Differential Distribution of the DNA-PKcs Inhibitor Peposertib Selectively Radiosensitizes Patient-derived Melanoma Brain Metastasis Xenografts

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Brain metastases from solid malignancies are a major cause of morbidity and mortality (1, 2). While molecularly targeted agents can be used to treat a subset of patients, neurosurgical resection, focal stereotactic surgery or whole-brain radiotherapy (WBRT) is necessary to achieve long-term local control for most patients with intracranial disease. To limit the neurocognitive effects of whole-brain radiation, a relatively gentle dose-fractionation schedule is used, which limits local control of brain metastases, especially for radioresistant histologies such as melanoma (3). Therefore, there is a compelling and unmet medical need to develop novel radiosensitizing strategies in conjunction with whole-brain radiation to enhance tumor control.

Therapeutic irradiation induces a multitude of DNA damages, but unrepaired double-strand DNA (dsDNA) breaks are critically lethal lesions. Repair of radiation-induced dsDNA breaks is primarily governed by two major repair pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). These pathways are modulated by two related protein kinases, ataxia-telangiectasia mutated (ATM) protein and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), respectively. Each kinase phosphorylates numerous downstream targets to coordinate comprehensive cellular DNA damage response, and consistent with prominent role of DNA-PKcs in NHEJ, multiple groups have developed DNA-PKcs inhibitors as radiosensitizing or chemosensitizing agents (4). In this article, we evaluated one of the more mature DNA-PKcs inhibitors, peposertib (5), as a possible radiosensitizer for melanoma brain metastases.

Peposertib is a potent, orally available small-molecule inhibitor of DNA-PKcs kinase with activity at nanomolar concentrations (6). Two studies have demonstrated compelling radiosensitizing effects of peposertib in clonogenic survival studies using colorectal and glioblastoma cell lines, and similar sensitizing effects in cell viability assays across many cancer cell lines (5–7). The initial description of peposertib also demonstrated remarkable in vivo radiosensitizing effects in FaDu and H460 lung carcinoma cell lines, while multiple subsequent studies reporting more limited in vivo radiosensitizing effects across numerous murine and human tumor cell lines grown as flank xenografts (5–9). With this apparent discrepancy between in vitro and in vivo radiosensitizing effects of peposertib, the current study focused on integrating time and concentration exposure relationships required for in vitro radiosensitization with pharmacokinetic parameters describing drug exposure to identify an efficacious peposertib dosing regimen that significantly radiosensitizes human melanoma brain metastasis patient-derived xenografts (PDX) to WBRT.

Animal studies were approved by Mayo Clinic and University of Minnesota (Minneapolis, MN) Institutional Animal Care and Use Committee policies (IACUC). Athymic nude mice (Charles River) were used for PDX orthotopic efficacy studies, which were performed as described previously (10). FVB mice (Charles River) were used for normal tissue radiation studies. Pharmacokinetic studies at the University of Minnesota (Minneapolis, MN) were in FVB mice, (8–12 weeks old) from a breeding colony with breeder pairs obtained from Taconic Biosciences. Mice were dosed as indicated, observed daily, and euthanized at indicated timepoints. IACUC endpoint guidelines for euthanasia included weight loss ≥ 20%, inability to reach food/water, immobility, hunched posture, lethargy, spasticity, seizures, circling, and paralysis.

Metastatic melanoma PDX short-term explant cultures M12, M12-eGFP, M15, and M27 were maintained as described previously (10)—additional information in Supplementary Materials and Methods. M12 and M12-eGFP cells were cultured in Stem Pro cell media. CyQUANT, clonogenic survival and other assays were performed as described previously (11, 12).

