Fig 2: PPM1D mutant immortalized human astrocytes are sensitive to NAMPT inhibitors. a Previously identified (refs. 8–10) PPM1D truncation mutations in pediatric HGGs (blue circles). CRISPR-modified mutations in human astrocytes shown in red arrows. b Immunoblot of PPM1D full-length (full arrow) and truncated (arrowhead) protein expression across parental astrocytes (Par.), an isolated wild type astrocyte clone (WT iso.), and four different isolated CRISPR-modified, PPM1D-truncated (PPM1Dtrnc.) astrocytes. c Immunoblot of PPM1D expression post cycloheximide (CHX) and MG132 treatment. d Quantification of the experiment in (c), (n = 3 biologically independent experiments, *p < 0.05, **p < 0.01 by Student’s T test). e Representative images of cellular γH2AX foci, +/− treatment with 10 Gy ionizing radiation (IR). f Quantification of γH2AX foci in untreated, IR-treated, and concurrent IR plus 50 nM PPM1D inhibitor GSK2830371 treatment (PPM1Di); (n = 4 biologically independent samples, **p < 0.001 by Student’s T test). g Calculated IC50 ratios (Parental/PPM1Dtrnc.) for a library of tested small molecule inhibitors. h Viability assessment of wild type (Par. Astros. and WT iso.) and three PPM1Dtrnc. cell lines, 72hrs post FK866 treatment (n = 3 biologically independent samples). i Calculated IC50 values of parental (black highlight) and PPM1Dtrnc. (red highlight) astrocytes for different NAMPT inhibitors; length of bar represents selectivity window of the given drug for PPM1D mutant cells (n = 2 biologically independent experiments). j Viability analysis of cell lines in response to 72 h of FK866 treatment (n = 3 biologically independent samples). All error bars represent standard deviation of the mean

Fig 3: A schematic of AMPK-mediated NAMPT activation under ionizing radiation. Ionizing radiation causes rapid activation of AMPK, which in turn phosphorylates NAMPT S314 and enhances NAMPT enzymatic activity. NAMPT activation promotes NAD+ synthesis, thereby facilitating DNA repair and cell viability.

Fig 4: AMPK-dependent S314 phosphorylation activates NAMPT by facilitating NAMPT/PRPP association. (a) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the NAMPT enzymatic activity in the precipitates was measured. ***p < 0.001. (b) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the NAMPT enzymatic activity in the precipitates was measured. IR, ionizing radiation; WCL, whole cell lysate; **p < 0.01. (c) The NAMPT catalytic domain in human NAMPT protein (shown in cyan) structure was boxed by dotted line, and enlarged to show the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple, PDB code: 2E5C), and nicotinamide (oxygen atom shown in purple, PDB code: 2E5D). (d) Human NAMPT protein structure (PDB code: 2E5C) shows the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple), and H247 (side chain shown in orange). (e) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the binding affinity between NAMPT protein and PRPP was measured. ***p < 0.001. (f) HOK cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01. (g) HOK cells with expression of WT Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and cells were then treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS and the binding affinity between NAMPT protein and PRPP was measured. **p < 0.01.

Fig 5: NAMPT inhibitors are effective in vivo agents against PPM1D mutant xenografts. a Fold change in tumor growth for serially-transplanted PPM1Dtrnc. xenografts in NSG mice treated with vehicle or 20 mg/kg FK866 BID for 3 cycles of: 4 days on, followed by 3 days off (n = 7 animals, ***p < 0.001 by Mann–Whitney U test, error bars represent standard deviation of the mean). Arrows indicate initiation of treatment cycle. b Kaplan–Meier plot of xenograft tumor growth from (a), with arrows indicating initiation of treatment cycle (p < 0.0001 by Log rank (Mantel-Cox) test). c NAPRT expression levels for PNOC003 DIPG cohort31 samples. d Model depicting the mechanism of mutant PPM1D-induced dependence on NAMPT for NAD production, and synthetic lethality with NAMPT inhibitors, such as FK866