Citations

Abstract Radiation is a cornerstone of glioblastoma (GBM) treatment, however, the majority of tumors progress within the radiation field due to inherent radiotherapy (RT) resistance. The integration of highly potent radiosensitizers, such as the ATM inhibitors AZD1390 or WSD0628, could reverse resistance and significantly improve local control of these tumors. However, the achievable brain tumor exposure of these inhibitors may be limited by drug exposure in skin and mucosa, leading to enhanced radiation toxicity. To support the clinical development of WSD0628, we developed pharmacokinetic (PK)-efficacy models to help interpret ongoing PK analyses. A model based on in vitro cell studies was based on the in vitro observations that 30 nM WSD0628 provided maximum ATM inhibition and the extent of radiosensitization was directly related to the duration of drug incubation after irradiation, up to a maximum of 24 hours. The unbound WSD0628 concentration, measured by rapid equilibrium dialysis, was 66% of the total concentration in cell culture media, and was used to define an optimal radiosensitizing target of 20 nM free WSD0628 for 4 to 24 hours. Previous PK analyses showed that the free, unbound fraction (fu) of WSD0628 in plasma and brain tumor tissue are 0.02 and 0.054, respectively, and the brain tumor-to-plasma ratio is 0.32 in GBM43 patient-derived xenografts (PDXs). From these data, a total drug level of 1160 nM in plasma is predicted to achieve unbound drug levels above the 20 nM WSD0628 target unbound concentration. To validate this prediction, parallel in vivo dose-ranging studies were performed in orthotopic PDXs. Mice with orthotopic GBM43 were randomized to treatment with RT (8 Gy x1) alone or in combination with a range of WSD0628 doses. RT combined with 0.25 mg/kg and 1 mg/kg WSD0628 had no impact on survival, compared to RT alone, while median survival increased by 1.3-fold for RT combined with 2.5 mg/kg, 1.7-fold with 5 mg/kg, and 3.2-fold with 10 mg/kg WSD0628. A similar dose-response was observed in an M12 melanoma brain metastasis PDX. Based on PK modeling of these drug doses, 2.5 mg/kg dosing will maintain total WSD0628 plasma levels above 1160 nM for 11.9 hours, 5 mg/kg for 23.6 hours, and 10 mg/kg for 40 hours. Taken together, we have developed a model to predict efficacious dosing of a brain tumor based on the free drug hypothesis, which allows integration of multiple aspects of in vitro modeling with measured drug levels in animal models to predict total plasma levels associated with robust sensitizing effects. Assuming similar tissue to plasma partitioning in human GBM, this model could be directly applied to interpret the ongoing Phase 1 PK analysis of WSD0628 in recurrent GBM. Citation Format: Ann Catherine Mladek, Juhee Oh, Sneha Rathi, Lily Liu, Danielle M. Burgenske, Brett L. Carlson, Katrina K. Bakken, Lauren L. Ott, Zeng Hu, Wei Zhong, William F. Elmquist, Jann N. Sarkaria. Modeling pharmacokinetics and efficacy of the ATM inhibitor WSD0628 in GBM and melanoma metastasis PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4629.

