Burgenske, Danielle M.

2071 Background: Brigimadlin (BI 907828) is a potent small molecule inhibitor of the p53-MDM2 pathway currently in a phase 0/1 clinical trial in combination with radiotherapy in newly diagnosed MGMTunmethylated glioblastoma (GBM). To inform development of brigimadlin for GBM, we developed a pharmacokinetic (PK)/ pharmacodynamic (PD)/ efficacy model using GBM patient derived xenografts (PDXs). Methods: Brigimadlin PK parameters were evaluated in FVB wild-type and knockout (Mdr1a/b-/-Bcrp1-/-) mice and in athymic nude mice. Drug binding was measured by rapid equilibrium dialysis, and drug levels quantified by LC/MS-MS. Dose-response relationships were evaluated in subcutaneous and orthotopic PDXs across a range of doses. Response to brigimadlin (2 mg/kg weekly) +/- RT (2 Gy x 10 fractions) was evaluated in orthotopic PDXs. Results: Brigimadlin significantly delayed median time to regrowth of GBM108 ( MDM2amplified) subcutaneous tumors (vs. placebo; p<0.05) at dose levels of 0.25 mg/kg (1.3-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (2.6-fold), and 2 mg/kg (5.3-fold). A single dose of brigimadlin resulted in corresponding intra-tumoral drug levels 24 hours post-dose: 84 nM (0.25 mg/kg), 175 nM (0.5 mg/kg), 398 nM (1 mg/kg) and 924 nM (2 mg/kg) and dose-dependent upregulation of p53 target genes (p21 and PUMA). In contrast, GBM14 ( MDM2non-amplified) showed regrowth delay (1.6-fold) only at 2 mg/kg (p=0.008). Brigimadlin was highly bound (fraction unbound = 0.0006 in brain; 0.0017 in GBM tumor tissue), and CNS distribution was limited by efflux; in wild-type mice Kp,uu_brain = 0.002 ± 0.003; in Mdr1a/b-/-Bcrp1-/- mice Kp,uu = 0.043 ± 0.025. Efficacy of brigimadlin was compared in orthotopic GBM108 PDXs grown in athymic nude mice vs. immuno/efflux-deficient (Rag2-/-Mdr1a-/-Bcrp1-/-) mice. In nude mice, median survival was extended at 2 mg/kg brigimadlin (1.4-fold, p=0.009), with corresponding intra-tumoral drug levels of 29 nM. In efflux deficient mice, median survival was extended (p<0.05) at 0.25 mg/kg (1.5-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (1.8-fold), and 2 mg/kg (>4-fold). Measurement of intra-tumoral drug levels is in progress. In combination with RT in GBM108, brigimadlin (2 mg/kg x 2 weeks) extended median survival vs. vehicle (66 vs. 50 days; p=0.012) and enhanced median survival following RT (128.5 vs. 82 days; p=0.009). In GBM14, 2 mg/kg brigimadlin (2 mg/kg x 12 weeks) extended median survival (39.5 vs. 32.5 days; p=0.0004) and enhanced survival following RT (97 vs. 81 days; p<0.0001). Conclusions: Efficacy of brigimadlin is dependent on adequate CNS delivery. Studies in subcutaneous PDX demonstrated intra-tumoral total drug levels in the mid-nanomolar range provide meaningful tumor effects in a highly sensitive, MDM2 amplified PDX. In orthotopic PDXs with and without MDM2 amplification, brigimadlin provided further benefit in combination with RT. These data support ongoing clinical evaluation of brigimadlin in 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 Purpose: Antibody-drug conjugates (ADC) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given GBM’s molecular heterogeneity, the bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs with similar auristatin toxins, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), were compared in GBM patient-derived xenografts (PDX) and normal murine brain following direct infusion by convection-enhanced delivery (CED). Experimental Design: EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Nontumor-bearing mice were used to evaluate the pharmacokinetics (PK) and toxicity of ADCs using LC-MS/MS and immunohistochemistry. Results: CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to &gt;300 days vs. 20–49 days with isotype control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221 with the cell-permeable monomethyl auristatin E (MMAE), compared with Depatux-M with the cell-impermeant monomethyl auristatin F. CED infusion of ABBV-221 into the brain or incubation of ABBV-221 with normal brain homogenate resulted in a significant release of MMAE, consistent with linker instability in the brain microenvironment. Conclusions: EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intratumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

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.

