Sarkaria, Jann

ABSTRACT Truncating mutations in the tumor suppressor STAG2 , which encodes a component of the cohesin complex, are prevalent across diverse human cancers. Here, we report that acute reconstitution of physiological STAG2 levels in STAG2 -mutant human glioblastoma multiforme cancer cells triggers a rapid reduction in the size of chromatin loops genome-wide. Despite this global change in chromatin loop size, early transcriptional responses to STAG2 restoration are limited to a small number of genes, most of which are induced by STAG2 reconstitution. Notably, the growth-suppressor EFEMP1 (Fibulin-3), a secreted glycoprotein that functions as an extracellular matrix-associated inhibitor of glioblastoma growth and invasion, was the only gene consistently induced across all experimental models. The most robust and conserved STAG2-induced genes all reside within intense chromatin loops whose anchors and overall intensity did not appear to change in response to STAG2 reconstitution. These findings suggest that inactivating mutations of STAG2 promote neoplastic transformation by alleviating a restriction on chromatin loop size, allowing for an expanded range of chromatin interactions that disrupts the maintenance of a tumor-suppressive transcriptome.

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor characterized by invasive growth, genetic heterogeneity and resistance to therapy. Alterations in the epithelial growth factor receptor (EGFR) are frequently detected in GBM, including gene amplifications, mutations and exon deletions, exemplified by the expression of EGFR variant III (EGFRvIII). To target both wild-type and EGFRvIII, we generated the humanized antibody A10, which reacts with the 287-302 loop that is fully exposed on the extracellular domain of EGFRvIII and conditionally exposed on cells expressing gene-amplified EGFR. However, A10 showed little or no reactivity for cells expressing normal levels of wild type EGFR. We engrafted antibody fragments derived from A10 on to ARTEMIS® Cell Receptor Platform, which introduces two separate receptors into engineered T cells to provide activation and co-stimulatory signals. Specifically, the A10 antigen-binding fragment (Fab) was fused to the N-terminus of a γδTCR-signaling domains (A10-AbTCR) while the A10 single chain variable fragment (scFv) was fused to a costimulatory signaling receptor (A10-CSR). The resulting A10 ARTEMIS® T cells were cytotoxic for tumor cell lines displaying amplified or overexpressed EGFR and EGFRvIII. Further, glioblastoma PDXs were sensitive to A10 ARTEMIS® T cell killing. This prompted an in vivo evaluation in EGFR-driven cancers. GBM39 PDX tumors (expressing amplified EGFRvIII) were implanted into the brains of NSG mice. Mice received one or two infusions. Treatment with one infusion of A10 ARTEMIS® cells resulted in prolonged survival of tumor bearing mice with 40 % of mice were long-term survivors (74 days) compared to a median survival of 32 days (control) or 52 days (mock T-cells). For mice receiving two A10 ARTEMIS® T cell infusions, there was 100% survival of mice beyond 60 days, while all control and mock treated mice were dead by 28 days. Further, treatments of EGFR-amplified MDA-MB-468 tumors resulted in complete tumor regression with one treatment of A10 ARTEMIS® cells. Thus, this new format of A10 engineered T-cells appears to be a particularly promising for treating cancers with high levels of EGFR or EGFRvIII including GBM. Citation Format: Antonella Antignani, Danielle Burgenske, Guangyan Xiong, Hongbing Zhang, Cheng Liu, Jann Sarkaria, David FitzGerald. Engineered T-cells, targeting overexpressed EGFR and EGFRvIII, produce complete remissions in a PDX model of human glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B056.

Abstract Background Acquired resistance to temozolomide (TMZ) chemotherapy due to DNA mismatch repair (MMR) enzyme deficiency is a barrier to improving outcomes for isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) patients. KL-50 is a new imidazotetrazine-based therapeutic designed to induce DNA interstrand cross-links, and subsequent double-stranded breaks, in an MMR-independent manner in cells with O-6-methylguanine-DNA methyltransferase (MGMT) deficiency. Previous research showed its efficacy against LN229 glioma cells with MMR and MGMT knockdown. Its activity against patient-derived GBM that model post-TMZ recurrent tumors is unclear. Methods We created MMR-deficient GBM patient-derived xenografts through exposure to TMZ, followed by treatment with additional TMZ or KL-50. We also generated isogenic, MSH6 knockout (KO) patient-derived GBM and tested them for sensitivity to TMZ and KL-50. Results KL-50 extended the median survival of mice intracranially engrafted with either patient-derived TMZ-naïve GBM6 or TMZ-naïve GBM12 by 1.75-fold and 2.15-fold, respectively (P < 0.0001). A low dose (4 Gy) of fractionated RT further extended the survival of KL-50-treated GBM12 mice (median survival = 80 days for RT + KL-50 vs. 71 days KL-50 alone, P = 0.018). KL-50 also extended the median survival of mice engrafted with post-TMZ, MMR-deficient GBM6R-m185 (140 days for KL-50 vs. 37 days for vehicle, P < 0.0001). MSH6 KO increased TMZ IC50 for GBM6 and GBM12 cultures by >5-fold and >12-fold for cell death and live cell count outputs, respectively. In contrast, MSH6-KO actually decreased KL-50 IC50 by 10–80%. Conclusion KL-50-based compounds are a promising new strategy for the treatment of MGMT-deficient, MMR-deficient GBM that recurs after frontline TMZ.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

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.

