Sarkaria, Jann N.

Abstract Current transcriptomic classification of Glioblastoma Multiforme (GBM) has been ineffective to predict survival and therapeutic vulnerabilities. Recently, we proposed a four-group functional classification of GBM that included proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic subtypes with prognostic and therapeutic implications as the mitochondrial subtype carries the best survival and exhibits distinct sensitivity to mitochondrial OXPHOS inhibitors. To uncover novel therapeutic targets for each functional GBM subtype, we focused on protein kinases for their attractive features as both drivers and drug targets, with current availability of 62 FDA-approved inhibitors available for cancer precision therapeutics. We designed an unbiased integrative, machine learning-based proteomics/phosphoproteomics network for the identification of Master Kinases (MKs) responsible for effecting key phenotypic hallmarks of each of the four GBM subtypes. Here we report the identification and validation of protein kinase C delta (PRKCd) and DNA-PKcs as MKs that sustain the glycolytic/plurimetabolic and proliferative/progenitor GBM subtypes, respectively. Genetic and pharmacologic inactivation of PKCd in GBM patient-derived organoids of the glycolytic/plurimetabolic subtype blocked glucose uptake and lipid accumulation, resulting in marked anti-tumor effects. We also validated the role of PKCd in oncometabolic processes at the intersection between insulin, IGF, and lipid metabolism. DNA-PKcs was qualified as MK for the proliferative/progenitor GBM subtype, which is characterized by high basal level of replication stress. Biochemical experiments showed activation of DNA-PK in GBM patient-derived organoids of the proliferative/progenitor subgroup. Targeting DNA-PK in proliferative/progenitor GBM organoids with the clinically tested DNA-PKcs inhibitor nedisertib in association with gamma irradiation, the essential component of the standard of care for patients with GBM, led to an unsustainable level of DNA damage and massive GBM cell death selectively in this GBM subtype. As DNA-PKcs inhibitors have been introduced into clinical trials, our findings suggest that pre-selection of patient with PPR tumors is likely to enhance therapeutic success.

Abstract Glioblastoma (GBM) remains the most common and lethal adult primary brain cancer. Two of the most significant issues preventing the development of effective GBM treatments are inter- and intra-tumor heterogeneity. To address these issues, we developed a novel platform termed ISOSCELES (Inferred cell Sensitivity Operating on the integration of Single-Cell Expression and L1000 Expression Signatures). ISOSCELES integrates single-cell gene expression data in individual GBM tumors with perturbation-response data derived from the NIH Library of Integrated Network-Based Cellular Signatures (LINCS) L1000 dataset to predict sensitive and resistant tumor cell populations. Importantly, we analyzed the predictive power of ISOSCELES in an in vivo xenograft model and demonstrated that ISOSCELES reveals the GBM cell identities primed for lineage expansion during treatment with the aurora kinase inhibitor alisertib. These studies suggest that ISOSCELES can be used to identify sensitive and resistant cell populations to targeted therapies in GBM, which can inform treatment decisions in ongoing and future clinical trials.

Abstract Tumor heterogeneity and broad therapeutic resistance are major challenges when treating patients with glioblastoma (GBM). Moreover, different from other cancer types, the absence of clinically useful classifiers for glioblastoma has hampered the translation of genomic and proteomic information to improve diagnosis and design precision therapeutics for patients. We analyzed a large dataset of human GBM to determine the multi-omics features characterizing the four GBM subtypes that we have recently identified through single-cell RNA sequencing and validated in bulk tumor analysis. Each subtype exhibits activation of unique functional traits that confer distinct therapeutic vulnerabilities. The examination of proteomics, phosphoproteomics, metabolomics, lipidomics and acetylomics data revealed that each GBM subtype has a coherent molecular structure driving the dominant function traceable in each analytical platform. Functional classes are not a specific attribute of GBM as we identified the same subtypes in breast and lung cancer. To test the translational impact of proteomic data in GBM, we developed an unbiased protein kinase signaling network approach for the selection of master kinases (MKs) aberrantly activated in each GBM subtype. We identified therapeutically actionable MKs and novel phosphorylation substrates that we experimentally validated. To provide rapid translation of the functional classifier for precision medicine in GBM, we developed a probabilistic classification tool which determines the probability that a patient’s GBM belongs to one of the four subtypes, exhibiting optimal performance when using RNA extracted from either frozen and paraffin-embedded tissues. The algorithm is publicly accessible and can be used to evaluate the association of therapeutic response with GBM subtypes and as tool for selection criteria in prospective clinical trials.

