 cells overexpressing full-length PPM1D (Full-Length PPM1D) or truncated PPM1D (Truncated PPM1D) cDNA. (D) Vector map of the degradation reporter vector. Different PPM1D cDNA constructs are cloned in-frame with EGFP, allowing for monitoring of PPM1D expression levels through EGFP expression. mCherry is expressed following an IRES sequence and provides an internal control for vector expression in each cell. (E) EGFP-to-mCherry ratio in Molm13 cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D, and provide an estimate for the level of degradation. Experiments were done using biological triplicates and data are shown as the means ± SD. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (F) EGFP-to-mCherry ratio in Molm13 p53^−/− cells with overexpression of full-length PPM1D, truncated PPM1D or the C-terminal end of PPM1D. The EGFP-to-mCherry ratios are normalized to the expression level of full-length PPM1D. Unpaired Student t tests were used to calculate the association between the different vectors and P values were corrected for multiple hypothesis testing. (G) EGFP-to-mCherry ratio in Molm13 cells before and after exposure to MG132 (10 μM, 6 hours), normalized to pretreatment values. Paired Student t tests were used to compare between treatment groups. Values represent means ± SD of biological replicates. (H) Cell viability analysis in Molm13 control cells (control), Molm13 PPM1D-truncating mutant cells (PPM1D-mutant) and Molm13 cells with overexpression of wild-type PPM1D (WT overexpression). Cells were exposed to increasing concentrations of cytarabine for 72 hours. Experiments were performed with biological replicates and data are shown as the means ± SD. Nonlinear logistic regression analyses and a sum of squares F test were performed to compare the inhibitory response to cytarabine in Molm13 WT overexpression cells and Molm13 PPM1D-truncating mutant cells to the inhibitory response in Molm13 control cells.

Figure 3.

PPM1D plays a central role in the DDR pathway. (A) Whole-cell lysates of Molm13 PPM1D-mutant and Molm13 isogenic control single-cell clones exposed to 400 nM cytarabine for 4 hours were probed with anti-p53 Ser15 and anti-CHEK1 Ser345. (B) The log fold-change of different peptides for Molm13 PPM1D-mutant cells compared with Molm13 control cells. The starting amino acid for each peptide is shown. The peptides range from 8 to 22 aa in length. (C) Heatmap of phosphosites that are significantly downregulated with an FDR < 0.05 in Molm13 PPM1D-mutant/control at baseline (Mut/cntrl) or after cytarabine (AraC) treatment (Mut AraC/cntrl AraC), and upregulated (FDR < 0.05) after 4 hours of treatment with 400 nM cytarabine and GSK2830371 (PPM1D inhibitor) (Mut AraC + PPM1Di/Mut AraC). Results shown are for biological replicates. (D) Heatmap of phosphosites belonging to the KEGG_P53 pathway that are significantly regulated (FDR < 0.1) in Molm13 PPM1D-mutant/control after 4-hour treatment with 400 nM cytarabine (AraC) (Mut AraC/cntrl AraC) or in Molm13 PPM1D-mutant cells treated with 400 nM AraC + 1 μM PPM1D inhibitor GSK2830371 compared with Molm13 PPM1D mut cells treated with 400nM AraC (Mut AraC + iPPM1D/Mut AraC) for 4 hours. Results shown are for biological replicates. (E) Conserved amino acid residues flanking PPM1D-dependent phosphorylation sites based on 43 substrate candidates that are downregulated (FDR < 0.1) in Molm13 PPM1D-mutant/cntrl for either the baseline or the AraC-treated comparisons and at the same time upregulated (FDR < 0.1) after PPM1D inhibitor GSK2830371 treatment. The combined occurrence of glutamine at +1 and ≥2 acidic residues in the flanking region is statistically significant (P = 3.595e-13, Fisher exact test). (F) Schema illustrating the components of the DDR pathway that are targeted by PPM1D in leukemia cells, based on the results from the phosphoproteomic analysis. Phosphorylation targets of PPM1D identified by mass spectrometry (FDR < 0.1) are shown in green. Predicted PPM1D target sites that were based on the identified consensus sequence with a glutamine at +1 and ≥2 acidic residues are shown in blue.

Figure 4.

PPM1D-mutant cells have an abrogated apoptotic and cell-cycle response to chemotherapy. (A) Annexin V staining and flow cytometric analysis of Molm13 PPM1D-mutant and control single-cell clones exposed to 400 nM cytarabine or vehicle treatment of 24 hours. Data are shown as the means ± SD for biological replicates, and unpaired Student t tests were used for statistical analyses. (B) BrdU staining and flow cytometric analysis of Molm13 PPM1D-mutant and control single-cell clones after 24 hours of exposure to 100 nM cytarabine. Data are shown as the means ± SD for biological replicates and unpaired Student t tests were used to compare the cell-cycle progression in Molm13 PPM1D-mutant and control cells. (C-D) Molm13 PPM1D-mutant and control cells were exposed to cytarabine (0.25 µM) (C) or etoposide (0.5 µM) (D) for 16 hours. For each peptide, the cytochrome c release of the no-drug (DMSO)-treated cells are subtracted from the drug-treated samples to derive the drug-induced change in priming (percent δ priming).

Figure 5.

PPM1D inhibition with GSK2830371 reverses chemotherapy resistance and selectively targets PPM1D-mutant cells. (A) Whole-cell lysate of Molm13 PPM1D-mutant single-cell clones 1 hour pretreated with the indicated concentrations of GSK2830371 and exposed to 400 nM cytarabine or vehicle were probed for p53 Ser15 and Actin as a loading control (B) Annexin V staining and flow cytometric analysis of Molm13 PPM1D-mutant and control single-cell clones exposed to 3 μM GSK2830371. Experiments were performed in triplicate and data are shown as the means ± SD unpaired Student t tests were used to compare means. (C) Molm13 PPM1D-mutant and control cells were exposed to GSK2830371 0.25 µM for 16 hours. δ priming (%) consists of the cytochrome c release of the no-drug (DMSO)-treated cells subtracted from the drug-treated samples for each peptide. (D) Seventy-two-hour cell viability analysis in Molm13 PPM1D-mutant and control single-cell clones after exposure to increasing doses of GSK2830371. Data are shown as the means ± SD for biological triplicates and nonlinear logistic regression analyses and a sum of squares F test were performed for statistical analysis. (E) Viability analysis of Molm13 PPM1D-mutant cells and Molm13 control single-cell clones pretreated for 1 hour with 3 μM GSK2830371 or vehicle and exposed to increasing doses of 72-hour cytarabine treatment. Data are shown as the means ± SD for biological replicates. Nonlinear logistic regression analyses and a sum of squares F test were performed to compare the inhibitory response to cytarabine between Molm13 PPM1D-truncating mutant cells pretreated with GSK2830371 and Molm13 control cells, and Molm13 PPM1D-mutant cells. (F) Competition experiment with Molm13 PPM1D-mutant pooled cells and isogenic control pooled cells mixed in respectively a 1:9 ratio and exposed to 100 nM cytarabine, 100 nM cytarabine plus 100 nM GSK2830371, or vehicle treatment. Data are shown as the means ± SD for biological triplicates.