Abstract Glioblastoma (GBM) is a highly aggressive and infiltrative primary malignant brain tumor. Current standard-of-care for newly diagnosed GBM patients includes maximal safe surgical resection, followed by concurrent radiation with temozolomide (TMZ), and adjuvant TMZ chemotherapy. However, despite aggressive treatments, GBM patients have a poor prognosis, with a median survival of 14.6 months and a 5-year survival rate of 6.8%. The p53 protein is a critical tumor suppressor in the cell cycle. The expression level of p53 is tightly controlled by its negative regulator, murine double minute 2 (MDM2). Amplification of the MDM2 gene, found in 14 % of GBMs, can inactivate p53, leading to loss of tumor suppressor functions. Therefore, we are investigating a potent MDM2 antagonist – BI-907828 in GBM to restore the p53 tumor suppressor function by inhibiting the p53/MDM2 interactions. We aim to evaluate the PK/PD of BI-907828 and demonstrate a new translational paradigm to rethink the “Delivery – Potency – Efficacy” relationship in drug discovery and development for central nervous system (CNS) tumors. BI-907828 was evaluated for cytotoxicity and the inhibition effect to MDM2-p53 pathway. Systemic PK and transport mechanisms controlling CNS distribution were evaluated in wild-type and transgenic mice that lack of efflux transporters P-gp and BCRP. BI-907828 unbound fractions in cell media, mouse and human specimens were determined by using rapid equilibrium dialysis to evaluate “active” concentrations. Efficacy measures, including survival and free drug distribution in tumor were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2-p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nM. The CNS distribution of BI-907828 is limited by BBB efflux mediated by P-gp, resulting in a brain free partition coefficient Kp,uu of 0.002. Despite this seemingly “poor” BBB penetration, weekly administration of 10 mg/kg BI-907828 significantly extended median survival of orthotopic GBM108 xenografts to 218 days compared to 28 days with vehicle treatment (P &lt; 0.0001). These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is severely limited by BBB efflux. In another words, in this case, this little drug distribution to brain is “enough” for extending survival of orthotopic GBM108 xenografts. Therefore, a comprehensive understanding of drug “Delivery – Potency – Efficacy” relationship is warranted in drug discovery development, especially for treatment of CNS tumors. Citation Format: Wenjuan Zhang, Rachael Vaubel, Juhee Oh, Ann Mlade, Surabhi Talele, Wenqiu Zhang, Katie Waller, Danielle Burgenske, Jann Sarkaria, William Elmquist. How much is enough? BI-907828: a MDM2-p53 antagonist with limited BBB penetration but potent efficacy in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B025.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of TP53 mutation status in a panel of IDH1 wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs). AZD1390 suppressed radiation-induced ATM signaling, abrogated G 0 -G 1 arrest, and promoted a proapoptotic response specifically in p53-mutant GBM in vitro. In a preclinical trial using 10 orthotopic GBM models, AZD1390/RT afforded benefit in a cohort of TP53 -mutant tumors but not in TP53 -WT PDXs. In mechanistic studies, increased endogenous DNA damage and constitutive ATM signaling were observed in TP53 -mutant, but not in TP53 -WT, PDXs. In plasmid-based reporter assays, GBM43 ( TP53 -mutant) showed elevated DNA repair capacity compared with that in GBM14 (p53-WT), whereas treatment with AZD1390 specifically suppressed homologous recombination (HR) efficiency, in part, by stalling RAD51 unloading. Furthermore, overexpression of a dominant-negative TP53 (p53DD) construct resulted in enhanced basal ATM signaling, HR activity, and AZD1390-mediated radiosensitization in GBM14. Analyzing RNA-seq data from TCGA showed up-regulation of HR pathway genes in TP53 -mutant human GBM. Together, our results imply that increased basal ATM signaling and enhanced dependence on HR represent a unique susceptibility of TP53 -mutant cells to ATM inhibitor–mediated radiosensitization.

