Citations

Abstract Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study, we used GBM patient-derived xenografts (PDX) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300 nmol/L and above, with a significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knockdown of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with a delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. Although the mechanism underpinning this discordant effect in vitro versus in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wild-type GBM lines significantly delayed growth and decreased nonhomologous end joining efficiency (but not homologous recombination), after peposertib exposure. See related commentary by Buchsbaum, p. 840

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 Glioblastoma (GBM) is an aggressive and highly infiltrative brain tumor with poor prognosis. Radiation therapy induces DNA damage, and DNA damage response (DDR) pathways modulate resistance to radiation therapy. Inhibiting DDR pathways by targeting ATM, a key enzyme involved in the repair of DNA double strand breaks, leads to radiosensitization and enhances cytotoxicity. Clinical translation of radiosensitizers for GBM may be influenced by a lack of effective drug delivery across the blood brain barrier (BBB). The purpose of this study is to determine the key preclinical pharmacokinetic parameters and mechanisms that influence the CNS delivery of WSD0628, a novel ATM inhibitor. In vitro clonogenic survival assays using U251 GBM cells indicate that WSD0628 shows significant radiosensitization, with a &gt;70% decrease in surviving colonies in the presence of 10nM or higher drug concentrations in combination with 2.5 to 10 Gy radiation as compared to radiation alone. We evaluated the systemic pharmacokinetics and CNS distribution of WSD0628 in FVB wild-type mice and FVB mice lacking Pgp and Bcrp (Mdr1a/b−/−Bcrp1−/−, triple knockout) after a 1, 5, 10 mg/kg intravenous (IV) or oral (PO) dose. Clearance decreased with increase in dose and a greater than dose proportional increase in exposure was observed. WSD0628 is rapidly absorbed and is bioavailable after oral administration. Tissue distribution coefficients were determined as the ratio of exposure in tissue to plasma (Kp = AUCtissue/AUCplasma). Kp,brain was 0.22 ± 0.05, and Kp, spinal cord was 0.30 ± 0.09 in the wild-type mice. For wild-type mice, Kp,uu,brain was 0.20 ± 0.05 and Kp,uu,spinal cord was 0.26 ± 0.08, determined by correcting for binding to CNS tissues and plasma. The brain distribution of WSD0628 was only modestly increased (1.4 times) in the triple knockout mice with Kp,brain of 0.32 ± 0.06 and Kp,spinal cord of 0.41 ± 0.08. Organ distribution of WSD0628 at steady-state was studied after intraperitoneal implantation of Alzet osmotic pumps primed to release WSD0628 at 7.5 ug/hr for 25 hours. Tissue distribution coefficients, determined by organ-to-plasma steady-state concentration ratios, were highest for liver and kidneys, followed by heart, lungs and CNS. Survival studies conducted in mice bearing orthotopic GBM patient derived xenografts show a significant improvement in survival in mice receiving a combination of WSD0628 and radiation as compared to radiation alone. In conclusion, WSD0628 is a robust radiosensitizer and has moderate brain penetration that is not limited by the efflux transporters Pgp and Bcrp. Based on preliminary efficacy studies in mice, WSD0628 shows potential for treatment of GBM tumors. Evaluations are underway to determine tumor free concentrations and quantification of corresponding biomarkers. Combining this knowledge of PK of WSD0628 with the dynamics of the targeted biological pathway will inform a PK-PD-efficacy model and predict effective dosage regimens for future preclinical and clinical trials. Citation Format: Sneha Rathi, Ann Mladek, Zhiyi Xue, Ju-Hee Oh, Wenjuan Zhang, Wenqiu Zhang, Jiayan Le, Jann S. Sarkaria, William F. Elmquist. Systemic and CNS distributional pharmacokinetics of WSD0628, a novel radiosensitizer: Implications for the treatment of brain tumors [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 B028.

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.

Abstract Important challenges in developing drugs that target central nervous system (CNS) tumors include overcoming barriers for CNS delivery and reducing systemic side effects. Alisertib, an aurora A kinase inhibitor, has been examined for treatment of several CNS tumors in preclinical and clinical studies. In this study, we investigated the distribution of alisertib into the CNS, the site of efficacy for brain tumors, and into the bone marrow, the site of dose-limiting toxicity leading to myelosuppression. Mechanisms influencing site-specific distribution, such as active transport mediated by the efflux proteins, p-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), were examined. Alisertib exposure to the brain in wild-type mice was less than 1% of that in the plasma and was evenly distributed throughout various brain regions and the spinal cord. Studies using transporter knockout mice and pharmacological inhibition show that alisertib CNS distribution is influenced by P-gp, but not Bcrp. Conversely, upon systemic administration, alisertib distribution to the bone marrow occurred rapidly, was not significantly limited by efflux transporters, and reached higher concentrations than in the CNS. This study demonstrates that, given an equivalent distributional driving force exposure in plasma, the exposure of alisertib in the brain is significantly less than that in the bone marrow, suggesting that targeted delivery may be necessary to guarantee therapeutic efficacy with minimal risk for adverse events. Therefore, these data suggest that, to improve the therapeutic index when using alisertib for brain tumors, a localized regional delivery, such as convection-enhanced delivery, may be warranted. Citation Format: Juhee Oh, Erica A. Power, Wenjuan Zhang, David J. Daniels, William F. Elmquist. Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: Pharmacokinetics and exposure at the sites of efficacy and toxicity [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 A033.

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 MDM2–p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified “Delivery – Potency – Efficacy” relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2–p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine “active” unbound concentrations. Efficacy measures, including overall survival and target expression 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 nmol/L. The CNS distribution of BI-907828 is limited by blood–brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly “poor” BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P &lt; 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the “Delivery – Potency – Efficacy” relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

AbstractBrain tumours have a poor prognosis and lack effective treatments. The blood–brain barrier (BBB) represents a major hurdle to drug delivery to brain tumours. In some locations in the tumour, the BBB may be disrupted to form the blood–brain tumour barrier (BBTB). This leaky BBTB enables diagnosis of brain tumours by contrast enhanced magnetic resonance imaging; however, this disruption is heterogeneous throughout the tumour. Thus, relying on the disrupted BBTB for achieving effective drug concentrations in brain tumours has met with little clinical success. Because of this, it would be beneficial to design drugs and drug delivery strategies to overcome the ‘normal’ BBB to effectively treat the brain tumours. In this review, we discuss the role of BBB/BBTB in brain tumour diagnosis and treatment highlighting the heterogeneity of the BBTB. We also discuss various strategies to improve drug delivery across the BBB/BBTB to treat both primary and metastatic brain tumours. Recognizing that the BBB represents a critical determinant of drug efficacy in central nervous system tumours will allow a more rapid translation from basic science to clinical application. A more complete understanding of the factors, such as BBB–limited drug delivery, that have hindered progress in treating both primary and metastatic brain tumours, is necessary to develop more effective therapies.

Abstract Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.