Abstract Therapeutic resistance limits the efficacy of radiation therapy in many tumors and provides a compelling rationale to develop novel treatments to enhance radiation sensitivity. DNA-dependent protein kinase (DNA-PK) is a major mediator of DNA repair following radiation, and DNA-PK inhibitors are effective radiosensitizers. WNC0901 is a potent DNA-PK inhibitor in both a cell-free kinase assay (IC50 = 0.073 nM) and a cell-based assay (IC50 = 76 nM). WNC0901 has a 30-fold specificity for DNA-PK relative to other PI3K-related kinases. In a broader kinome screen (363 kinases), 1 μM WNC0901 resulted in >25% inhibition of only five other kinases. There is robust inhibition of radiation-induced DNA-PK autophosphorylation (Ser-2056), as analyzed by Western blotting, with maximal effect at 300 nM WNC0901 in A549 (lung carcinoma), U2OS (osteosarcoma), and HT29 (colorectal) cells. The efficacy of WNC0901 as a radiosensitizer was evaluated in clonogenic assays across these same three radioresistant tumor models. Following treatment with graded concentrations of WNC0901 (0-1000 nM) combined with 2.5 Gy radiation, maximal effects were observed at a 300 nM concentration in A549 (88-fold reduction in relative clonogenic survival compared to RT alone, p<0.001), U2OS (5.8-fold decrease, p=0.002), and HT29 (59-fold decrease, p=0.008) In a full radiation clonogenic, the sensitizer enhancement ratio at 10% survival (SER10) was 3.8 (A549), 2.3 (U2OS), and 1.8 (HT29). In vitro metabolism studies have identified demethylated WNC0901 (WNC0901-M) as a major metabolite. Similar to the parent drug, WNC0901-M is a potent DNA-PK inhibitor with maximal radiosensitizing effects of the metabolite observed at 300nM with SER10 of 2.4 (A549), 1.9 (U2OS), and 1.8 (HT29). The in vivo efficacy of 50 mg/kg WNC0901 combined with 8 Gy was evaluated and compared to another DNA-PK inhibitor, 50 mg/kg peposertib, in A549 tumor heterotopic xenografts. Compared to placebo (median time to endpoint 63 days) or RT alone (median 72 days), the combination of WNC0901 and RT significantly delayed tumor regrowth (median 169 days; p=0.002 compared to RT). Interestingly, peposertib + RT was ineffective (median 83 days; p=0.332 compared to RT). In a second study, similarly robust sensitizing effects of WNC0901 was observed in HT29 xenografts (placebo - median 45 days; RT - median 75 days; RT + WNC0901 - median >110 days; p=.03 relative to RT). Together, these findings demonstrate the robust radiosensitizing effects of WNC0901 in radioresistant tumors and provide a rationale for continued development of this drug. Citation Format: Jessica L. Abraham, Ann C. Mladek, Sonia Jain, Shiv K. Gupta, Paul A. Decker, Matthew L. Kosel, Katrina K. Bakken, Brett L. Carlson, Lauren L. Ott, Danielle M. Burgenske, Jeanette E. Eckel-Passow, Wei Zhong, Jann N. Sarkaria. WNC0901 is a novel DNA-PK inhibitor and potent radiosensitizer of otherwise radioresistant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6608.

Abstract Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers—short, synthetic, single-stranded DNA or RNA molecules—can be ∼6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast (∼100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo , cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

Abstract Background Murine double minute 2 (MDM2) inhibitors reactivate wild-type p53 and are a promising therapy for glioblastoma, IDH-wildtype (GBM). Brigimadlin is a highly potent MDM2 inhibitor being tested in a phase 0/1 clinical trial in combination with radiation in GBM. Methods Brigimadlin pharmacokinetics, pharmacodynamics, and efficacy were evaluated in GBM patient-derived xenografts (PDXs). Results In vitro, brigimadlin impaired viability in TP53 wild-type GBM with an IC50 of 0.8-6.6 nmol/L, but sensitivity did not correlate with MDM2 amplification. In vivo, MDM2 amplification was highly correlated with efficacy. In subcutaneous PDXs, 1 or 2 mg/kg brigimadlin dosed weekly was highly effective in 2 MDM2-amplified PDXs. At 2 mg/kg, brigimadlin delayed tumor regrowth by >5-fold in the MDM2-amplified PDXs compared to 1.5-fold in a non-amplified PDX. In orthotopic PDXs, efficacy was more limited, but 2 mg/kg brigimadlin enhanced the response to fractionated radiation in MDM2-amplified PDXs. Consistent with blood-brain barrier efflux limiting drug distribution, 2 mg/kg brigimadlin extended survival by >5-fold in an MDM2-amplified orthotopic PDX established in Rag1−/−Abcb1a−/− Abcg2−/− mice. In pharmacodynamic studies, p53 target genes were upregulated at both subtherapeutic and therapeutic dose levels, and the extent of activation did not correlate with MDM2 status. Concentrations of brigimadlin in tumor tissue were approximately 10-fold higher in MDM2-amplified tumors, and intracellular drug levels directly correlated with drug-dependent MDM2 upregulation, suggesting target binding affects drug accumulation. Conclusions Brigimadlin is highly effective in MDM2-amplified GBM when adequate drug levels are achieved in tumor tissue. MDM2 amplification impacts both treatment efficacy and intratumoral drug accumulation.

