Mladek, Ann C.

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 >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 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<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<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<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.

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 < 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.

Abstract Background Although the epidermal growth factor receptor (EGFR) is a frequent oncogenic driver in glioblastoma (GBM), efforts to therapeutically target this protein have been largely unsuccessful. The present preclinical study evaluated the novel EGFR inhibitor WSD-0922. Methods We employed flank and orthotopic patient-derived xenograft models to characterize WSD-0922 and compare its efficacy to erlotinib, a potent EGFR inhibitor that failed to provide benefit for GBM patients. We performed long-term survival studies and collected short-term tumor, plasma, and whole-brain samples from mice treated with each drug. We utilized mass spectrometry to measure drug concentrations and spatial distribution and to assess the impact of each drug on receptor activity and cellular signaling networks. Results WSD-0922 inhibited EGFR signaling as effectively as erlotinib in in vitro and in vivo models. While WSD-0922 was more CNS penetrant than erlotinib in terms of total concentration, comparable concentrations of both drugs were measured at the tumor site in orthotopic models, and the concentration of free WSD-0922 in the brain was significantly less than the concentration of free erlotinib. WSD-0922 treatment provided a clear survival advantage compared to erlotinib in the GBM39 model, with marked suppression of tumor growth and most mice surviving until the end of the study. WSD-0922 treatment preferentially inhibited phosphorylation of several proteins, including those associated with EGFR inhibitor resistance and cell metabolism. Conclusions WSD-0922 is a highly potent inhibitor of EGFR in GBM, and warrants further evaluation in clinical studies.

Abstract Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

Abstract The Mayo Clinic collection of patient-derived xenograft (PDXs) has been developed from the direct implantation of glioblastoma (GBM) samples into immunodeficient mice. Over 105 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated infiltration of tumor cells into the brain parenchymal in 90% of models with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed the PDX collection captures the genetic heterogeneity of GBM, with the majority of typical molecular alterations seen at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations (86%) and homozygous deletion of CDKN2A (70%) were the most frequent. Additional alterations in the p53 pathway included TP53 mutations (36%) and MDM2/4 amplification (10%/2%). IDH mutations were found in three models while MGMT promoter methylation was observed in 45% of PDXs. The available molecular sequencing results are viewable on cBioPortal. These genetically diverse models are extremely useful for biomarker discovery and efficacy evaluations. Using an innovative 1x1x1 PDX preclinical trial design, we are establishing a workflow to evaluate control and drug treatment in an individual animal across PDX lines in the flank. As our first use of this strategy, a small molecule MDM2 inhibitor was evaluated in 17 PDX lines with variable MDM2 and TP53 statuses. Tumors harboring MDM2 amplification without TP53 mutation (n=5) showed profound response, with 4 PDXs showing a complete response that lasted >150 days. Response in TP53 wild-type tumors lacking MDM2 amplification was variable, with a 1.9 to 4.8 fold longer time to endpoint (mean 3; median 2.7). A more traditionally powered intracranial efficacy study performed with one MDM2 amplified (GBM108) and two non-amplified (GBM14, GBM10) PDX lines confirmed these findings of reduced and more variable response to therapy in MDM2 non-amplified TP53 wild-type tumors. Future studies will aim to examine novel biomarkers indicative of therapeutic response to better understand molecular features associated with MDM2 inhibitor efficacy. With over 20 years of experience, our group has extensive expertise in development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Our diverse collection of clinically relevant GBM PDX models provides a highly sought-after resource which has been utilized by the pharmaceutical community with 58 working relationships to date (26 contracts, 32 collaborations). Through these collaborations, we aim to expand the use of our highly characterized GBM PDX panel for biomarker discovery using similar strategies. Citation Format: Danielle M. Burgenske, Ann C. Mladek, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Rachael Vaubel, Jann N. Sarkaria. Evaluation of novel therapeutics using the Mayo Clinic GBM Patient-Derived Xenograft (PDX) collection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4001.

Abstract Introduction: EGFR targeted antibody-drug conjugates (ADCs) show promise as a novel treatment in a subset of glioblastoma (GBM). Two EGFR targeting ADCs include first generation Depatux-M, with an antimitotic toxin monomethyl auristatin F (MMAF), and ABBV-321, with a DNA crosslinking agent pyrrolobenzodiazepine dimer (PBD) toxin. Due to the large molecular weight, poor drug distribution across the blood-brain barrier significantly limits the efficacy in EGFR-amplified GBM. We studied whether convection enhanced delivery (CED) can be used to safely infuse these two EGFR-targeted ADCs in patient-derived xenograft (PDX) models of EGFR-amplified GBM. Methods: The efficacy of Depatux-M and ABBV-321 was evaluated in vitro and in vivo in two EGFRviii-amplified PDXs (GBM6 and GBM108). Immunofluorescence staining was used to evaluate drug distribution along with pharmacodynamics of the ADCs. CED was performed by stereotactic placement of an infusion catheter in the same location as the original tumor implantation. Immunohistochemistry was used to explore mechanisms of normal cell toxicity. Results: Despite potent activity in vitro (high ng/ml IC50), systemic administration of either ADC conferred minimal extension in survival for either GBM6 or GBM108. In contrast, CED significantly enhanced ADC delivery to tumor and peri-tumoral regions and extended survival. In a pilot study in GBM6 with n=3 mice per group, a single CED infusion of 0.002mg ABBV-321 resulted in extended survival beyond 365 days for two mice, while other doses and placebo were associated with shorter median survival (0 mg - 39 days; 0.03 mg - 105 days; 0.3 mg - 49 days). In a subsequent trial evaluating serial infusions performed every 21 days, four infusions at 0.002 mg ABBV-321 resulted in lethal toxicity. In contrast, limiting ABBV-321 to only two serial CED infusions in GBM108 was associated with extended survival of more than 300 days vs. 53 days for AB095 antibody control infusion. In contrast, serial infusion with Depatux-M was much better tolerated. In a single infusion in GBM6, a 0.06 mg dose was well tolerated and associated with a 49-day extension in median survival. Further, four serial infusions at 0.06 mg given every 21 days in GBM6 and GBM108 was associated with 100 day and more than 250-day extension in survival as compared to AB095 antibody control infusion. To investigate the mechanism of toxicity, infusion of non-tumor bearing C57Bl6 mice with ABBV-321 resulted in a marked loss in NeuN staining and elevated CD68 and GFAP staining 7 days later; Depatux-M infusion was only associated with modest elevation in GFAP without loss of NeuN staining or elevated CD68-positive cells. Conclusion: Depatux-M is well tolerated when infused into normal brain and results in extended survival in orthotopic GBM PDXs. In contrast, ABBV-321, with a distinct PBD toxin, had a much narrower therapeutic window when delivered by CED. Citation Format: Kendra A. Porath, Ann Mladek, Rachael A. Vaubel, Michael S. Regan, Sonia Jain, Danielle Burgenske, Katrina Bakken, Brett Carlson, Margaret Connors, Zeng Hu, Lihong He, Paul A. Decker, Gaspar Kitange, Shiv Gupta, Nathalie Y. Agar, Thomas M. Feldsien, Didier R. Lefebvre, Jann N. Sarkaria. Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 334.

Abstract Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.