Targeting synthetic lethality between non-homologous end joining and radiation in very-high-risk medulloblastoma

Abstract

Specific and biologically informed treatments for medulloblastoma, especially for the highly lethal TP53-mutant SHH subgroup, remain elusive, where radiotherapy is the primary treatment modality. Leveraging genome-wide CRISPR-Cas9 dropout screening in combination with lethal doses of radiotherapy, we identify loss of p53 as the main driver of radiation resistance in SHH medulloblastoma. A negative-selection CRISPR-Cas9 screen across multiple models of Trp53-deficient SHH medulloblastoma reveals a strong synthetic lethal interaction between components of the non-homologous end-joining pathway and radiation, particularly DNA-dependent protein kinase (DNA-PK) and its binding partners. Both genetic and pharmacological perturbation of DNA-PK enhance radiosensitivity in TP53-deficient SHH medulloblastoma, leading to cell death. In vivo treatment of both somatic and germline TP53-mutant SHH medulloblastoma models with peposertib, a small-molecule inhibitor of DNA-PK, significantly improves survival when combined with radiotherapy, strongly supporting further clinical investigation.

Keywords: DNA repair; DNA-protein kinase; medulloblastoma; non-homologous end joining; p53; peposertib; radiation; subgroup.

Graphical abstract

Figure 1

p53 deficiency confers radiation resistance in SHH-MB (A) Radiation dose-response curve (left) across a panel of SHHtrp53 (gray) and SHHwt (red). Cell viability was measured using alamarBlue and normalized to 0 Gy and a positive control (puromycin). Analysis of the area under the curve (AUC) from a non-linear regression of all models and replicates (right) demonstrates significantly increased (p = 0.0002) radiation resistance in SHHtrp53. Data were analyzed by Mann-Whitney test. Data are represented as mean ± SD across three biological replicates per model. (B) Screen methodology. The mTKO library contains 94,528 gRNAs that target 19,069 protein-coding genes and are analogous to the human TKO version 3 (TKOv3) library, providing full coverage of the mouse genome. This screen is performed in three experimental arms with three technical replicates per arm, comparing no radiation with a sublethal and lethal dose of radiation to identify context-specific genes in SHH-MB. Created in BioRender. Decarlo, A. (2025) https://BioRender.com/jcd6d4f. (C) Volcano plots of normZ scores of gene essentiality versus −log10(FDR) in SHHwt treated with lethal doses of radiation. A positive normZ score infers genes that cause resistance to radiotherapy. The shaded area represents the 10% FDR. (D–F) Western blot analysis of yH2AX expression following radiation among SHHtrp53 (D), SHHwt (E), and isogenic model (F) of SHH-MB over 48 h. Data were normalized to protein loading control and T0 and analyzed by one-way ANOVA with Dunnett’s test for multiple comparisons to assess relative expression levels. Data are represented as mean ± SD across three biological replicates.

Figure 2

Trp53-deficient SHH-MB relies on NHEJ following radiation (A) Screen methodology as described in Figure 1A. Created in BioRender. Decarlo, A. (2025) https://BioRender.com/jcd6d4f. (B) NormZ scores at midpoint across all screened SHHtrp53 models and one SHHwt model with genes highlighted in pink being involved in NHEJ and below the FDR threshold of 0.1 (10%). (C) Venn diagram of normZ score of −1 or less with 10% FDR threshold at midpoint, representing essential genes for response to radiation across all three SHHtrp53, highlighting key three genes involved in NHEJ. (D) Schematic diagram of NHEJ DNA DSB repair. Genes and the corresponding protein highlighted in black represent enriched targets from the CRISPR screen. (E) Heatmap of quantile normalization of normZ scores at endpoint divided into different DNA repair pathways across both Trp53-deficient and wild-type SHH-MB models.