Peposertib was provided by Cancer Therapy Evaluation Program at the NCI via EMD Serono. Drug was dissolved in DMSO and stored at −20°C. For in vivo studies, peposertib was suspended in 0.25% hydroxypropyl methylcellulose and 0.25% Tween 20 in sodium citrate buffer (500 mmol/L, pH2.5) and administered by oral gavage. Radiation was delivered to anesthetized mice using a 10 mm circular collimator with opposing lateral beams (225 kVp, 20 mA, 0.3 mm Cu filter) delivered with a PXI X-RAD SmART+ irradiator. Oral cavity irradiation was performed with a 10 mm circular collimator and single anterior-posterior beam. Analysis of peposertib concentration by LC/MS was performed as published previously (13).

All Western blots were performed as reported previously (14).

Human-induced pluripotent stem cells (iPSC; 004-BIOTR-0002) were obtained from the Biotrust at Mayo Clinic Center for Regenerative Medicine. The undifferentiated iPSCs culture described method previously (15) was used to derive differentiated cortical neurons.

Immunofluorescence staining of dsDNA breaks was evaluated using γH2AX foci as described in ref. 14 and in Supplementary Materials and Methods.

In vitro data presented are the mean ± SEM from three or more experiments unless otherwise indicated. The two-sample t test (rank-sum) was used to make comparisons across groups. Analysis of covariance was used to compare weight change across the groups. For this analysis, follow-up weight was the outcome; group and weight at day 0 were the independent predictors. Cumulative survival distributions were estimated using the Kaplan–Meier method. The log-rank test was used to make comparisons across groups. In all cases, P value <0.05 were considered statistically significant.

The data generated in this study are available within the article and its Supplementary Data files.

The effects of peposertib on radiation-induced DNA damage signaling pathways was assessed to define drug concentrations that could affect DNA repair. Short-term explant cultures from M15 melanoma PDXs were pretreated with graded concentrations of peposertib followed by exposure to 0 or 5 Gy. Concentrations of 100, 300, and 1,000 nmol/L peposertib effectively suppressed radiation-induced autophorylation of DNA-PKcs (phospho-S2056; Fig. 1A). Consistent with enhanced activation of parallel signaling pathways, 300 and 1,000 nmol/L peposertib combined with radiation resulted in elevated phospho-S824-KAP1. A similar dose–response was observed in M27 melanoma cells (Fig. 1B). Using 300 nmol/L peposertib, the temporal effect of peposertib on DNA damage signaling induced by 5 Gy radiation was evaluated in M12 cells. Consistent with inhibition of DNA repair, resolution of phospho-S139 H2AX (γH2AX) was delayed with peposertib cotreatment, and phospho-S824-KAP1 remained elevated 8 hours after irradiation (Fig. 1C). These data are consistent with inhibition of DNA-PKcs and suppression of efficient DNA repair at concentrations above 100 nmol/L peposertib.

The effect of peposertib on radiation survival was evaluated in M12 cells in a clonogenic assay. M12 cells were treated with peposertib and then treated with 0 or 2.5 Gy. In comparison with radiation alone (45% ± 1.7% survival), clonogenic survival was significantly reduced with peposertib cotreatment of 100 nmol/L (9% ± 2%, P = 0.0001), 300 nmol/L (4% ± 0.4%, P = 0.0007), and 1,000 nmol/L (2% ± 0.2%, P = 0.0008) but not 30 nmol/L (41% ± 3%, P = 0.21).

Stepwise comparisons between peposertib concentrations in combination with radiation also identified significant differences in survival between 30 and 100 nmol/L (P = 0.002), 100 and 300 nmol/L (P = 0.03), but not 300 and 1,000 nmol/L (P = 0.11; Fig. 1D). The radiosensitizing effect of 300 nmol/L peposertib also was confirmed in a second study with robust sensitizing effects observed at 2.5 and 5 Gy radiation (P = 0.03, P = 0.002, respectively; Fig. 1E) and a calculated sensitizer enhancement ratio at 10% survival (SER₁₀) of 1.9 ± 0.1, which reflects potent radiosensitizing effects.