Radiation exposure to the eye during cancer treatment can lead to ocular radiation injury (ORI), a devastating condition that can have a profound and permanent impact on vision-related quality of life. Rodent models do not have adequate ocular anatomy to accurately simulate human ORI, and modeling in non-human primates is limited by logistical and ethical concerns. To improve future translational research investigating ways to treat or prevent ORI, we developed protocols for a tree shrew model of ORI. Northern tree shrews (Tupaia belangeri) were obtained by our laboratory. Custom housing and handling methods were developed, including custom body suits to maintain the tree shrew’s body temperature during procedures. Radiation delivery was optimized to accurately deliver radiation, and imaging was performed to observe fundus changes from ORI. Optimization of tree shrew handling, housing, anesthesia approaches, radiation delivery, and clinically-relevant ocular imaging permitted successful induction and assessment of ORI in tree shrews. With these protocols, tree shrews can be used as a highly relevant model organism with key anatomic features similar to humans to study ORI.

Abstract Failure to repair radiation (RT)-induced DNA damage results in cell lethality, and combined treatment with DNA repair inhibitors can markedly enhance tumor control achieved with radiation. These inhibitors also can potentiate acute RT toxicities by suppressing DNA repair. Capitalizing on this normal tissue radiosensitizing effect, we determined effective radiosensitizing dosing regimens in mice for the ATM inhibitor WSD0628 and the DNAPK inhibitor WNC0901 in mice. Drugs were administered via oral gavage prior to each RT dose, and RT was delivered using an anterior/posterior 225 kVp beam. In an initial iteration of this model, various combinations of WSD0628 and RT were administered to cohorts of mice (n=5/group) and body weight was followed for 2 weeks. The resulting data were modeled to define the total dose of radiation estimated to cause 50% of mice to lose >20% body weight (D50). There was an inverse relationship between the WSD0628 and D50 doses: 0 mg/kg WSD0628/24.0 Gy (95% confidence interval (CI) 24.5–24.7); 0.25 mg/kg/19.2 Gy (95% CI 16.7–21.8); 1 mg/kg/13.7 Gy (95% CI 13.2–14.2); 2.5 mg/kg/11.0 Gy (95% CI 10.5–11.5); 5 mg/kg/11.3 Gy (95% CI 6.7–15.9) and 7.5 mg/kg/7.8 Gy (95% CI 7.8–7.9). Histological review of tongues from animals treated with 15 Gy RT (5Gy x 3) + 7.5 mg/kg WSD0628 demonstrated epithelial denudation and acute inflammation consistent with oral mucositis, while these features were not seen with the same dose as RT alone. Similar WSD0628 doses were evaluated in combination with 8 Gy supratentorial RT (via lateral beams) in orthotopic GBM43 PDX tumors. By treatment arm, the median time to a moribund state was: sham (29 days (d)), RT (39d), 1 mg/kg (42d), 2.5 mg/kg (49d), 5 mg/kg (65d), and 10 mg/kg (123d, overall p<0.0001). A second mucositis model was used for evaluation of WNC0901. Cohorts of 5 mice/group were treated with a set dose of RT (4Gy x 3) to the oral cavity alone and in combination with various doses of WNC0901, and the fraction of mice with >20% weight loss within 2 weeks was determined. The results of this study are as follows: placebo (0 of 5 mice with >20% weight loss); 15 mg/kg (3 of 5 mice); 25, 50, or 100 mg/kg (5 of 5 mice). Based on these data, we defined 25 mg/kg WNC0901 as the minimally effective dose and predicted that 50 mg/kg would provide robust radiosensitization. Consistent with this prediction, co-treatment of B16 melanoma flank tumor xenografts with RT (8Gy x 1) and WNC0901 (50 mg/kg) more than doubled the median tumor control duration (median 42 [range 24-45] days; time from implantation for tumor to exceed 1500 mm3) as compared to RT alone (24 [13-30] days) or sham treatment (17 [13-17] days; overall p<0.0001). This study describes how a radiation-induced oral mucositis model can be used to identify effective radiosensitizing regimens faster and with fewer resources than relying on tumor control studies alone. Citation Format: Danielle Burgenske, Darwin Garcia, Jiayan Le, Wenjuan Zhang, Sneha Rathi, Katrina K. Bakken, Lauren L. Ott, Zeng Hu, Brett L. Carlson, Ann Mladek, Jessica L. Abraham, Paul A. Decker, Matthew L . Kosel, Jeanette E. Eckel-Passow, William F. Elmquist, Wei Zhong, Jann N. Sarkaria. Radiation-induced oral mucositis to rapidly determine effective radiosensitizing dosing regimens for ATM and DNAPK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B090.

