PPM1D modulates hematopoietic cell fitness and response to DNA damage and is a therapeutic target in myeloid malignancy

Key Points • Ppm1d activity is a key regulator of hematopoietic cell fitness in the absence and presence of exogenous genotoxic stresses.• Inhibition of Ppm1d sensitizes malignant cells to cytotoxic therapies and is dependent of p53 activity.


In Vitro Drug Exposure of Mouse Leukemia Cells
In vitro exposure experiments were performed as follows.For the three-day viability analyses, each genotype, 5,000 cells were plated in 50 ul of media per well of 384-well plates.The indicated drugs (Selleck Chemicals) were diluted in DMSO and added to the appropriate wells o using an automated drug dispenser (Tecan) or the plates were exposed to the indicated dose of radiation.Three days later, the 10 ul of CellTiter-Glo (Promega) were added to each well and the luminescence signal was measured using an EnVision plate reader (PerkinElmer).
For the in vitro resistance experiments, RFP657 + Ppm1d T476-fl/fl and BFP + Ppm1d +/+ leukemia cells were mixed in a 1:20 ratio then plated at 50,000 cells per well in 200 ul of media into a 96 well plate.The indicated dose of drugs (Selleck Chemicals) were added to each well using the automated drug dispenser (Tecan).Every 3-4 days the cells were replated in fresh media and analyzed for the ratio of RFP657:BFP using a BD FACSCanto II.

In Vitro Drug Exposure of Human Patient Derived Xenograft Models
Primary patient samples were acquired following written informed consent per the Declaration of Helsinki, and patient-derived xenografts (PDX) models were established under protocols approved by Dana-Farber Cancer Institute and Cincinnati Children's Hospital Medical Center institutional review boards.For short-term in vitro culture, PDX cells were maintained in IMDM containing 20% FBS and 1% PS and supplemented with 10 ng/mL human SCF, TPO, FLT3L,  IL3, and IL6 (PeproTech 300-07, 300-18, 300-19, 200-03 and 200-06).Cells were exposed to drugs at the indicated concentrations for 72 hours and viability was measured using the CellTiter-Glo reagent.

Dynamic BH3 Profiling of Human Patient Derived Xenograft Models
All animal studies were performed in accordance with approved IACUC guidelines at Dana-Farber Cancer Institute animal facility (IACUC protocol #14-038) and at National University of Singapore facility (IACUC protocol # R20-0342).AML PDX models are available from the Center for Patient-Derived Models at Dana-Farber Cancer Institute (https://www.pdxfinder.org/source/dfci-cpdm/). Female NSG mice at 6-8 weeks of age (Jackson Labs) were injected with 0.6 million human leukemia cells intravenously (IV).Following transplant, mice were bled weekly, and treatment was initiated when circulating leukemia burden was >5% as assessed by flow cytometry staining for hCD45 (clone HI30, BD Biosciences) and hCD33 (clone WM53, BD Biosciences).Animals were humanely euthanized, and the spleen and bone marrow were processed into single cell suspensions.Red blood cell lysed cells were plated with heat-inactivated RPMI-1640 (Invitrogen) and 10% fetal bovine serum in 24-well plates at 1 million/mL and exposed to a GSK compound for 14 h or DMSO (control) followed by BH3 profiling (Ryan et al., 2016).Briefly, cells were exposed to synthetic BH3 peptides after plasma membrane permeabilization by 5% digitonin.The gating strategy included antibody markers for CD45 (Fisher Scientific, Clone HI30), CD34 (BioLegend, Clone 581), CD38 (BioLegend, Clone HIT2), CD33 (Fisher Scientific, Clone WM53), c-kit (BioLegend, Clone 104D2), and live/dead fixable zombie yellow stain (BioLegend).AML blasts were identified by CD45 lo-mid / SCC-low.Sensitivity to BH3 peptides was measured as percent cytochrome c release (BioLegend, 6H2.B4) loss as determined by FACS.DMSO was used as a negative control for cytochrome c release, whereas a control without the cytochrome c antibody was used as a positive control for 100% cytochrome c release.The read-out of dynamic BH3 profiling is drug-induced change in priming defined as "delta priming" (calculation: delta priming = cytochrome c loss[drug] -cytochrome c loss [DMSO]).An acceptable delta priming threshold (<15%) calculated by cytochrome c release caused by 3(mean ± SD) of DMSO treated wells was used to determine significance.

CRISPR/Cas9 Screen
Human K562 cells were grown in RPMI (Gibco) supplemented with 10% FBS.K562 cells were engineered to carry a wild-type copy of TP53 and a truncated version of PPM1D as previously described. 12,13The cells were infected with a custom library of sgRNAs encoded by lentivirus obtained from the Broad Institute.Two days after infection the cells were selected in 2 ug/ml puromycin for three days then cultured for the following three weeks (media change every three days) in the presence of either DMSO (Sigma Aldrich), daunorubicin (Selleck Chemicals), or GSK2830371 (Selleck Chemicals).An aliquot of cells was isolated immediately following puromycin selection and after 21 days of culture.DNA was isolated from the cells and the relative representation of each sgRNA was quantified using next generation sequencing as previously described. 14,15man Cell Line Drug Exposures The human cell lines were cultured in the RPMI (Gibco) + 10% FBS (TC32, TC71, SIMA) or DMEM/F12 (Gibco) +10% FBS (SKNBE2).For the drug or radiation treatments, 5,000 cells were plated in 50 ul of media per well of 384-well plates.The indicated drugs (Selleck Chemicals) were diluted in DMSO and added to the appropriate wells o using an automated drug dispenser (Tecan) or the plates were exposed to the indicated dose of radiation.Three days later, the 10 ul of CellTiter-Glo (Promega) were added to each well and the luminescence signal was measured using an EnVision plate reader (PerkinElmer).

PRISM Screen and Analysis of Cancer Dependency Map Data
750 DNA-barcoded cell lines grown in pools of approximately 100 cell lines per pool were exposed for five days to 8 different doses of daunorubicin (range from 0.1nM to 2.5 uM), 8 doses of GSK2830371 (range from 1nM to 10 uM), or 8 doses of daunorubicin (range from 0.1nM to 2.5 uM) with 2.5 uM GSK2830371.The relative viability of each treatment was assessed by comparing the representation of each cell line, assessed by quantification of DNA barcodes, in the treated wells compared to the negative control (DMSO) and positive control (Bortezomib) wells.Using these normalized viability values, the area under the curve for each treatment condition was then calculated. 16The publicly available 22Q4 release was used.DNA mutation data, RNA-sequencing expression data (Log2(Total counts per million + 1), and CRISPR/Cas9 gene knockout dependencies (CERES scores) were obtained for all available cell lines and analyzed, stratified based on TP53 mutation status, as defined as any mutation present. 19
expression levels of PPM1D across 989 cell lines profiled as part of the Cancer Dependency Map, stratified by TP53 mutational status.**** p-value < 0.0001 (B) Sensitivity of cell lines to CRISPR-Cas9 mediated knockout of PPM1D and TP53, as assessed by the CERES score which corrects for copy number differences between cell lines.A negative CERES score reflects impaired cell growth with knockout of the target gene.The TP53 mutant lines are highlighted in red.(C) Comparison of PPM1D RNA expression levels and sensitivity to PPM1D knockout, as measured by the CERES score, in TP53 wildtype (left) and mutant (right) cell lines.