The impact of peposertib on DNA repair was indirectly assessed by evaluating the kinetics of γH2AX foci resolution. Following 2.5 Gy irradiation, the majority of γH2AX foci were resolved 8 hours after treatment. While peposertib did not significantly affect the extent of γH2AX foci induction with similar levels of foci at 0.5 and 2 hours after irradiation, at later timepoints, the number of cells with unresolved γH2AX was significantly greater (Fig. 2A and B). Similarly, delay in γH2AX foci resolution was mediated by peposertib cotreatment in M15 and M27 melanoma PDX lines, with greater number of cells with unresolved γH2AX foci at 4 and 16 hours (M15 and M27) after irradiation (Supplementary Fig. S1 and S2). The optimal duration of peposertib exposure was evaluated in a clonogenic assay. M12 cells were treated with 300 nmol/L peposertib, irradiated, then drug removed through a media change at different timepoints (2, 4, 8, 16, 20, 24 hours) after radiation exposure. Significant sensitizing effects were observed even with only 2 hours of exposure (P = 0.02), while maximal sensitizing effect was observed with exposures of 16 hours or longer (P = 0.0004; Fig. 2C). Notable, to facilitate comparisons of drug levels between in vitro and in vivo studies, the fraction of unbound, free peposertib in the culture media was previously determined to be 50% in the cells culture media used (13). On the basis of the totality of the in vitro data, this defines a target tissue exposure in animals for maximal radiosensitizing effects of 150 nmol/L free peposertib for 16 hours, and minimally effective concentration of 50 nmol/L free peposertib.

We have previously evaluated the pharmacokinetics of peposertib. Following a single oral dose of 20 mpk, the terminal t_1/2 was 2.4 hours, T_max was 2 hours, and a free fraction in mouse plasma and brain was 11.4% ± 0.9% and 4.1% ± 0.6%, respectively (13). Assuming dose-linearity, these data were used to simulate plasma exposure of the published radiosensitizing regimen of 50 mpk daily. This demonstrated drug levels in blood plasma would drop below the target for maximal sensitizing effects (150 nmol/L free peposertib) within 5 hours of dosing, and a minimally effective free drug concentration of 50 nmol/L free peposertib would be maintained only for 7 hours (Fig. 3A). Even 150 mpk dosed once had limited exposure at times greater than 12 hours. In contrast, 75 mpk dosed twice daily, 7 hours apart, was required to maintain 50 nmol/L free peposertib for the 16-hour target duration (Fig. 3A).

Genetic deficiency in DNA-PKcs is associated with significantly elevated normal tissue toxicities following radiation, and similar enhanced normal tissue effects with small-molecule DNA-PKcs inhibitors are expected. Therefore, the impact of different peposertib dosing regimens were evaluated in a radiation model of oral mucositis (16). In this model, the oral cavity and oropharynx of non–tumor-bearing mice were exposed to three daily radiation doses. Ensuing mucosal injury limits oral intake and results in significant weight loss approximately 1.5 weeks after radiation exposure. Using this model, radiation alone (3.6 Gy × 3 fractions) was well tolerated with no body weight loss [1.3% ± 0.6% maximal change (max Δ) in weight] at day 10 after the start of radiation and cotreatment with radiation and peposertib at 50 mpk daily similarly had minimal impact on body weight loss (max Δ −0.4% ± 2.1%, P = 0.14 vs. radiation). In contrast, radiation combined with peposertib dosed either 150 mpk daily (max Δ −15.2% ± 5.0%, P < 0.001 vs. radiation + 50 mpk), or 75 mpk × 2, 7 hours apart, (max Δ −12.2% ± 5.9%, P < 0.001 vs. radiation + 50 mpk) was associated with significant weight loss. Notably, 2 of 7 mice that received 75 mpk dosed twice daily were euthanized because of >20% weight loss, while none of the mice that received 150 mpk daily met this endpoint (Fig. 3B). These data highlight the importance of considering drug exposure in developing novel radiosensitizer strategies and defining a rational dosing schedule for subsequent studies.

