Citations

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 Purpose: Pharmacologic ascorbate (P-AscH−) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH− with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH− in humans. Patients and Methods: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan–Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker. Results: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7–26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems. Conclusions: P-AscH− combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255

Abstract BACKGROUND The microenvironment of glioblastoma is complex and defined in part by protumorigenic metabolic changes. Within this microenvironment, blood-brain barrier disruption is often associated with the most aggressive portions of the tumor. To date, the metabolic tumor microenvironments of enhancing, blood-brain barrier (BBB) disrupted portions of gliomas, as compared to BBB-intact tumor, remain poorly understood. Specifically, it is unclear if the associated blood-brain barrier disruption merely reflects or functionally supports tumor aggressiveness. METHODS Intra-operative high-molecular weight microdialysis was utilized under an investigational device exemption (NCT NCT04047264) to sample the extracellular metabolome of radiographically diverse regions during fifteen neurosurgical resections. The global extracellular metabolome of recovered microdialysate from contrast-enhancing and non-enhancing tumor, as well as brain adjacent to tumor, was evaluated via ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) on the Metabolon platform and assessed using enrichment and correlation analyses. RESULTS Among 162 named metabolites identified via ultra-performance liquid chromatography tandem mass spectrometry, a novel glioma-associated metabolite, guanidinoacetate (GAA), was 126.32x higher in enhancing tumor than in adjacent brain and 13.63x higher in non-enhancing tumor than brain. Forty-eight additional metabolites were 2.05-10.18x more abundant in enhancing tumor than brain, including carnitine family members and all nine large neutral amino acids. Except for GAA and 2-hydroxyglutarate in IDH-mutant gliomas, differences between non-enhancing tumor and brain microdialysate were comparatively modest and less consistent, suggesting heterogeneity in the glioma metabolic microenvironment of blood-brain barrier (BBB) intact areas as compared to BBB-disrupted portions of gliomas. Interestingly, the enhancing but not the non-enhancing glioma metabolome was significantly enriched for plasma-associated metabolites, largely comprising amino acids and carnitines. CONCLUSION Our patient-derived data suggest that the BBB-disrupted metabolome of high-grade gliomas is characterized by metabolite diffusion from circulation into the tumor microenvironment. Further studies are ongoing to determine how this alteration in the glioma extracellular metabolome impacts glioma behavior and how it could be therapeutically targeted via BBB-modulating drugs.

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options, due in part to the low blood-brain-barrier (BBB) penetrance of many anti-cancer compounds. Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term cell cultures established from four patient-derived xenografts with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by RNA sequencing and mass spectrometry (proteomics) to characterize and compare cargo profiles for each condition. We found that depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Mass spectrometry of protein cargo further demonstrated both upregulated and downregulated peptide abundances with treatment compared to vehicle. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow for the selective interrogation of patient GBM-derived EVs, we have simultaneously pursued the identification of biomarkers to allow for immunoprecipitation of EVs from patient biofluids. Potential biomarkers, including PTPRZ1, B7H3, and IL13RA2, were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.

Abstract WSD0922-Fu is an oral, central nervous system (CNS)-penetrant, small molecule, ATP non-competitive, reversible EGFR inhibitor which potently inhibits EGFR aberrations specific to non-small cell lung cancer (NSCLC) and high-grade astrocytoma (HGA). MC1914 (NCT04197934, sponsored by Wayshine Biopharma and FDA OOPD) is a first-in-human phase 1 dose-escalation trial investigating the safety and tolerability of WSD0922-Fu in patients with EGFR aberrant recurrent HGA and NSCLC. Adult patients with either recurrent EGFR mutant NSCLC with CNS metastases or recurrent EGFR mutant and/or EGFR amplified HGA were initially treated with oral WSD0922-FU, with doses escalated in cohorts based on a standard 3 + 3 design to determine the maximum tolerated dose (MTD). After establishing the MTD, this trial has now activated three dose expansion cohorts. Two of these cohorts evaluate different dose/schedule strategies in independent populations. The NSCLC cohort includes patients with EGFR mutant NSCLC and CNS metastases, and examines the safety, tolerability, plasma pharmacokinetics and preliminary efficacy of continuous WSD0922-Fu dosing (twice daily, seven days per week). The HGA cohort includes patients with EGFRvIII mutant HGA at first recurrence after prior radiation/temozolomide and examines the impact of higher doses of WSD0922-Fu administered intermittently (twice daily, five days per week). The HGA cohort also includes a food-effect study which evaluates the effect of the fed and fasted states on plasma PK. The third cohort (Brain tumor penetration cohort) includes patients with HGA (any EGFR mutation and/or EGFR amplification) who need a surgical resection for recurrent disease. Patients enrolled will be treated pre-operatively with WSD0922-Fu for five days, and tumor PK/PD will be examined in samples obtained from image-registered brain tumor biopsies. WSD0922-Fu therapy will be resumed after post-operative recovery. Patient enrollment to all three dose expansion cohorts is ongoing.