Abstract Purpose: Navtemadlin is a potent small-molecule inhibitor of MDM2, which has completed a phase 0 window-of-opportunity study in glioblastoma (GBM). To optimally interpret the clinical data, a detailed analysis of navtemadlin pharmacokinetics (PK), pharmacodynamics, and efficacy was performed in GBM patient-derived xenografts (PDX). Experimental Design: Response to navtemadlin was characterized in vitro and in vivo in GBM PDXs with and without MDM2 amplification. Efficacy in vivo was integrated with measured plasma and intra-tumoral drug levels to develop a translational PK/efficacy model comparing exposure effective in PDX to exposure achieved in phase 0 patient samples. Results: In vitro, navtemadlin showed robust on-target activity in TP53–wild-type GBM. In vivo efficacy strongly correlated with MDM2 amplification status. In subcutaneous PDXs, navtemadlin significantly extended survival when dosed at 25 mg/kg in an MDM2-amplified PDX compared with 100 mg/kg in a non-amplified PDX. Central nervous system distribution was limited by blood–brain barrier efflux (Kp_brain = 0.009). In an MDM2-amplified orthotopic PDX model, navtemadlin was ineffective at 100 mg/kg; when established in mice with deficient blood–brain barrier efflux (Rag−/−Abcb1a−/−Abcg2−/−), 25 mg/kg doubled survival. A tumor PK/efficacy model was built to define target exposure for efficacy in GBM, using the effective 25 mg/kg dose. Modeled exposures exceeded this threshold in three (of 16) tumor samples from phase 0 study patients at the 240 mg dose level. Conclusions: Navtemadlin efficacy was highly dependent on adequate brain penetration. Our translational PK/efficacy model suggests that the minimum effective tumor exposures were achieved only in a minority of patients with GBM.

Abstract Background The ataxia-telangiectasia mutated (ATM) protein coordinates the cellular response to therapeutic radiation, and ATM inhibitors can potentially enhance the efficacy of radiation in otherwise radiation-resistant tumors. Methods The small molecule ATM kinase inhibitor WSD0628 was specifically designed for enhanced distribution across the blood-brain barrier to more effectively treat glioblastoma (GBM) and brain metastases in combination with radiation therapy (RT). GBM and brain metastasis patient-derived xenograft models were used to understand target inhibition, radiosensitization, inhibition of the DNA damage response, and in vivo efficacy. Results Initial in vitro characterization of WSD0628 demonstrates a high level of selectivity across kinase families, limited aldehyde oxidase liability, and low risk of hERG interactions. Consistent with a central role for ATM in radiation response, WSD0628 blocked radiation-induced signaling and enhanced radiosensitivity in U251 glioma cells and brain tumor PDXs GBM120 and M12. In comparison to control or RT alone in orthotopic PDXs, the combination of WSD0628 with RT markedly prolonged median survival—GBM12 (19, 55, and 408 days, respectively); GBM43 (26, 44, and 143 days, respectively); GBM120 (51, 89, and 231 days, respectively); M12 (17, 39, and 190 days, respectively). Pharmacokinetic and pharmacodynamic testing after treatment in orthotopic GBM43 tumors showed inhibitory levels of WSD0628 and a reduction of γH2AX foci in the combination-treated tumors. Conclusions Collectively, these results suggest a promising role for WSD0628 in combination with RT in brain tumors and provide the rationale for an ongoing Phase 0/1A clinical trial testing this combination in recurrent GBM.