The relationship between plasma and brain tumor drug levels was evaluated to further develop an optimal dosing schedule. Total drug levels were measured in orthotopic M12-eGFP tumors; animals were dosed with 125 mpk peposertib, and tissues were collected 2 or 6 hours after dosing (n = 4–5/timepoint). A fluorescence dissecting microscope was used to guide ex vivo isolation of tumor and adjacent normal brain (Fig. 4A). At 2 and 6 hours after dosing, total drug levels of peposertib in M12 tumors were 1,419 ± 436 nmol/L and 774 ± 305 nmol/L, respectively, and plasma levels were 3,588 ± 733 and 1,616 ± 635 nmol/L, respectively (Fig. 4B). To estimate the free fraction in PDX tissue, rapid equilibrium dialysis was performed on M12, M15, and M27 tumor tissue. This demonstrated free fractions of 9.1% ± 2.3%, 4.5% ± 0.8%, and 5.6% ± 0.9%, respectively (Fig. 4C). The free fraction of peposertib for M12 was then used in conjunction with previously reported free fraction values for mouse brain (11.4% ± 0.9%) and plasma (4.1% ± 0.6%) to compute the concentrations of free peposertib. At the 2 and 6 hours timepoints, free drug levels of peposertib in M12 tumors were 129 ± 44 and 71 ± 31 nmol/L, respectively. These measured data were integrated into our pharmacokinetic simulation model to extrapolate an anticipated drug exposure in plasma and intracranial tumor over a 24-hour timeframe (Fig. 4D). These data support the concept that peposertib distribution into M12 brain metastases is near a 50 nmol/L target free concentration, but that at 6 or 7 hours, the drug concentration is falling below target, which supports the concept of split dosing 7 hours apart.

The radiosensitizing effects of different peposertib dosing schedules were evaluated in orthotopic M12 tumors. First, free peposertib exposures were simulated in M12 orthotopic tumor tissues following 60, 90, and 125 mpk, dosed twice, 7 hours apart (Fig. 5A), and then the efficacy of this regimen was evaluated in a survival study. Animals (n = 10 per group) with established M12 tumors were randomized and treated with vehicle, 60, 90, or 125 mpk peposertib dosed just prior to and then 7 hours after each dose of 2.5 Gy radiation × 5 fractions (12.5 Gy total). As seen in Fig. 5B, all three peposertib dosing regimens significantly extended survival, compared with radiation alone with the most efficacious peposertib dosing regimen being 125 mpk twice daily (P = 0.005 vs. 60 and P = 0.01 vs. 90 mpk). In a second, confirmatory study using GFP-transduced orthotopic M12 tumors (n = 5 per group), 125 mpk peposertib combined with radiation (median survival 43 days) significantly extended survival compared with radiation alone (21 days; 105% extension, P = 0.002; Fig. 5C). A similar potentiation of radiation efficacy was observed in M15 (n = 7 per group) with radiation/peposertib (125 mpk twice daily; 36 days median survival) compared with radiation (24 days; 50% extension, P = 0.03) or placebo/sham radiation (15 days; Fig. 5D). More limited benefit was observed in M27 (n = 6 per group): median survival was 72 days with radiation/peposertib, 62 days with radiation, and 37 days with sham treatment (16% extension, P = 0.04 for radiation vs. peposertib/radiation; Fig. 5E). These data demonstrate the potential for significant radiosensitization by peposertib in orthotopic melanoma brain metastases PDX models, and are consistent with a model in which greater drug exposure is associated enhanced efficacy.

The potentiation of brain toxicity is an important consideration for brain tumor therapies. To assess the potential for radiosensitization of normal brain tissue, a clonogenic assay was performed with the human astrocyte cell line, SVG-A. Similar to our results with M12, 300 nmol/L peposertib significantly enhanced the radiosensitivity of SVG-A cells with a SER₁₀ of 1.9 ± 0.1 (Fig. 6A). As a second model for normal tissue sensitization, γH2AX foci resolution was evaluated in human-iPSCs. Consistent with impairment of DNA damage repair, neuronal-differentiated iPSCs cotreated with peposertib and radiation (2.5 Gy) had a larger fraction of cells with unresolved γH2AX foci 4 hours after irradiation as compared with radiation alone (Fig. 6B and C, P = 0.03). These data suggest that if adequate peposertib concentrations are achieved within the brain, this could enhance radiation-induced neurotoxicity.

