Mladek, Ann C.

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 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 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.

Temozolomide (TMZ) is the main chemotherapy for GBM, but resistance develops in most patients, highlighting the need for sensitizing strategies. PARP inhibitors (PARPi), which disrupt DNA repair, are being investigated as TMZ-sensitizing agents. Here we evaluated the efficacy and explored molecular mechanisms of pamiparib-mediated TMZ sensitization. In vitro, low concentrations of pamiparib (0.1-1.0 μM) synergized with TMZ and enhanced DNA damage, particularly during the S- and G2/M phases. In vivo, pamiparib monotherapy (0.75 or 3 mg/kg, twice daily) was ineffective, however, TMZ afforded survival benefit in orthotopic patient-derived xenograft (PDX) models (GBM12, GBM22, and GBM84) of MGMT-methylated primary GBM. Pamiparib combined with TMZ (25 or 50 mg/kg daily) prolonged survival in GBM12 [median survival 272 vs. 114 days with TMZ (P=0.02), 30 days with placebo (P<0.001)] and GBM22 (median survival 88 vs. 60 days with TMZ (P=0.05), 26 days with placebo, (P<0.001)] but not in GBM84 [median survival 355 vs. 404 days with TMZ (P=0.44), 37 days with placebo (P<0.001)]. These findings align with prior studies indicating that PARPi can potentiate TMZ efficacy in a subset of primary GBM models sensitive to TMZ. TMZ induces cytotoxic DNA adducts, N7MeG, N3MeA, and O6MeG. While N7MeG and N3MeA are repaired by base-excision repair (BER), MGMT removes O6MeG. Suppressed MGMT expression, due to promoter methylation, drives TMZ sensitivity in ∼40% of primary GBM cases. In MGMT-methylated tumors, O6MeG-thymine mispairs trigger futile mismatch repair, causing replication stress. Although BER is PARP-dependent, BER inhibition did not replicate the TMZ-sensitizing effects of pamiparib. Beyond DNA repair, PARP regulates replication stress by recruiting translocases like SMARCAL1 and controlling endonucleases such as MRE11 and EXO1, protecting stressed forks and nascent DNA. We targeted these components using siRNA and small-molecule inhibitors to elucidate their roles in TMZ sensitization. Unexpectedly, silencing SMARCAL1 or MRE11, or inhibiting MRE11 with mirin, did not affect pamiparib-mediated sensitization in U251 cells. While EXO1 silencing attenuated DNA damage signaling, it had minimal impact on TMZ sensitivity or pamiparib-mediated sensitization. These results suggest that fork protection mechanisms may be epistatic to PARP but redundant for TMZ sensitivity. Alternatively, replication stress may be mitigated by lesion bypass via translesion synthesis (TLS), where replicative polymerases are replaced with low-fidelity polymerases. Consistent with this, silencing RAD18, involved in TLS, enhanced sensitivity to both TMZ and pamiparib, with the combination being more effective than either drug alone. In conclusion, pamiparib enhances TMZ efficacy in a subset of primary GBM. While further in vivo validation is needed, TLS deficiency may be a key determinant of pamiparib-mediated sensitization. Citation Format: Shiv K. Gupta, Ann C. Mladek, Sonia Jain, Katrina K. Bakken, Zeng HU, Brett L. Carlson, Danielle M. Burgenske, Jann N. Sarkaria. Pamiparib-induced replication stress augments the efficacy of temozolomide in GBM PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2910.

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.