Figure 3

Genetic and therapeutic targeting of PRKDC and DNA-PK sensitizes Trp53-deficient SHH-MB to radiation (A) Radiation dose-response curve (left) testing radiation sensitivity between two PRKDC genetic knockouts (green and orange), 250 nM of peposertib (pink), and a non-targeting control (blue). Analysis of the AUC from a non-linear regression of all replicates demonstrating a significant increase in sensitivity between the non-targeting control and gRNA#2 (p = 0.0028), gRNA#3 (p = 0.0056), and peposertib (p = 0.046) with no significant difference in sensitivity between peposertib and gRNA#2 and gRNA#3 (p = 0.21, 0.43, respectively). Data are represented as mean ± SD across three biological replicates. (B–D) Three shRNA knockdowns of PRDKC in human DAOY MB. Top and bottom graphs demonstrate that combining peposertib with the shRNA knockdown does not decrease viability compared to knockdown alone. Bottom graphs demonstrate that all three knockdowns sensitize DAOY to 6 Gy of radiation (p < 0.0001). Data analyzed by Mann-Whitney tests. Data are represented as mean ± SD across three biological replicates. (E) 12-point dose-response curves with peposertib with and without a sublethal dose of 4 Gy of radiation for Trp53-deficient SHH-MB. Solid colors represent no radiation, and dashed lines represent sublethal radiation. Data are represented as mean ± SD across three biological replicates. (F) 12-point dose-response curve with peposertib with and without a sublethal dose of 4 Gy of radiation for SHH^wt;MSCV-DDP53-GFP. Solid colors represent no radiation, and dashed lines represent sublethal radiation. Data are represented as mean ± SD across three biological replicates. (G) 12-point dose-response curves with peposertib with and without a sublethal dose of 6 Gy of radiation for Tp53-mutated human DAOY MB. Solid colors represent no radiation, and dashed lines represent sublethal radiation. Data are represented as mean ± SD across three biological replicates.

Figure 4

Therapeutic inhibition of DNA-PK sustains radiation sensitization over several cell doublings (A) Cell growth assays over 7 days with and without radiation in combination with 250 nM of peposertib. Cell images were taken by Incucyte from four different fields/well every 2 h over 7 consecutive days to infer cell growth. Cell confluence plotted as percent of the confluence determined in the DMSO controls (set as 100%). Top graph shows combined mean across multiple SHHtrp53 models. Bottom graph represents AUC of each independent assay, analyzed by one-way ANOVA with Tukey’s multiple comparisons test demonstrating a significant decrease in cell growth between radiation alone and radiation in combination with peposertib (p = 0.037). Data are represented as mean ± SD across three biological replicates. (B) Cell growth assay in human SHH-MB Daoy cells over 7 days as described above. Top graph shows combined mean across four replicates. Bottom graph represents AUC of each independent assay analyzed by one-way ANOVA with Tukey’s multiple comparisons test demonstrating a significant decrease in cell growth between radiation alone and radiation in combination with peposertib (p = 0.0017). Data are represented as mean ± SD across three biological replicates. (C) 14-day clonogenic assay combined analysis of multiple SHHtrp53 models with and without radiation in combination with 250 nM of peposertib. Surviving fraction is calculated based on the number of colonies formed normalized to seeding density and plating efficiency. Data are analyzed with multiple unpaired t tests of means of all replicates demonstrating significant decrease in clonogenic potential (p < 0.0001) when cells are treated with a combination of radiation and peposertib. Data are represented as mean ± SD across three biological replicates. (D) 14-day clonogenic assay of SHHwt models with and without radiation in combination with 250 nM of peposertib. Surviving fraction is calculated based on the number of colonies formed normalized to seeding density and plating efficiency. Data are analyzed with multiple unpaired t tests of means of all replicates demonstrating no significant decrease in clonogenic potential (p = 0.97) when cells are treated with a combination of radiation and peposertib. Data are represented as mean ± SD across three biological replicates. (E) 12-point dose-response curves with peposertib with and without 1 Gy of radiation for high-risk metastatic group 3 MB002. Solid colors represent no radiation, and dashed lines represent sublethal radiation. Data are represented as mean ± SD across three biological replicates. (F) 12-point dose-response curves with peposertib with and without 1 Gy of radiation for high-risk primary group 3 2112FH. Solid colors represent no radiation, and dashed lines represent sublethal radiation. Data are represented as mean ± SD across three biological replicates.