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due, at least in part, to increased phosphorylation, cytoplasmic retention, and reduced activity of the YAP/TAZ transcriptional coactivators. Furthermore, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells, irrespective of their inherent response to TMZ. The data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.

Abstract Background Glioblastoma (GBM) is the most common malignant brain tumor and has a poor prognosis. Imaging findings at diagnosis and in response to treatment are nonspecific. Developing noninvasive assays to augment imaging would be helpful. Plasma extracellular vesicles (EVs) are a promising biomarker source for this. Here, we develop spectral flow cytometry techniques that demonstrate differences in bulk plasma EV phenotype between GBM patients and normal donors that could serve as the basis of a liquid biopsy. Methods Plasma EVs were stained for EV-associated tetraspanins (CD9/CD63/CD81), markers indicating cell of origin (CD11b/CD31/CD41a/CD45), and actin/phalloidin (to exclude cell debris). EVs were analyzed using spectral flow cytometry. Multiparametric analysis using t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) was performed comparing GBM and normal donor (ND) plasma EVs. Results Size exclusion chromatography plus spectral-based flow cytometer threshold settings enriched plasma EVs while minimizing background noise. GBM patients had increased CD9+, CD63+, CD81+, and myeloid-derived (CD11b+) EVs. Multiparametric analysis demonstrated distinct surface marker expression profiles in GBM plasma EVs compared to ND EVs. Fifteen plasma EV sub-populations differing in size and surface marker expression were identified, six enriched in GBM patients and two in normal donors. Conclusions Multiparametric analysis demonstrates that GBM patients have a distinct nonneoplastic plasma EV phenotype compared to ND. This simple rapid analysis can be performed without purifying tumor EVs and may serve as the basis of a liquid biopsy.

Inhibition of the interaction between MDM2 and p53 has emerged as a promising strategy for combating cancer, including the treatment of glioblastoma (GBM). Numerous MDM2 inhibitors have been developed and are currently undergoing rigorous testing for their potential in GBM therapy. Encouraging results from studies conducted in cell culture and animal models suggest that MDM2 inhibitors could effectively treat a specific subset of GBM patients with wild-type TP53 or functional p53. Combination therapy with clinically established treatment modalities such as radiation and chemotherapy offers the potential to achieve a more profound therapeutic response. Furthermore, an increasing array of other molecularly targeted therapies are being explored in combination with MDM2 inhibitors to increase the effects of individual treatments. While some MDM2 inhibitors have progressed to early phase clinical trials in GBM, their efficacy, alone and in combination, is yet to be confirmed. In this article, we present an overview of MDM2 inhibitors currently under preclinical and clinical investigation, with a specific focus on the drugs being assessed in ongoing clinical trials for GBM patients.

Glioblastoma (GBM) is a lethal brain cancer with a five-year survival rate of <5%. Approximately half of GBM tumors lack the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), which reverses O6-alkylguanine (O6G) lesions. Patients presenting MGMT– GBM are treated with surgery followed by radiation therapy and temozolomide (TMZ), an imidazotetrazine prodrug that produces O6-methylguanine (O6MeG) lesions. However, ~50% of these patients will develop TMZ resistance by silencing of the DNA mismatch repair (MMR) pathway. We recently reported that the novel N3-(2-fluoroethyl)imidazotetrazine “KL-50” is efficacious and well-tolerated in murine models of TMZ-resistant GBM (Lin et al. Science 2022, 377, 502). Herein, we rigorously establish that KL-50 generates DNA interstrand crosslinks (ICLs) by DNA alkylation to generate O6-(2-fluoroethyl)guanine (O6FEtG), displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. 2-Chloroethylating agents, such as lomustine and mitozolomide (MTZ), generate the same ICL by an analogous mechanism. However, DNA ICLs form >10-fold more slowly from O6FEtG than O6ClEtG, and this slower rate of cross-linking allows MGMT to reverse the initial O6FEtG in healthy tissue while also reducing MGMT–DNA cross-links arising from addition of MGMT to the N1,O6EtG intermediate. KL- 50 is efficacious in an intracranial patient-derived murine xenograft of TMZ-resistant, MGMT–/MMR– GBM (mOS = 205, 28, and 26 d for KL-50, TMZ, and vehicle-treated control, respectively) and in murine models of newly-diagnosed MGMT–/MMR+ GBM, suggesting its use in recurrent and up-front settings, respectively. These studies underscore the significance of considering the rates of chemical DNA modification and biochemical DNA repair in the design of systemic DNA alkylation agents.