Elmquist, William F.

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 Current assessments of brain permeability rely predominantly on drug delivery to contrast-enhancing tumor regions. However, substantial portions of central nervous system (CNS) tumors reside within non-enhancing brain (NEB), where drug concentrations frequently remain subtherapeutic. This collaborative Adult Brain Tumor Consortium and Food and Drug Administration workshop aimed to identify criteria for defining NEB permeability to accomplish 2 critical objectives: (1) allocate clinical trial resources toward agents achieving therapeutic NEB concentrations and (2) minimize systemic toxicity when CNS benefit is improbable. The workshop systematically evaluated permeability assessment modalities, including drug physicochemical properties, in vitro blood-brain barrier models, and penetration into cerebrospinal fluid and normal rodent brain. Methodological approaches to determine requisite NEB drug concentrations and approaches to measuring NEB pharmacokinetics and pharmacodynamics were examined. This culminated in developing the Non-Enhancing Brain Permeability Index (NEBPI), which assigns therapeutic agents to 3 categories: sufficiently permeable, insufficiently permeable, or impermeable. The NEBPI provides a standardized framework to assist investigators and regulatory agencies to evaluate NEB penetration before human efficacy studies are initiated for agents that require direct tumor contact. Assessing NEB drug penetration is critical to improving outcomes in CNS tumors and reducing the incidence of brain metastases in systemic malignancies.

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.

Abstract Despite intensive research over the past two decades, all glioblastoma (GBM) Phase 3 drug trials have been negative. The utility of the preclinical studies used to inform development for 10 Phase 3 trials was evaluated by four criteria: 1) drug distribution to the brain, 2) tumor location (flank vs. intracranial), 3) GBM xenograft source (patient-derived (PD) vs. cell line-derived (CD)), and 4) magnitude of survival extension. For the trials evaluated, no preclinical brain:plasma (B/P) data were reported for enzastaurin, cilengitide, nimotuzumab, Depatux-M, or bevacizumab, although IgG typically has a B/P ≈1%. B/P for imatinib was 1-3% and the other four drugs ranged from 25% to 51%. Except for enzastaurin, all drugs were studied in orthotopic tumor survival studies. The best response for each drug compared to relevant control (relative increase in median survival) was 22% (nimotuzumab), 31% (marizomib), 36% (sunitinib CD) / 5% (sunitinib PD), 63% (bevacizumab/temozolomide), 140% (imatinib), 157% (Depatux-M), 220% (veliparib/temozolomide), 300% (cediranib); median survival was not reached for cilengitide. However, only veliparib and sunitinib were studied in orthotopic PD models, and a single CD model (U87 or D54) was used exclusively for four drugs (enzastaurin, nimotuzumab, sunitinib, marizomib). In summary, 6 of 10 drugs had unknown or very poor brain distribution, 8 of 10 drugs were tested exclusively in CD models, which have questionable translational value, and 3 of 10 drugs had nominal activity (<50% survival prolongation) in preclinical survival studies. The stunning lack of clinical progress in GBM is deeply discouraging, but is consistent with underwhelming preclinical testing and the contextual interpretation of those results. Acknowledging multifaceted reasons for failed clinical trials, a more rigorous and critical approach should be used in preclinical studies, coupled with follow-up surgical window of opportunity studies, to identify and promote only the most promising therapies into advanced clinical testing.

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 Background Disruption of the blood–brain barrier (BBB) in high-grade brain tumors is characterized by contrast accumulation on diagnostic imaging. This window of opportunity study correlates contrast imaging features with the tumor distribution of BBB-permeable (levetiracetam) and -impermeable (cefazolin) drugs. Methods Patients with a clinical diagnosis of a high-grade brain tumor underwent MRI for surgical planning. Cefazolin and levetiracetam were administered prior to skin incision, and serial plasma and image-registered tumor samples were collected during the operation. Drug levels were measured by LC-MS/MS, tissue drug levels were corrected for residual blood, and tumor-to-plasma concentration ratios were calculated. Intraoperative microdialysis was performed in a subset of patients to measure the same two drugs. Results Tumor (n = 125) and plasma (n = 261) samples were available for analysis from 42 operative cases. Across all samples, the tumor-to-plasma ratio was significantly lower for cefazolin (marginal mean [MM]: 0.15, 95% CI: 0.11–0.19) as compared to levetiracetam (MM: 0.70, 95% CI: 0.64–0.75; P < .001). When compared between contrast-enhancing and non-enhancing regions, tumor-to-plasma ratios for cefazolin varied by 4.4-fold (0.27, 95% CI: 0.20–0.35 vs. 0.06, 95% CI: 0.04–0.08, respectively; P < .001), and varied for levetiracetam by 1.4-fold (0.88, 95% CI: 0.78–0.97 vs. 0.61, 95% CI: 0.55–0.66, respectively; P < .001). These results were confirmed with the intra-operative microdialysis and a population pharmacokinetic analysis. Conclusions This study demonstrates significant inter- and intra-tumoral heterogeneity in drug delivery for both levetiracetam and cefazolin within high-grade brain tumors that is not necessarily predicted by clinical MR imaging and may reflect tumor-induced changes in both perfusion and BBB integrity.

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.