Adult-type diffusely infiltrating gliomas, of which glioblastoma is the most common and aggressive, almost always recur after treatment and are fatal. Improved understanding of therapy-driven tumor evolution and acquired therapy resistance in gliomas is essential for improving patient outcomes, yet the majority of the models currently used in preclinical research are of therapy-naïve tumors. Here, we describe the development of therapy-resistant IDH-wildtype glioblastoma patient-derived xenografts (PDX) through orthotopic engraftment of therapy naïve PDX in athymic nude mice, and repeated in vivo exposure to the therapeutic modalities most often used in treating glioblastoma patients: radiotherapy and temozolomide chemotherapy. Post-temozolomide PDX became enriched for C>T transition mutations, acquired inactivating mutations in DNA mismatch repair genes (especially MSH6), and developed hypermutation. Such post-temozolomide PDX were resistant to additional temozolomide (median survival decrease from 80 days in parental PDX to 42 days in a temozolomide-resistant derivative). However, temozolomide-resistant PDX were sensitive to lomustine (also known as CCNU), a nitrosourea which induces tumor cell apoptosis by a different mechanism than temozolomide. These PDX models mimic changes observed in recurrent GBM in patients, including critical features of therapy-driven tumor evolution. These models can therefore serve as valuable tools for improving our understanding and treatment of recurrent glioma.

PURPOSE Patients with progressive or recurrent meningiomas have limited systemic therapy options. Focal adhesion kinase (FAK) inhibition has a synthetic lethal relationship with NF2 loss. Given the predominance of NF2 mutations in meningiomas, we evaluated the efficacy of GSK2256098, a FAK inhibitor, as part of the first genomically driven phase II study in recurrent or progressive grade 1-3 meningiomas. PATIENTS AND METHODS Eligible patients whose tumors screened positively for NF2 mutations were treated with GSK2256098, 750 mg orally twice daily, until progressive disease. Efficacy was evaluated using two coprimary end points: progression-free survival at 6 months (PFS6) and response rate by Macdonald criteria, where PFS6 was evaluated separately within grade-based subgroups: grade 1 versus 2/3 meningiomas. Per study design, the FAK inhibitor would be considered promising in this patient population if either end point met the corresponding decision criteria for efficacy. RESULTS Of 322 patients screened for all mutation cohorts of the study, 36 eligible and evaluable patients with NF2 mutations were enrolled and treated: 12 grade 1 and 24 grade 2/3 patients. Across all grades, one patient had a partial response and 24 had stable disease as their best response to treatment. In grade 1 patients, the observed PFS6 rate was 83% (10/12 patients; 95% CI, 52 to 98). In grade 2/3 patients, the observed PFS6 rate was 33% (8/24 patients; 95% CI, 16 to 55). The study met the PFS6 efficacy end point both for the grade 1 and the grade 2/3 cohorts. Treatment was well tolerated; seven patients had a maximum grade 3 adverse event that was at least possibly related to treatment with no grade 4 or 5 events. CONCLUSION GSK2256098 was well tolerated and resulted in an improved PFS6 rate in patients with recurrent or progressive NF2-mutated meningiomas, compared with historical controls. The criteria for promising activity were met, and FAK inhibition warrants further evaluation for this patient population.