The distribution of peposertib across the blood–brain barrier (BBB) is limited. Simulation of brain exposure with 125 mpk dosed twice, 7 hours apart, suggests peak free peposertib concentrations in the brain will only reach 9.3 nmol/L (Fig. 6D). The hippocampus is a key site of adult neurogenesis and is a radiation-sensitive structure associated with radiation-induced neurocognitive decline. Therefore, we tested the impact of peposertib on resolution of γH2AX foci in orthotopic M12 tumors and contralateral normal hippocampus following brain radiation. Four hours after irradiation, mice co-dosed with peposertib had higher residual γH2AX foci in M12 tumors as compared with radiation alone (P = 0.05; Fig. 6E–G). In contrast, in the hippocampus, there was no evidence for delayed γH2AX foci resolution with drug cotreatment; radiation and radiation+ peposertib (P = 0.17; Fig. 6F). These data are consistent with differential peposertib accumulation within tumor versus normal brain and supports the concept that limited peposertib accumulation within the brain is insufficient to radiosensitize normal brain tissue.

Unrepaired radiation-induced dsDNA breaks often result in cell death. The repair of radiation-induced cytotoxic dsDNA breaks is performed by two dominant pathways: NHEJ and HR. The trimer of DNA-PKcs, Ku70 and Ku80 is a core complex required for NHEJ and recovery from DNA damage (17). Radiosensitizing effects have been uniformly reported for small-molecule inhibitors of DNA-PK kinase activity (18, 19). However, successful deployment of potent radiosensitizers in living organisms requires critical integration of understandings of target inhibition, DNA damage repair kinetics, drug pharmacokinetics, and differential sensitizing effects in normal and tumor tissues. The current study describes our strategy for developing the DNA-PKcs inhibitor peposertib for brain metastases from melanoma and other radioresistant histologies and provides a general paradigm for developing potent radiosensitizers that target DNA repair pathways.

Efficacy of any medical therapy is dependent on achieving drug concentrations sufficient to suppress the pharmacologic target. For small-molecule kinase inhibitors, monitoring inhibition of direct phosphorylation targets using phosphospecific antibodies coupled with biological readouts is commonly used to assess effective target inhibition. Regarding peposertib, suppression of DNA-PKcs autophosphorylation at serine-2056 is an established biomarker (20). Coupled with robust radiosensitizing effects measured in clonogenic assays, our data established a minimal radiosensitizing concentration in cell culture media of 100 nmol/L. With increasing monotherapy toxicities at 1,000 nmol/L, we identified 300 nmol/L total drug as being an optimal target peposertib concentration. In previous reports, a broader range of total peposertib concentrations were required for effective in vitro radiosensitization, ranging from 100 to 15,000 nmol/L (5–7, 21, 22). We previously demonstrated that peposertib is a substrate for the P-glycoprotein (P-gp) and breast cancer resistance protein 1 (BCRP1) drug efflux pumps active within the BBB (13), and variable expression of these efflux pumps in different tumor cell lines likely is a major contributor to this wide range of peposertib concentrations required for effective sensitization. Thus, delineation of a target tissue concentration for effective radiosensitization is not only dependent on the potency of the inhibitor, but also the equilibrium of cell influx versus efflux that can be modulated by drug efflux transporters.