2012 Background: Navtemadlin (nvtm) is a potent, selective, orally available small molecule inhibitor of murine double minute 2 homologue (MDM2), which has completed a Ph 0 surgical window of opportunity study in glioblastoma, IDH-wildtype (GBM; Lee SNO 2022). To optimally interpret the clinical data, an extensive evaluation of nvtm PK, PD, and efficacy was performed in GBM patient derived xenografts (PDXs). Methods: Response to nvtm +/- radiotherapy (RT) was characterized in vitro and in vivo across a panel of GBM PDXs with various TP53 and MDM2 alterations. Efficacy, PK, and PD were evaluated in flank or orthotopic PDX models. p53 pathway activation was evaluated by qPCR, Western blot, and IHC. Nvtm brain penetration was evaluated in immunodeficient abcb1a/b-/- (P-glycoprotein; P-gp), abcg2-/- (BCRP), or double-knockout mice. Binding was assessed by rapid equilibrium dialysis, and nvtm concentrations by LC/MS-MS and MALDI-MSI. Results: Nvtm decreased in vitro viability of TP53WT GBM PDX explant cultures with IC50s <100 nM, but was ineffective in TP53-mutant GBM. In flank PDX models, nvtm (100 mg/kg daily for 4 weeks) delayed tumor regrowth by 1.9 to 4.8-fold in TP53 WT PDXs without MDM2amplification (n=9 models), and more markedly by 20.1 to 29.4-fold in MDM2amplified models (n=4; 2 PDXs did not recur). In a flank PDX dose-response study, nvtm delayed the growth of MDM2amplified GBM108 at 25 mg/kg vs. placebo (61 vs. 11 days; p=0.014; mean intra-tumoral nvtm = 590 nM). In contrast, growth of MDM2 non-amplified GBM14 was delayed only at 100 mg/kg (37 vs. 15 days; p=0.082; mean intra-tumoral nvtm = 2990 nM). In both flank models, p53 target gene upregulation was similar between 25 and 100 mg/kg and did not correlate with regrowth. Nvtm was highly bound (fraction unbound = 0.090 in culture media and 0.014 in GBM tissue homogenate). In mice after oral dosing, distribution to CNS was limited by P-gp, with a brain to plasma ratio of 0.009 and unbound ratio of 0.005, whereas BCRP deficiency did not affect CNS distribution. With orthotopic GBM108, nvtm efficacy was evaluated in nude vs. efflux-deficient mice. In nude mice, nvtm was ineffective at doses up to 100 mg/kg; however, in efflux deficient mice, the mean intra-tumoral nvtm level at 25 mg/kg was similar to flank tumors (690 nM) and survival was significantly extended (134 vs. 62 days, p=0.0002) with additional benefit at 100 mg/kg (>300 vs. 62 days, p=0.002). Conclusions: Nvtm showed significant efficacy in TP53 WT GBM flank PDX models but efficacy in orthotopic PDX was highly dependent on CNS penetration. Targeting nvtm levels based on the in vitroIC50 may significantly underestimate the in vivo effective concentration . In vivo studies showed lower intra-tumoral nvtm levels are required to significantly inhibit growth of MDM2amplified compared with non-amplified tumors.

