James, C. David

ABSTRACT Bispecific T cell engagers (BTEs) induce MHC‐independent cytotoxicity by bridging T cells to tumor cells via binding a T cell–activating receptor and a tumor‐associated antigen. BTEs have proven effective in hematologic malignancies and some solid tumors, yet their potential in glioblastoma (GBM) is largely unexplored. We developed a fully humanized BTE (hBTE) targeting interleukin‐13 receptor alpha 2 (IL13RA2), a tumor‐associated antigen widely expressed in GBM and associated with poor prognosis. In vitro, hBTE activated T cells and induced antigen‐dependent cytokine release and cytotoxicity against IL13RA2‐positive GBM cells. In vivo, hBTE showed robust target‐specific activity and markedly prolonged survival in primary and recurrent GBM xenograft models, without detectable off‐target local or systemic toxicity. Beyond GBM, hBTE also exhibited antitumor activity in IL13RA2‐expressing solid tumors, demonstrating selective tumor accumulation and therapeutic efficacy in models of breast cancer brain metastases and extracranial lung cancer. This work highlights the therapeutic potential of BTEs in IL13RA2‐expressing tumors and establishes a strong preclinical rationale for advancing hBTE therapy toward clinical translation in GBM and other tumors.

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.

Abstract Background Advanced age is a major risk factor for the development of many diseases including those affecting the central nervous system. Wild-type isocitrate dehydrogenase glioblastoma (IDHwt GBM) is the most common primary malignant brain cancer and accounts for ≥90% of all adult GBM diagnoses. Patients with IDHwt GBM have a median age of diagnosis at 68–70 years of age, and increasing age is associated with an increasingly worse prognosis for patients with this type of GBM. Methods The Surveillance, Epidemiology, and End Results, The Cancer Genome Atlas, and the Chinese Glioma Genome Atlas databases were analyzed for mortality indices. Meta-analysis of 80 clinical trials was evaluated for log hazard ratio for aging to tumor survivorship. Results Despite significant advances in the understanding of intratumoral genetic alterations, molecular characteristics of tumor microenvironments, and relationships between tumor molecular characteristics and the use of targeted therapeutics, life expectancy for older adults with GBM has yet to improve. Conclusions Based upon the results of our analysis, we propose that age-dependent factors that are yet to be fully elucidated, contribute to IDHwt GBM patient outcomes.

Abstract Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Abstract PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

1509 Background: Lack of MGMT methylation in GBM tumors is associated with a worse outcome with combined radiation (RT) and temozolomide (TMZ) therapy, and it is unclear whether non-methylated tumors benefit from TMZ treatment. Methods: The potential benefits of concomitant or adjuvant TMZ relative to methylation status were evaluated in 10 GBM tumors established from patient samples and maintained by serial passage in mice. For each tumor line, 32 mice with established orthotopic xenografts were randomized to A) placebo, B) TMZ PO daily × 5 (120 mg/kg or 66 mg/kg, as indicated) C) RT, 2 Gy bid × 10 fx., D) combined TMZ and RT, and mice were followed until moribund. Efficacy was evaluated by regression modeling and Kaplan-Meier statistical analyses. Results: Six lines with non-methylated MGMT (GBM10, 14, 26, 28, 36, 43) and 4 with methylated MGMT (GBM8, 12, 16, 34) were evaluated, and complete results are available in 6 lines. In all tumor lines and at both dose levels, there was a significant benefit for TMZ relative to placebo (p<0.003). Both methylated tumors (GBM8 and 12) and one non-methylated tumor (GBM14) were markedly sensitive to TMZ with greater than a 50 day extension in survival with monotherapy. Individually and in a pooled analysis, there was no evidence for a synergistic interaction between TMZ and RT in the non-methylated tumors (p=0.91 for pooled analysis). In contrast, there was a striking degree of synergy in GBM12 (p<0.001) and at least additive effects of combined treatment in GBM8 (5 of 8 mice still surviving in the RT/TMZ arm). In a pooled analysis of these 2 MGMT methylated tumors, there was a trend towards synergy (p=0.11). Conclusions: TMZ treatment did not sensitize MGMT non-methylated tumors to radiation, and with one exception, these non-methylated tumors were only modestly sensitive to TMZ alone. In contrast, treatment with TMZ appears to sensitize MGMT-methylated tumors to radiation and monotherapy is associated with marked and significant benefit. [Table: see text] No significant financial relationships to disclose.

Abstract Dysregulation of ErbB-mediated signaling is observed in up to 90% of solid tumors. ErbB family cross-talk is implicated in the development of resistance and metastasis, including CNS metastases. Inhibition of multiple ErbB receptors may result in improved patient outcomes. EO1001 is a novel, patented, oral, brain-penetrating, irreversible pan-ErbB inhibitor targeting EGFR (ErbB1), HER2 (ErbB2) and HER4 (ErbB4). METHODS: (1) In vitro testing. EO1001 demonstrates high specificity for the ErbB family of receptors with excellent, balanced equipotent activity against EGFR, HER2 and HER4 (0.4 to 7.4 nM). EO1001 inhibits signaling downstream of wild type EGFR, mutant EGFR (T790M, L858R and d746-750) and HER2. (2) PK and toxicity. In rodent studies in vivo, EO1001 exhibited a half-life of 16–20 hours. EO1001 rapidly enters the CNS and penetrates tumor tissue at higher concentrations relative to plasma. Safety of EO1001 was evaluated by repeat-dosing studies in SD rats and beagle dogs. Toxicities typical of the ErbB inhibitor class, including gastro-intestinal effects, weight loss and decreased activity were observed at higher dose groups in both species. Mortality was observed in SD rats at higher dose groups. (3) In vivo efficacy studies. EO1001 was studied following oral administration in several erbB-positive mouse xenograft models including N87 (Her2+), H1975 (EGFR/T790M), GBM12 (EGFR+), GBM39 (EGVRvIII+). Following oral administration, treatment with EO1001 resulted in a statistically significant improvement in outcomes compared to positive and negative controls in both CNS and systemic tumor models. EO1001 was well-tolerated with no gastrointestinal side effects observed at efficacious doses in these models. CONCLUSION: Based on research to date, EO1001 has the potential to be a best-in-class CNS-penetrating pan-ErbB inhibitor with a safety and pharmacokinetic profile amenable for use as a single agent and in combination with other agents. EO1001 is poised to enter phase 1-2a clinical testing in the second-half of 2019.

Abstract Purpose: Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas. Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. Results: A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of β-catenin. Conclusions: These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.