Abstract Background Most oncology therapeutics have limited distribution into the brain, and developing strategies to overcome this limitation would be clinically impactful. While therapeutic radiation is often cited as a strategy to accomplish this, there are no published studies demonstrating the effect of radiation on drug distribution into the brain or brain tumors. Methods Mice were treated with brain radiation (6 Gy × 5, 4 Gy × 10; 40 Gy × 1) and dosed with drugs (levetiracetam, cefazolin, nedisertib, brigimadlin, apitolisib, or GNE-317) at times ranging from just prior to months after radiation. Plasma and tissue drug concentrations were measured by LC-MS/MS. Results Radiation did not significantly enhance drug delivery into brain tissue for levetiracetam, cefazolin, GNE-317, apitolisib, or nedisertib at any time post-radiation. Even a single, supra-therapeutic dose of radiation (40 Gy) did not significantly affect brain distribution of GNE-317 or apitolisib (P ≥ .07) from 16 to 160 hours post-radiation. For brigimadlin, radiation (6 Gy × 5) was associated with a modest but significant increase in drug accumulation only at 72 hours post-radiation (brain-to-plasma ratio 0.014 ± 0.006 vs. 0.025 ± 0.010, respectively; P = .04), but not at any other timepoint (24 hours, 15, 28, 94, 133, 183 days; P > .05). Similarly, radiation (6 Gy × 5) of orthotopic tumors did not increase levels of brigimadlin in GBM10 or GBM108 or nedisertib in GBM108 (P > .05). Conclusions Radiation had no meaningful impact on drug delivery into brain or brain tumors for the drugs tested.

Abstract Ataxia Telangiectasia–mutated (ATM) inhibitors are being developed as radiosensitizers to improve the antitumor effects of radiotherapy, but ATM inhibition can also radiosensitize normal tissues. Therefore, understanding the elevated risk of normal tissue toxicities is critical for radiosensitizer development. This study focused on modeling the relationship between acute mucosal toxicity, radiation dose, fractionation schedule, and radiosensitizer exposure. The ATM inhibitor WSD0628 was combined with single or fractionated doses of radiation delivered to the oral cavity or esophagus of Friend Leukemia virus B (FVB) mice. The potentiation by WSD0628 was quantified by a sensitizer enhancement ratio (SER), which describes the changes in radiation tolerance for radiation combined with WSD0628 relative to radiation-only regimens. WSD0628 profoundly enhanced radiation-induced acute oral and esophageal toxicities. For oral mucosal toxicity, the enhancement by WSD0628 with 3 fractions of radiation resulted in an SER ranging from 1.3 (0.25 mg/kg) to 3.1 (7.5 mg/kg). For the 7.5 mg/kg combination, the SER increased with increasing number of fractions from 2.2 (1 fraction) to 4.3 (7 fractions) for oral toxicity and from 2.2 (1 fraction) to 3.6 (3 fractions) for esophageal toxicity, which reflects a loss of the normal tissue sparing benefit of fractionated radiation. These findings were used to develop a modified biologically effective dose model to determine alternative radiation schedules with or without WSD0628 that result in similar levels of toxicity. Successful radiosensitizer dose escalation to a maximally effective therapeutic dose will require careful deliberation of tumor site and reduction of radiation dose volume limits for organs at risk.