Abstract Background Response to targeted therapy varies between patients for largely unknown reasons. Here, we developed and applied an integrative platform using mass spectrometry imaging (MSI), phosphoproteomics, and multiplexed tissue imaging for mapping drug distribution, target engagement, and adaptive response to gain insights into heterogeneous response to therapy. Methods Patient-derived xenograft (PDX) lines of glioblastoma were treated with adavosertib, a Wee1 inhibitor, and tissue drug distribution was measured with MALDI-MSI. Phosphoproteomics was measured in the same tumors to identify biomarkers of drug target engagement and cellular adaptive response. Multiplexed tissue imaging was performed on sister sections to evaluate spatial co-localization of drug and cellular response. The integrated platform was then applied on clinical specimens from glioblastoma patients enrolled in the phase 1 clinical trial. Results PDX tumors exposed to different doses of adavosertib revealed intra- and inter-tumoral heterogeneity of drug distribution and integration of the heterogeneous drug distribution with phosphoproteomics and multiplexed tissue imaging revealed new markers of molecular response to adavosertib. Analysis of paired clinical specimens from patients enrolled in the phase 1 clinical trial informed the translational potential of the identified biomarkers in studying patient’s response to adavosertib. Conclusions The multimodal platform identified a signature of drug efficacy and patient-specific adaptive responses applicable to preclinical and clinical drug development. The information generated by the approach may inform mechanisms of success and failure in future early phase clinical trials, providing information for optimizing clinical trial design and guiding future application into clinical practice.

ABSTRACT Various types of radiation are utilized in the treatment of cancer. Equal physical doses of different radiation types do not always result in the same amount of biological damage. In order to account for these differences, a scaling factor known as the relative biological effectiveness (RBE) can be used. 137Cesium (137Cs) has been used as a source of radiation in a significant body of radiation therapy research. However, high-energy X-rays, such as 6 MV X-rays, are currently used clinically to treat patients. To date, there is a gap in the literature regarding the RBE comparison of these two types of radiation. Therefore, the purpose of this study was to investigate the RBE of 137Cs relative to that of 6 MV X-rays. To determine the RBE, five cell lines were irradiated [Chinese hamster ovary (CHO); human lung adenocarcinoma (A549); human glioma (U251); human glioma (T98); and human osteosarcoma (U2OS)] by both types of radiation and assessed for cell survival using a clonogenic assay. Three of the five cell lines resulted in RBE values of ~1.00 to within 11% for all survival fractions, showing the physical and biological dose for these two types of radiation were equivalent. The other two cell lines gave RBE values differing from 1.00 by up to 36%. In conclusion, the results show the range in biological effect seen between cell lines, and therefore cell type must be considered when characterizing RBE.

AbstractBackgroundGlioblastoma (GBM) exhibits profound intratumoral genetic heterogeneity. Each tumor comprises multiple genetically distinct clonal populations with different therapeutic sensitivities. This has implications for targeted therapy and genetically informed paradigms. Contrast-enhanced (CE)-MRI and conventional sampling techniques have failed to resolve this heterogeneity, particularly for nonenhancing tumor populations. This study explores the feasibility of using multiparametric MRI and texture analysis to characterize regional genetic heterogeneity throughout MRI-enhancing and nonenhancing tumor segments.MethodsWe collected multiple image-guided biopsies from primary GBM patients throughout regions of enhancement (ENH) and nonenhancing parenchyma (so called brain-around-tumor, [BAT]). For each biopsy, we analyzed DNA copy number variants for core GBM driver genes reported by The Cancer Genome Atlas. We co-registered biopsy locations with MRI and texture maps to correlate regional genetic status with spatially matched imaging measurements. We also built multivariate predictive decision-tree models for each GBM driver gene and validated accuracies using leave-one-out-cross-validation (LOOCV).ResultsWe collected 48 biopsies (13 tumors) and identified significant imaging correlations (univariate analysis) for 6 driver genes: EGFR, PDGFRA, PTEN, CDKN2A, RB1, and TP53. Predictive model accuracies (on LOOCV) varied by driver gene of interest. Highest accuracies were observed for PDGFRA (77.1%), EGFR (75%), CDKN2A (87.5%), and RB1 (87.5%), while lowest accuracy was observed in TP53 (37.5%). Models for 4 driver genes (EGFR, RB1, CDKN2A, and PTEN) showed higher accuracy in BAT samples (n = 16) compared with those from ENH segments (n = 32).ConclusionMRI and texture analysis can help characterize regional genetic heterogeneity, which offers potential diagnostic value under the paradigm of individualized oncology.