AbstractGlioblastoma (GBM) tumor cells are found in the perivascular niche microenvironment and are believed to associate closely with the brain microvasculature. However, it is largely unknown how the resident cells of the perivascular niche, such as endothelial cells, pericytes, and astrocytes, influence GBM tumor cell behavior and disease progression. A 3D in vitro model of the brain perivascular niche developed by encapsulating brain‐derived endothelial cells, pericytes, and astrocytes in a gelatin hydrogel is described. It is shown that brain perivascular stromal cells, namely pericytes and astrocytes, contribute to vascular architecture and maturation. Cocultures of patient‐derived GBM tumor cells with brain microvascular cells are used to identify a role for pericytes and astrocytes in establishing a perivascular niche environment that modulates GBM cell invasion, proliferation, and therapeutic response. Engineered models provide unique insight regarding the spatial patterning of GBM cell phenotypes in response to a multicellular model of the perivascular niche. Critically, it is shown that engineered perivascular models provide an important resource to evaluate mechanisms by which intercellular interactions modulate GBM tumor cell behavior, drug response, and provide a framework to consider patient‐specific disease phenotypes.

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 Isocitrate dehydrogenase (IDH) 1 or 2 mutations confer a favorable prognosis compared to IDH-wildtype in astrocytoma, frequently denoting a lower grade malignancy. However, recent molecular profiling has identified specific aggressive tumor subgroups with clear clinical prognostic implications that are independent of histologic grading. The homozygous deletion of CDKN2A/B is the strongest implicated independent indicator of the poor prognosis within IDH-mutant astrocytoma, and the identification of this alteration in these lower histologic grade tumors transforms their biology toward an aggressive grade 4 phenotype clinically. CDKN2A/B homozygous deletion is now sufficient to define a grade 4 tumor in IDH-mutant astrocytomas regardless of histologic appearance, yet there are currently no effective molecularly informed targeted therapies for these tumors. The biological impact of CDKN2A/B homozygous deletion in IDH-mutant tumors and the optimal treatment strategy for this molecular subgroup remains insufficiently explored. Here we review the current understanding of the translational significance of homozygous deletion of CDKN2A/B gene expression in IDH-mutant astrocytoma and associated diagnostic and therapeutic implications.

Abstract INTRODUCTION High grade gliomas (HGG) are devastating diseases with largely unchanged survival outcomes despite decades of research. Recent studies suggest the interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is selectively upregulated in up to 80% of HGG, including glioblastoma (GBM) and diffuse midline gliomas (DMG) harboring H3K27 alterations. Immunotoxins targeting IL-13Rα2 have been demonstrated as safe and have shown some benefit for patients with HGG in previous phase I/II and III clinical trials. We hypothesized that by using GB-13 (IL13.E13K-PE4E), a novel peptide-toxin that binds IL-13Rα2 with high specificity and possesses a Pseudomonas exotoxin moiety, we would enhance the anti-tumor effects of this immunotherapy for HGG in vitro and in vivo while decreasing off-target toxicity. METHODS We examined the pharmacological effects of GB-13 in multiple patient-derived cell lines and rodent models of HGG. GBM and DMG lines were used to confirm IL-13Rα2 expression and sensitivity towards GB-13. Tumor naïve rats were evaluated for toxicity, and orthotopic PDX mice were used to monitor tumor size and survival following loco-regional infusion of GB-13. RESULTS GB-13 induced a potent cytotoxic response strongly predicated on IL-13Rα2 expression in vitro. No treatment-related adverse effects were noted after 7-day continuous intracranial infusion of GB-13 in tumor naïve rats. Further, in IL-13Rα2-upregulated orthotopic PDX mice, direct intratumoral administration of GB-13 via convection-enhanced delivery abrogated tumor growth and prolonged survival. CONCLUSIONS Given these promising results as well as the critical need for novel therapies in CNS malignancies, we are progressing to human trials using GB-13 targeting recurrent HGG. Ongoing safety studies in tumor-bearing animals will be able to define dose levels for the initial adult study-arm and the following pediatric study-arm. In this Phase 1 clinical trial, we hypothesize that loco-regional infusion of GB-13 will safely enhance tumor clearance by causing selective killing of IL-13Rα2-upregulated HGG cells.