Figure 5

Therapeutic inhibition of DNA-PK induces necrosis in Trp53-deficient SHH-MB (A) Representative images of the comet assay with lysed SHHtrp53 cells stained with SYBR gold following radiation with or without peposertib collected at 1 or 24 h following treatment. White scale bar represents 100 μm. (B) Analysis of comet tail length radiation alone and radiation combined with peposertib via open comet software through ImageJ. Data were analyzed by multiple Mann-Whitney tests and demonstrate that peposertib significantly increases the amount of DNA damage following 1 h (p = 0.00037) and 24 h (p = 0.011) of a single 4 Gy dose of radiation. (C) Representative images of annexin V/PI flow cytometry cell viability assay in one SHHtrp53 model at 48 and 96 h time points. (D) Combined analysis of two SHHtrp53 models, each with three independent technical and biological replicates of annexin V/PI assay at multiple time points representing total percentage of cell death demonstrating a significant increase in cell death after 96 h with combination therapy compared to radiation alone (p = 0.0031). Data analyzed by a two-way ANOVA with the Geisser-Greenhouse correction with matched values and Tukey’s multiple comparisons test across six biological replicates. (E) Analysis of senescence with β-galactosidase via flow cytometry in one SHHtrp53 model at multiple time points.

Figure 6

Inhibition of DNA-PK with peposertib results in potent radiosensitization in vivo (A) Experimental schema (top). PDX models were orthotopically injected into the hindbrain of 6-week-old male NRG mice. Following 10 days post intracranial injections, mice were divided into four treatment cohorts treated with peposertib with and without radiation for 5 consecutive days. Field of radiation (bottom) with 10 × 20 mm rectangular collimator. 2 Gy was delivered in a bilateral beam arrangement. (B) Craniospinal radiation: field of radiation with 10 × 20 mm rectangular collimator (brain), C10 collimator (cervical spine), and 10 × 30 mm rectangular collimator (thoracic spine). 2 Gy was delivered in a bilateral beam arrangement. (C) Kaplan-Meier survival plot of TP53-mutated SHH-MB PDX in four treatment cohorts: vehicle (n = 3), peposertib (n = 3), whole-brain radiation (n = 9), and whole-brain radiation with peposertib (n = 9). Results demonstrate a significant increase in tumor-free survival (p < 0.0001) when treated in combination with peposertib and radiation. (D) Kaplan-Meier survival plot of TP53-mutated SHH-MB PDX in four treatment cohorts: vehicle (n = 2), peposertib (n = 2), craniospinal radiation (n = 5), and craniospinal radiation with peposertib (n = 4). Results demonstrate a significant increase in tumor-free survival (p < 0.0001) when treated in combination with peposertib and radiation. (E) Ki-67 immunoreactivity (brown) of brain and spinal cord (SC) of mice treated with whole-brain radiation. Circles represent bulk tumors, and arrows represent metastasis demonstrating a mouse which died of spinal metastasis (top) compared to a mouse dying from the primary tumor (bottom). Black scale bar represents 2 mm. (F) MRI images of all mice treated with craniospinal radiation in combination with peposertib. Images represent before treatment, 2 weeks following treatment, and 1 month following treatment. Yellow contouring highlights bulk tumor. (G) MRI images of all mice treated with craniospinal radiation alone. Images represent before treatment, 2 weeks following treatment, and 1 month following treatment. Yellow contouring highlights bulk tumor.

Figure 7

Inhibition of DNA-PK with peposertib results in potent radiosensitization in vivo (A) Kaplan-Meier survival plot of TP53-mutated SHH-MB PDX in four treatment cohorts: vehicle (n = 5), peposertib (n = 5), whole-brain radiation (n = 9), and whole-brain radiation with peposertib (n = 9). Results demonstrate a significant increase in tumor-free survival (p = 0.001) when treated in combination with peposertib and radiation. (B) H&E (purple) and Ki-67 immunohistochemistry (IHC) (brown) of brain and spinal cord (SC) of mice treated with whole-brain radiation. Circles represent bulk tumors, and arrows represent metastasis. (C) MRI images of all mice treated with whole-brain radiation alone following 9 and 11 weeks after treatment. Yellow contouring highlights bulk tumor. (D) MRI images of all mice treated with whole-brain radiation in combination with peposertib following 9 and 11 weeks after treatment. Yellow contouring highlights bulk tumor. (E) MRI (top row), H&E (middle row), and Ki-67 IHC (bottom row) of vehicle alone (left) and peposertib alone (right)-treated mice. Black scale bare represents 2 mm. Yellow contouring highlights bulk tumor.