The kinetics of DNA repair have been extensively studied. The majority of radiation-induced dsDNA breaks in cells with intact DNA repair machinery are repaired rapidly within the first several hours following damage, and a second, slower, phase of repair processes more complex lesions (23). Our study is the first to evaluate the duration of peposertib exposure required for effective radiosensitization, and consistent with the established kinetics of dsDNA break repair, suppression of DNA-PKcs activity for a minimum of 16 hours was associated with maximal in vitro radiosensitizing effects following irradiation. An important consideration in evaluating the duration of required repair inhibition is the radiation dose. While DNA repair following standard to moderate hypofractionated radiation doses of 2 to 5 Gy used in the current study likely is completed within 16 to 24 hours, complete DNA repair following much larger radiation doses of 12 to 24 Gy commonly used during radiosurgery approaches likely extend beyond 24 hours and would require extended drug exposure for maximal radiosensitizing effects. Furthermore, the genetic and epigenetic differences between tumors can dictate the reliance on HR versus NHEJ for repair of dsDNA breaks. Although beyond the scope of this study, we speculate that M12 is more heavily reliant on NHEJ as compared with M15 and M27, which could explain the greater sensitizing impact of peposertib in M12. Although beyond the scope of the current study, more limited drug uptake in M27 also could have contributed to the more modest sensitizing effects seen in this experiment. Thus, consideration of drug distribution, DNA repair kinetics, duration of DNA repair, and reliance on a specific repair pathway are all critical considerations in evaluating DNA repair inhibitors as radiosensitizing agents.

In vitro defined target drug exposure levels and duration must be integrated with in vivo drug pharmacokinetics to optimize an effective radiosensitizing regimen in animals and patients. Drug binding within culture media, plasma, and tumor tissue can be unique for each drug, and comparison of free drug concentrations measured in these substrates allows for a more direct translation from cell culture to animal and human dosing studies. Because DNA repair begins promptly following radiation exposure, peposertib was administered prior to radiation to ensure effective target inhibition at the time of irradiation. On the basis of early timepoint sampling after oral dosing, plasma concentrations of peposertib exceed our free concentration target of 150 nmol/L in plasma within 10 minutes of dosing. With a short half-life of 2.4 hours, we gave peposertib just 10 minutes prior to irradiation to maximize exposure after irradiation, instead of a more typical 60-minute pre-radiation time interval (5, 13). With a relatively short half-life, peposertib plasma levels will fall quickly and are approximately 1.8% of peak levels at 16 hours after dosing. Thus, superimposing the pharmacokinetic data on the common preclinical oral dosing regimen of 50 mpk peposertib daily delivered 60 minutes prior to radiation, plasma concentrations might be expected to remain above a nominal radiosensitizing concentration of 100 nmol/L total/50 nmol/L free drug for only 6 hours. While the distribution of peposertib in several tissues, including lung and intestine are much higher than plasma levels (13), failure to maintain optimal inhibitory concentrations of peposertib for a minimum duration (16 hours in the M12 melanoma PDX) may have led to suboptimal radiosensitizing effects in some of the prior reports that used a single peposertib dose with each radiation fraction (6–9).

A split dosing regimen with a 7-hour interval was used to maintain adequate radiosensitizing levels for a minimum of 16 hours in the studies reported here. Using radiation-induced DNA-PKcs autophosphorylation in flank tumors as a measure of DNA-PK activity, the original report of peposertib (5) demonstrated that a 25 mpk dose of peposertib only suppressed radiation-induced DNA-PK activity for 3 hours, and at 8 hours, DNA-PK activity had rebounded in association with a very low peposertib plasma level. A similar effect was reported in a phase I clinical trial with peposertib monotherapy in patients with advanced cancer; autophosphorylation of DNA-PKcs in peripheral blood mononuclear cells was only maintained for the first 6 hours after drug dosing, and as the peposertib levels fell, DNA-PKcs autophosphorylation rebounded to near baseline by the next, 11-hour timepoint (24). These data all support the recommended clinical dosing schedule of twice daily dosing of peposertib combined with radiation, and similar twice daily dosing should be considered for in vivo animal studies.