Abstract Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Abstract Radiation therapy (RT) is an important non-surgical clinical treatment for glioblastoma (GBM) and brain metastases, but inherent radio-resistance can limit the efficacy in many patients. The Ataxia Telangiectasia Mutated (ATM) protein critically regulates radiotherapy-mediated DNA damage repair pathways, and defects in this kinase can lead to profound radiosensitivity. In this study, the efficacy of the highly potent ATM inhibitor WSD0628 was evaluated in GBM and melanoma brain metastasis patient derived xenografts (PDXs). In short term explant cultures of three PDXs, WSD0628 robustly suppressed RT-induced autophosphorylation of ATM at serine 1981 and ATM-mediated phosphorylation of Chk2 at threonine-68 and KAP1 at serine-824, with maximal inhibition at 100 nM. Similarly, RT-induced gH2AX foci were significantly reduced when combined with WSD0628 in the GBM43 cells. Consistent with the importance of ATM in the DNA damage response after radiation, WSD0628 significantly increased the radiosensitivity of U251 cells in clonogenic survival assays (0nM vs. 30nM at 5Gy, p&lt;0.01). To evaluate the optimal duration of drug exposure, WSD0628 was removed from U251 cells at various intervals following irradiation with 5 Gy. Using clonogenic survival as a readout, a 10-fold increase in cytotoxicity was observed with an 8 hour drug exposure, with progressively increasing cytotoxicity gains with exposures up to 24 hours. In vivo efficacy of WSD0628 was evaluated in multiple intracranial PDX models. In an initial dose-ranging study, robust radiosensitization was observed in GBM43 treated with 2 Gy x 5 fractions combined with 5 mg/kg WSD0628 (20 day survival extension) or 10mg/kg WSD0628 (39 day with 10 mg/kg; p&lt;0.01 for both dose levels). Moreover, a single dose of 12.5 Gy combined with 10 mg/kg WSD0628 had profound impact on treatment efficacy in the melanoma brain metastasis M12 PDX: at 180 days post-treatment, all mice in the combination group were electively euthanized, while median survival for sham or RT alone was 17d and 49d, respectively. Histologic analysis identified large intracranial tumors in all sham and RT-only treated mice, but only a small accumulation of melanotic cells without obvious tumor in the combination-treated mice. Immunohistochemical staining of NeuN and GFAP in the combination-treated mice showed preserved neuronal density at the 180-post treatment timepoint and minimal reactive gliosis within the ‘tumor scar’. An ongoing study is comparing this single-fraction 12.5 Gy regimen to a 2.4 Gy x 10 fraction regimen alone or in combination with WSD0628. Collectively, this study demonstrates the potential for profound radiosensitizing effects of WSD0628 in combination without obvious neuronal toxicity and has provided the scientific rational for the first-in-man study of this combination in recurrent GBM (NCT05917145) at Mayo Clinic. Citation Format: Zhiyi Xue, Ann C. Mladek, Sneha Rathi, Danielle M. Burgenske, Shiv K. Gupta, Brett L. Carlson, Zeng Hu, Lauren L Ott, Katrina K. Bakken, Rachael A. Vaubel, William F Elmquist, Wei Zhong, Jann N Sarkaria. The radiosensitizing effects of the novel brain penetrant and potent ATM inhibitor WSD0628 in glioblastoma and melanoma patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A148.

Abstract Large PDX collections provide broad representation of tumor genetics and are commonly used to evaluate the clinical potential of novel therapeutic strategies. In many solid malignancies, profound benefit in PDX preclinical studies has foreshadowed eventual FDA approval of clinically impactful therapeutics. With a lack of corresponding progress in glioblastoma (GBM), the value of PDX testing has been questioned. Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients, and over the past 23 years, we have used this collection for extensive in vivo preclinical testing of 145 drugs in both orthotopic and heterotopic settings. As part of this work, the impact of standard of care therapy with temozolomide (TMZ) and bevacizumab was evaluated in orthotopic models using clinically relevant dosing regimens. For each animal study, the ratio of median survival for treated vs. control animals were used to define a ‘survival ratio’ as a normalized metric of benefit. TMZ efficacy was evaluated in 26 newly diagnosed GBM PDXs, with equal representation of MGMT methylated and unmethylated tumors. As expected, MGMT methylated PDXs were much more sensitive to TMZ as compared to unmethylated PDXs (median survival ratio 4.47 vs 1.48, respectively), and similar to clinical experience, 2 MGMT unmethylated PDXs were markedly sensitive to TMZ, while 1 MGMT methylated PDX was relatively resistant. In a more limited study, dose-dense vs. standard TMZ dosing was compared in 7 PDXs (4 MGMT methylated, 3 MGMT unmethylated). In a pooled analysis of all lines, both standard (HR=0.09; 95% CI: 0.06–0.14) and dose-dense TMZ therapy (HR=0.09; 95% CI: 0.06–0.14) were superior to vehicle controls (p&lt;0.001). Similar to the clinical results in the Phase III randomized RTOG 0525 trial, survival with the dose-dense TMZ was no different than standard dosing (p=0.88, HR=0.98; 95% CI: 0.72-1.32). In an analysis of bevacizumab across 32 PDXs, mice were dosed once a week until moribund, and a minimum overall median survival benefit of 20% was used to define ‘responders’. Thirteen PDXs met this criterion, although the overall survival ratio among this subset was limited (range 1.21-1.65). Only 2 models had a 50% or greater survival benefit. Stratification by disease state (24 newly diagnosed vs 8 recurrent) showed equivalent performance (median survival benefit 1.13 for both). These results are consistent with the limited survival benefit observed with bevacizumab and the RTOG 0825 clinical trial. In summary, the results from these relatively inexpensive PDX studies demonstrate the potential of pre-clinical PDX trials to evaluate the extent of benefit and fraction of responsive PDXs to a novel therapeutic. If analyzed across an ‘adequate’ number of PDXs, these results potentially can be used to identify predictive biomarkers or to compare various treatment regimens prior to clinical testing. 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. The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A002.