Sarkaria, Jann N.

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 < 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 OBJECTIVE: We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS: Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS: Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION: The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. Citation Format: Weihua Zhou, Zitong Zhao, Angelica Lin, John Yang, Jie Xu, Kari Wilder-Romans, Annabel Yang, Andrew J. Scott, Jing Li, Sumeet Solanki, Jennifer Speth, Natalie Walker, Ayesha U. Kothari, Yangyang Yao, Erik R. Peterson, Navyateja Korimerla, Christian K. Werner, Jessica Liang, Janna Jacobson, Sravya Palavalasa, Alexandra M Obrien, Ameer L Elaimy, Sean P. Ferris, Shuang G. Zhao, Jann N. Sarkaria, Balázs Győrffy, Shuqun Zhang, Wajd N. Al-Holou, Yoshie Umemura, Meredith A Morgan, Theodore S. Lawrence, Costas A. Lyssiotis, Marc Peters-Golden, Yatrik M. Shah, Daniel R. Wahl. GTP signaling links metabolism, DNA repair, and responses to genotoxic stress [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 A004.

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.

Glioblastoma (GBM), a highly lethal and aggressive central nervous system malignancy, presents a critical need for targeted therapeutic approaches to improve patient outcomes in conjunction with standard-of-care (SOC) treatment. Molecular subtyping based on genetic profiles and metabolic characteristics has advanced our understanding of GBM to better predict its evolution, mechanisms, and treatment regimens. Pharmacological ascorbate (P-AscH−) has emerged as a promising supplementary cancer therapy, leveraging its pro-oxidant properties to selectively kill malignant cells when combined with SOC. Given the clinical challenges posed by the heterogeneity and resistance of various GBM subtypes to conventional SOC, our study assessed the response of classical, mesenchymal, and proneural GBM to P-AscH−. P-AscH− (20 pmol/cell) combined with SOC (5 µM temozolomide and 4 Gy of radiation) enhanced clonogenic cell killing in classical and mesenchymal GBM subtypes, with limited effects in the proneural subtype. Similarly, following exposure to P-AscH− (20 pmol/cell), single-strand DNA damage significantly increased in classical and mesenchymal but not proneural GBM. Moreover, proneural GBM exhibited increased hydrogen peroxide removal rates, along with increased catalase and glutathione peroxidase activities compared to mesenchymal and classical GBM, demonstrating an altered H2O2 metabolism that potentially drives differential P-AscH− toxicity. Taken together, these data suggest that P-AscH− may hold promise as an approach to improve SOC responsiveness in mesenchymal GBMs that are known for their resistance to SOC.

Abstract Inactivating mutations of genes encoding the cohesin complex are common in a wide range of human cancers. STAG2 is the most commonly mutated cohesin complex subunit. Here we report the impact of stable correction of endogenous, naturally occurring STAG2 mutations on gene expression, 3D genome organization, chromatin loops, and Polycomb signaling in glioblastoma multiforme (GBM). In two GBM cell lines, correction of their STAG2 mutations significantly altered the expression of ~10% of all expressed genes. Virtually all the most highly regulated genes were negatively regulated by STAG2 (i.e., expressed higher in STAG2-mutant cells than in STAG2-corrected cells), and one of them – HEPH – was regulated by STAG2 in uncultured GBM tumors as well. While STAG2 correction had little effect on large scale features of 3D genome organization (A/B compartments, TADs), STAG2 correction did alter thousands of individual chromatin loops, some of which controlled the expression of adjacent genes. Loops specific to STAG2-mutant cells, which were regulated by STAG1-containing cohesin complexes, were very large, supporting prior findings that STAG1-containing cohesin complexes have greater loop extrusion processivity than STAG2-containing cohesin complexes and suggesting that long loops may be a general feature of STAG2-mutant cancers. Finally, STAG2-regulated chromatin loops were enriched for the H3K27me3 Polycomb epigenetic mark, revealing that inactivation of STAG2 can activate Polycomb signaling in GBM, identifying a potential target for therapeutic intervention in STAG2-mutant tumors. Together, these findings illuminate the landscape of STAG2-regulated genes, A/B compartments, chromatin loops, and pathways in GBM, providing important clues into the largely still unknown mechanism of STAG2 tumor suppression.

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 Purpose: We evaluated the properties and activity of AZD9574, a blood–brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. Experimental Design: AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. Results: AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)–methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. Conclusions: The combination of three key features—PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule—supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.