Abstract Radiation therapy (RT) is a cornerstone of oncologic therapy for the majority of solid malignancies, and unrepaired DNA double-strand breaks are responsible for radiation-induced cytotoxicity. RT-induced DNA lesions can be repaired by multiple mechanisms including homologous recombination (HR) and non-homologous end joining (NHEJ), and disruption of either of these pathways can enhance cytotoxicity. A key component of the NHEJ pathway is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and loss of kinase activity can significantly increase radiosensitivity. Herein we describe the results of our medicinal chemistry campaign to develop a potent and selective DNA-PKcs inhibitor, WNC0901. WNC0901 inhibits DNA-PKcs kinase activity in a cell free system with an IC50 of 0.071 nM and demonstrated at least 30-fold higher sensitivity than other family members (ATM, ATR, mTOR, PI3K). WNC0901 has limited aldehyde oxidase (AO) liability with a T1/2 &gt;2000 min in liver cytosol and is stable for over 120 minutes in the presence and absence of an AO inhibitor. A preliminary pharmacokinetic analysis in wista han rat (IV=2mg/kg, PO=20mg/kg) demonstrated 116% apparent absolute oral bioavailability, 2.6 hour terminal half-life, 33.0 mL/min/kg clearance, and a 2.6% unbound brain-to-plasma partition coefficient (Kpuu). WNC0901 also had favorable pharmacokinetic properties in beagle dog (IV=2mg/kg, PO=20mg/kg) with moderate clearance (8.5 mL/min/kg), high apparent absolute oral bioavailability (131%) with good exposure (AUC=36092 h*ng/mL), moderate half-life (4.28 h), and low protein binding (74.3% fraction unbound). The volume of distribution was moderate in both species (Vss = 1.32 L/kg in wistar han rat and 1.87 L/kg in beagle dog). In cell culture, WNC0901 inhibited autophosphorylation of DNA-PKcs in HT29 cells irradiated with 10 Gy with an IC50 of 32.7 nM and robustly inhibited autophosphorylation in both U251 glioma and A549 lung cancer cell lines at 300 nM in combination with 5Gy. In a clonogenic assay, 5Gy irradiation (10% survival) combined with 100nM WNC0901 demonstrated modestly enhanced cell killing (1.5% survival), and maximal effects were seen at 300nM (0.04% survival, p&lt;0.01). Similar radiosensitizing effects with 300 nM WNC0901 were seen in the A549 cell line (0.2% survival with combination compared to 19% with 5Gy alone, p&lt;0.01). In summary, WNC0901 inhibits DNA-PK kinase activity and provides potent radiosensitization in a GBM and lung cancer cell line. Future studies will assess the combination of WNC0901 and RT in GBM patient-derived xenograft models in vivo. Citation Format: Ann C. Mladek, Danielle L. Burgenske, William F. Elmquist, Wei Zhong, Jann N. Sarkaria. Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 701.

Abstract Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients. This large collection of PDXs provides a broad representation of genetic heterogeneity seen in patients. GBM PDXs that perform well in both in vitro and in vivo settings can be extensively utilized in the preclinical setting for downstream analysis. Based on 20 years of experience, we have defined the attributes that best enable preclinical use of our GBM PDX models. Among them are short-term in vitro culturing performance, large scale genomic evaluation data, benchmarked responses to standards of care, and median time to moribund for untreated orthotopic tumors. PDXs with prolonged survival timelines delay study readouts and, in some situations, confound efficacy results due to age-related animal decline. Setting median survival thresholds as part of the upfront experimental design allows for more timely study readouts that are unaffected by these confounding variables. Our preferred cutoff for the Mayo GBM PDX collection is 70 days median survival (n=64). Using this 64 PDX line panel, regimens expected to double survival would produce results within 4-5 months. This timeframe is well within the range of robust health for athymic nude mice. This 64-line collection features both newly diagnosed (nGBM; n=42) and recurrent (rGBM; n=22) tumors. MGMT promoter methylation status was determined by qMS-PCR (25 methylated, 37 unmethylated, 2 indeterminate). Available WES (n=62) and RNAseq (n=42) data, along with our tissue microarray (n=42), are critical resources for biomarker-based evaluations aimed at defining patient populations that are most likely to benefit from a given therapeutic. From these data, the molecular subtypes available include 26 classical, 14 proneural, and 3 mesenchymal. Twenty-seven of these 64 PDX models have also been successfully cultured from fresh as well as cryopreserved cell stocks to further expand their experimental value. Correlative clinical data including diagnosis and treatments are also available (n=61). Current standards of care for GBM include radiation, temozolomide, and bevacizumab. Defining sensitivity to these therapies is an important first step for evaluating the integration of novel therapeutics with these standards. Since the creation of our GBM PDX collection, efficacy of these therapies has been evaluated as follows: RT 26 PDXs, TMZ 20 PDXs, and Avastin 23 PDXs. The Mayo Clinic GBM PDX collection provides an important platform for preclinical evaluation of novel therapeutics. Through careful selection of PDX lines with favorable growth characteristics and known genetic features, researchers can select the most relevant models for their study. This approach will enable more meaningful preclinical work in the hopes of providing more tangible clinical benefits for GBM patients. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Katrina K. Bakken, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Defining critical features for successful preclinical use of glioblastoma patient-derived xenografts (PDX) models for therapeutic assessment and clinical trial development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6897.