Citations

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.

Abstract WSD0922-Fu is an oral, CNS-penetrant, small molecule EGFR inhibitor which potently inhibits EGFR aberrations specific to non-small cell lung cancer (NSCLC) and high-grade astrocytoma (HGA). MC1914 (NCT04197934) is a first-in-human phase 1 trial in patients with EGFR aberrant recurrent HGA and NSCLC. Adult patients with either recurrent EGFR mutant NSCLC with CNS metastases or recurrent EGFR aberrant HGA were enrolled and treated across six cohorts (40 mg once daily to 320 mg twice daily, 28-day cycles) to determine the maximum tolerated dose (MTD). 25 patients (17 HGA, 8 NSCLC) were enrolled (median age = 57 years; 24% female; 36% ECOG 0). All patients with HGA received prior radiation and temozolomide. All patients with NSCLC had previous disease progression on EGFR inhibitor therapy (prior osimertinib 88%). The median duration of WSD0922-Fu exposure among all patients was 55 days (range 3 to >279 days) [NSCLC median = 131 days; HGA median = 51 days]. Common treatment-related adverse events are comparable to other EGFR inhibitors (fatigue, acneiform rash, diarrhea, nausea, pruritis). At Cycle 1 Day 15 for patients treated at MTD (160 mg twice daily), the mean terminal elimination half-life (%CV) in the plasma was 31.6 h (102%) (median 14.5 h). The mean Cmax, Tmax, oral clearance at steady state, and AUC (0-12 h) (%CV) were 4513 ng/mL (50%), 2.0 hr (69%), 6.9 L/hr/m2 (42%), and 28084 h*ng/mL (52%), respectively. Among patients with NSCLC treated at MTD or higher dose levels and with available response data, 100% (5/5) of patients demonstrated clinical benefit (SD + PR) while 60% of patients had partial responses. Intracranial and extracranial efficacy was concordant. Furthermore, prolonged stable disease (9 cycles) was observed in a patient with EGFRvIII mutant GBM. Expansion cohorts are in progress to optimize WSD0922-Fu dose and schedule for future phase 2 development.

Abstract BACKGROUND In GBM, effective chimeric antigen receptor T-cell (CART) therapy remains a challenge; immunosuppressive components within the tumor microenvironment (TME) inhibit CART cells. EphA3 is a receptor tyrosine kinase with low expression in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. METHODS We performed RNA sequencing and Immunohistochemical staining for of EphA3 in a clinically annotated GBM PDX biobank and M01 M1 and M2 polarized macrophages. We then investigated the suppressive capability of M2 macrophage on EphA3-CART in an SNB-19 GBM cell co-culture. We tested the antiantitumor potential of EphA3-CART against luciferase+ SNB-19 GBM cells in vivo with flank and orthotopically engrafted NOD-SCID-ɣ-/- (NSG) mice. Engraftment was confirmed by bioluminescent imaging (BLI) and vernier caliber (flank) for 7-14 days post-injection. Mice were randomized to be treated with EphA3-CART or untransduced T-cells (UTD). Subsequently tumor size and mouse survival was monitored to determine the antitumor effect. RESULTS EphA3 was highly expressed on GBM PDX models as well as M2 macrophages. Compared to M0 and M1 macrophages, M2 macrophages significantly suppressed EphA3-CART However, despite the suppressive activity of the M2 macrophage, EphA3-CART exhibited significant antitumor activity . In both flank and orthotopically implanted mouse models, there was an observed reduction in tumor volume with EphA3-CART treated model compared to the UTD counterpart. EphA3-CART treatment resulted in significantly improved survival of the treated models compared to UTD models (hazard ratio = 0.053 **p = 0.005). CONCLUSION We demonstrate that EphA3 is expressed on GBM cells as well as on M2 macrophages. EphA3 CART is able to overcome and ameliorate the effect of a major component of the tumor microenvironment (TME), M2 macrophages. EphA3 CART appears effective in flank and orthotopic mouse models of GBM.