The risk of elevated radiation-induced normal tissue injury is a key factor that must be considered in developing DNA-PKcs inhibitors or other potent DNA repair inhibitors as radiosensitizers (25). Homozygous inactivation of DNA-PKcs in mice and similar disruption of the ATM gene, critically involved in HR, is associated with hypersensitivity to radiation (26, 27). Coupled with the marked enhancement of oral mucositis in our drug combination studies (Fig. 3), these observations highlight the importance of developing strategies to minimize radiosensitizing effects of these drugs in normal tissues. In fact, a recent phase I study of peposertib combined with palliative radiation (30 Gy in 10 fractions) was associated with a high rate of radiation-induced skin toxicity (53%) and a fatal case of esophageal perforation, which highlights the importance of sparing epithelial tissues when combining radiation with this potent radiosensitizer (28). Thus, for acutely responding normal tissues that are especially susceptible to DNA-PK inhibition, such as skin, oral mucosa, and gut, limiting the volume of normal tissue exposed to full dose radiation by using highly conformal radiation treatments and reducing the typical radiation dose limits for these tissues during the radiation therapy planning process will be important strategies to maintain an acceptable therapeutic window for these potent radiosensitizers.

Variation in drug accumulation across tissues is another critical consideration relevant to development of novel radiosensitizers. Potentially important for sparing normal brain tissue, peposertib is effectively excluded from the central nervous system by the activity of P-gp and BCRP1 with drug levels in brain and spinal cord only 1% of those in plasma (13). In contrast to orthotopic tumor tissues in which peposertib significantly delayed the resolution of DNA damage, marked by γH2AX foci, there was no significant impact on γH2AX resolution in the region of the hippocampus. Multiple clinical studies evaluating the adverse effects of brain radiation on cognitive ability have clearly defined the hippocampus as a critical brain region associated with radiation injury (29). Our studies of drug distribution and γH2AX resolution kinetics provide some reassurance that the combination of peposertib with brain irradiation will be safe if acutely responding tissues outside of the central nervous system are appropriately protected during the radiotherapy planning process.

The DNA-PKcs inhibitor peposertib is a potent radiosensitizer in melanoma brain metastasis models. Integration of the strategies used here coupled with human drug pharmacokinetics will be important for developing effective clinical radiosensitizing regimens with peposertib and other potent radiosensitizers.

J.E. Eckel-Passow reports grants from NIH during the conduct of the study. J.N. Sarkaria reports grants from KLT, grants from Rain Therapeutics, Sumitomo Dainippon Pharma Oncology, ABL Bio, ModifiBio, SKBP, Wugen, GSK, AbbVie, Bayer, AstraZeneca, and Karyopharm outside the submitted work. No disclosures were reported by the other authors.

J. Ji: Data curation, writing–review and editing. S. Dragojevic: Data curation, investigation, methodology, writing–original draft, writing–review and editing. C.M. Callaghan: Data curation, formal analysis, writing–review and editing. E.J. Smith: Data curation, investigation, writing–review and editing. S. Talele: Data curation, formal analysis, investigation. W. Zhang: Data curation, formal analysis, methodology, writing–review and editing. M.A. Connors: Data curation, formal analysis, validation, visualization, methodology. A.C. Mladek: Data curation, investigation. Z. Hu: Data curation, investigation. K.K. Bakken: Data curation, investigation. P.P. Sarkaria: Data curation, investigation. B.L. Carlson: Data curation, investigation. D.M. Burgenske: Project administration. P.A. Decker: Formal analysis. M.A. Rashid: Data curation, investigation, writing–review and editing. M.-h. Jang: Data curation, funding acquisition, writing–review and editing. S.K. Gupta: Conceptualization, data curation, validation, investigation, visualization, methodology, writing–review and editing. J.E. Eckel-Passow: Formal analysis. W.F. Elmquist: Data curation, formal analysis, funding acquisition, investigation, writing–review and editing. J.N. Sarkaria: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing.

This study was supported by NIH U01 CA227954, U19 CA256779, R01 CA242158 and Mayo Clinic National Brain Tumor Society.

The healthcare business of Merck KGaA reviewed the article for medical accuracy only before submission. The authors are fully responsible for the content of this article, and the views and opinions described in the article reflect solely those of the authors.