Abstract GB13 is a novel biologic therapy in the final stages of preclinical development for the treatment of glioblastoma (GBM) and H3 K27-altered diffuse midline glioma (DMG) that has received Orphan Drug Designation by the Food and Drug Administration for treatment of malignant gliomas. GB13 targets a specific tumor-restricted receptor of interleukin 13 called interleukin 13 receptor alpha 2 (IL13Rα2). IL13Rα2 is highly expressed in malignant gliomas and, importantly, is not expressed on non-cancerous cells of the brain. GB13 consists of an engineered proprietary mutant of IL13, which binds exclusively to IL13Rα2, and is fused to a highly cytotoxic exotoxin molecule. In vitro data demonstrate that GB13 potently kills IL13Rα2+ cancer cells while sparing non-cancerous cells. These characteristics make GB13 an attractive agent for safe and efficacious targeting of tumor cells expressing IL13Rα2 in upcoming clinical trials. In the current studies, we employed patient-derived GBM and DMG cell and animal models to provide key conclusions that support GB13 as an effective treatment option for gliomas in the clinic. First, we demonstrated that single acute infusions of GB13 by enhanced delivery (CED) is effective at decreasing orthotopic tumor volumes and increasing animal survival and that a long-term infusion can more significantly extend animal survival. Second, we determined that IL13Rα2 is requisite for GB13 activity as tumors that do not express the receptor are insensitive to the cytotoxic effects of GB13, demonstrating a precise and clear biomarker for clinical trial patient selection. Finally, we determined that GB13 works in concert with clinical standard-of-care radiation therapy to increase cell death, compared to radiation alone. Based on this work, we and our clinical collaborators are confident that the current work confirms GB13 has many important characteristics as an exciting therapy, which continues development towards a Phase I clinical trial for both GBM and pediatric DMG.

Abstract Glioblastoma is the most aggressive malignant brain tumor with the poor survival due to its invasive nature driven by cell migration. There is still no effective treatment targeting glioma cell migration based on their transcriptomic information, due to the lack of understanding in the links to the fundamental mechanics of cell migration. In the previous studies, we developed a physics-based motor-clutch model, the cell migration simulator (CMS), to identify the basic mechanisms of cell migration in various cell types. In this study, we found that glioblastoma patient-derived (xenograft) (PD(X)) cell lines across mesenchymal (MES), proneural (PN), classical (CL) subtypes and two institutions (N = 13 patients) had optimal motility and traction force on stiffnesses around 9.3kPa, with otherwise heterogeneous and uncorrelated motility, traction, and F-actin flow. We applied the CMS as a consistent framework to describe the physical parameters of glioblastoma patient cell migration. Using single cell migration and force generation data on compliant 2D surfaces, we found all glioma PD(X) lines (11 patients) had balanced myosin/adhesion ratios resulting in effective migration. Mesenchymal (MES) lines had enhanced F-actin polymerization resulting in higher motility. Adherent PDX lines had increased myosin-adhesion amount resulting in higher traction force. The CMS also predicted the differential cell migration under cytoskeletal drugs between glioma subtypes. Finally, we found 18 genes in actin cytoskeleton and focal adhesion pathways significantly correlated with the glioma physical parameters, which were being used to estimate the physical parameters of a larger cohort (66 patients) and to strategize targeting their cell migration. Overall, we described a consistent physics-based framework for parameterizing individual glioblastoma patients, connecting to clinical transcriptomic data, and predicting drug effects, that can potentially be used to develop subtype and patient-specific anti-migratory therapeutic strategies.

Abstract BACKGROUND New therapies are needed for meningioma since treatment options are limited once patients have exhausted surgery and radiation. FAK, CDK4/6 and RAF/MEK represent potential targets because of synthetic lethality with NF2, loss of CDKN2A, and activation of MAPK signaling pathway in meningioma, respectively. The objective of this study was to evaluate the efficacy of targeting FAK, CDK4/6 and RAF/MEK as monotherapies and combination therapies in traditional and patient-derived cell lines and xenografts. METHODS In vitro, cell viability and proliferation assays were used to test abemaciclib (CDK4/6 inhibitor), defactinib (FAK inhibitor) and VS-6766 (RAF/MEK inhibitor) as monotherapy and in combination. To better recapitulate the meningioma microenvironment, targeted agents were evaluated with Matrigel, an artificial extracellular matrix. The efficacy of VS-4718 (in vivo formulation of defactinib) and abemaciclib were evaluated in a subcutaneous IOMM-lee meningioma model (having CDKN2A loss) in nude mice. Furthermore, we are establishing cell cultures and patient-derived cell and xenograft (PDX) models using freshly-resected tissue from patients with recurrent or high-grade (WHO grade 2-3) meningiomas. RESULTS In vitro evaluation identified the combination of defactinib with abemaciclib resulted in a synergistic decrease in cell viability relative to each monotherapy alone, while the combination of defactinib+VS-6766 was not beneficial. The presence of Matrigel enhanced the sensitivity of all three agents. In vivo, we observed improved survival in mice treated with abemaciclib vs control (45 days vs 26 days; p=0.03), as well as combination of VS-4718 + abemaciclib vs control (44 days vs 26 days; p=0.04, Gehan-Breslow-Wilcoxon test). We did not observe survival extension with VS-4718 monotherapy. Lastly, we successfully established an orthotopic meningioma PDX using a WHO Grade 3 tumor for evaluation of novel therapies in meningioma. CONCLUSION Abemaciclib showed promising efficacy in preclinical meningioma xenografts with CDKN2A loss. Studies to evaluate these therapies in other orthotopic models are underway.

Abstract 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 PDX cultures with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by mass spectrometry (proteomics) and RNA sequencing to characterize and compare cargo profiles for each condition. We found that at the proteome level, FDFT1 and FARSB were detected only in temozolomide EVs across all cell lines. In addition, there were drug-agnostic indications of exposure, including decreased abundance in all treated EV conditions of SLC3A2, C3, PLG, and A1BG, and increased abundance of GSTP1. These cargo changes were further validated by western blotting and ExoView immunofluorescence on isolated EVs. At the EV-transcriptome level, 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. Thus, these results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow immunoprecipitation of patient GBM-derived EVs with the selective interrogation, the potential biomarkers, including PTPRZ1, B7H3, IL13RA2, and EGFR 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 Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Temozolomide (TMZ) is the only drug currently been used for treatment of newly diagnosed GBM patients but the benefit from this agent has been hampered by the primary and secondary resistance. Thus, there is a strong need for uncovering the mechanisms of TMZ resistance. The DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) is the main driver of primary TMZ resistance and lacking MGMT expression due to promoter hypermethylation has been associated with a significant survival prolongation. However, the MGMT-independent resistance mechanisms remain poorly understood. To investigate TMZ resistance mechanisms, we developed in vivo TMZ resistant models using GBM patient derived xenografts (PDXs) by treating animals bearing flank PDXs with clinically relevant 3-cyles of TMZ. The recurrent tumors were all resistant to TMZ through multiple mechanisms, including MGMT re-expression, elevated homologous recombination (HR) and/or non-homologous end joining (NHEJ) repair activities, and loss of mismatch repair (MMR). However, other tumors developed resistance due to unknown mechanisms. To identify novel modulators of TMZ sensitivity, we performed genome wide screening using shRNA library, and we identified over 600 druggable targets. Validation experiments identified RBBP4 gene as one of the candidates involved in TMZ sensitivity. Silencing RBBP4 in GBM cells sensitized both MGMT-methylated and -unmethylated GBM cells to TMZ. Mechanistically, RBBP4 interacts with p300 to form a chromatin remodeling complex that control expression of DNA damage repair genes, and silencing RBBP4 or p300 significantly blocked the recovery of TMZ induced DNA double strand breaks causing cell death. We also found the nuclear pore exporter XPO1 is the modulator of TMZ sensitivity, and the XPO1 inhibitor Selinexor is currently being evaluated for GBM treatment in a clinical trial. Collectively, our work has uncovered multiple mechanisms of TMZ resistance that are potential targets for GBM therapy.

ABSTRACT How cell metabolism regulates DNA repair is incompletely understood. Here, we define a GTP-mediated signaling cascade that links metabolism to DNA repair and has significant therapeutic implications. GTP, but not other nucleotides, regulates the activity of Rac1, a guanine nucleotide-binding protein, which promotes the dephosphorylation of serine 323 on Abl-interactor 1 (Abi-1) by protein phosphatase 5 (PP5). Dephosphorylated Abi-1, a protein previously not known to activate DNA repair, promotes nonhomologous end joining. In patients and mouse models of glioblastoma, Rac1 and dephosphorylated Abi-1 mediate DNA repair and resistance to standard-of-care genotoxic treatments. The GTP–Rac1–PP5–Abi-1 signaling axis is not limited to brain cancer, as GTP supplementation promotes DNA repair and Abi-1-S323 dephosphorylation in nonmalignant cells and protects mouse tissues from genotoxic insult. This unexpected ability of GTP to regulate DNA repair independently of deoxynucleotide pools has important implications for normal physiology and cancer treatment. SIGNIFICANCE: A newly described GTP-dependent signaling axis is an unexpected link between nucleotide metabolism and DNA repair. Disrupting this pathway can overcome cancer resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues. This article is featured in Selected Articles from This Issue, p. 5