Blomquist, Mylan

Abstract Background Glioblastoma (GBM) is driven, in part, by glioma stem cells (GSCs), which sustain therapeutic resistance and recurrence. OLIG2 is a key regulator of GSC programs but the signaling pathways that cooperate with Oligodendrocyte transcription factor 2 (OLIG2) to maintain GSC states remain incompletely defined. Although Signal transducer and activator of transcription (STAT) signaling has been implicated in GSC biology, work to date has focused primarily on STAT3, and the contribution of STAT5 remains poorly defined. Here, we investigated whether STAT5B functionally cooperates with OLIG2 in sustaining GSC phenotypes. Methods Transcriptomic analyses, proximity ligation assays and biochemical studies were utilized to define OLIG2-STAT5B interactions in patient-derived GSCs. Functional consequences of OLIG2, STAT5B, BIRC5, and NUSAP1 suppression were assessed using small interfering RNA/short hairpin RNA-knockdown, stemness, and migration assays in EGFRvIII-expressing GSCs. In vivo tumor growth studies were performed using orthotopic xenograft models. Results OLIG2 and STAT5B expression were positively correlated in GBM datasets and patient-derived models. OLIG2 physically associated with STAT5B in EGFRvIII-expressing GSCs, and loss of OLIG2 reduced STAT5 activation, supporting a functional link between these factors. OLIG2 or STAT5B suppression induced convergent transcriptional changes, including downregulation of genes associated with GSC maintenance and proliferation, notably stemness regulators BIRC5 and NUSAP1. Silencing BIRC5 or NUSAP1 impaired GSC self-renewal and migration. In orthotopic models, STAT5B knockdown prolonged survival and phenocopied OLIG2 suppression and stemness loss. Conclusions These findings identify STAT5B as a functional partner of OLIG2 in GBM and define anOLIG2-STAT5B transcriptional axis that sustains GSC maintenance and malignant behavior. Targeting this axis may represent a therapeutic strategy to disrupt treatment-refractory glioma cell states.

Abstract Purpose: Despite advances in molecular diagnostics, glioblastoma (GBM) remains incurable, with recurrence driven by residual invasive tumor cells that escape surgical resection and therapy. Current genomic profiling to guide therapy for recurrent disease typically relies on a single-site clinical biopsy (SSCB) obtained from the contrasting-enhancing (CE) tumor core - the region targeted for maximal resection and most visible on Magnetic Resonance Imaging (MRI). However, this region poorly represents the non-enhancing (NE) invasive tumor margins, which harbor treatment-resistant and genetically distinct cell populations responsible for disease recurrence. GBM exhibits extensive spatial heterogeneity, raising concerns that a single tissue may underrepresent the true actionable landscape of the tumor. Experimental Procedures: We analyzed 302 biopsies from 66 GBM patients who underwent multiregional whole exome and RNA sequencing across CE and NE tumor regions. Of these, 17 patients also had standard-of-care SSCB molecular profiling utilizing clinically available genomic sequencing panels outside of the multiregional protocol. Variants that have been targeted in ongoing or successful human clinical trials were annotated as actionable. We compared actionability profiles between SSCB and multiregional sampling, to determine the gain of actionable variant detection across the two sampling strategies. We performed an in-silico biopsy simulation to model the coverage of these alterations across the tumor landscape with increasing sampling frequency in the multiregional protocol, versus the coverage from clinical sequencing panels on SSCBs. Results: Clinical sampling identified a median of one actionable mutation per patient (range 0-3). In contrast, multiregional profiling revealed additional potentially actionable variants in over 70% of patients, particularly within NE infiltrative regions that are not captured by conventional resection. Notably, nine potentially actionable copy-number variants, including PDGFRA amplification, were consistently absent from SSCB results and underrepresented in most clinical panels. In silico biopsy simulations demonstrated that actionable variant detection continues to rise even after sampling five additional regions, reflecting spatial segregation of therapeutic targets. Conclusions: These findings demonstrate that a single sample from the CE tumor core fails to capture the biologically diverse invasive tumor populations responsible for GBM recurrence. Multiregional sequencing uncovers clinically meaningful alterations that would otherwise remain undetected with conventional sampling, highlighting the risk of incomplete molecular characterization in current clinical biopsy workflows. Citation Format: Enmar Harfoush, Taylor Weiskittel, Mylan Blomquist, Dustin Grief, Aadel A. Chaudhuri, Leland S. Hu, Nhan L. Tran, Shannon Fortin-Ensign. Beyond a single core biopsy: Spatial genomic profiling improves therapeutic target detection in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 706.

Abstract Glioma stem cells (GSCs) are key drivers of glioblastoma (GBM) progression, therapy resistance, and recurrence, making them critical therapeutic targets. While the lineage-defining transcription factor OLIG2 is critical for GSC maintenance, the molecular co-regulators and downstream pathways it engages remain poorly defined. Here, we identify a previously unrecognized, functionally significant interaction between OLIG2 and STAT5B, uncovering a novel regulatory axis in GSCs. Using proximity ligation assay (PLA), we demonstrate that OLIG2 interacts with STAT5 in human and mouse GSC models, particularly in EGFRvIII-mutant cells. We find that OLIG2 preferentially partners with STAT5B and not STAT5A. Knockdown studies in GSCs reveal that OLIG2 and STAT5B co-regulate critical oncogenes BIRC5 and NUSAP1, both of which are required for sphere formation and migration capacity of EGFRvIII positive GSCs. In orthotopic xenograft models, knockdown of either STAT5B or OLIG2 significantly prolongs survival reduces expression of stemness and invasion markers, SOX2, VIMENTIN, and NUSAP1. In contrast, STAT5A knockdown fails to recapitulate these phenotypes, underscoring the role of OLIG2-STAT5B signaling in GSCs. These findings reveal a novel OLIG2-STAT5B transcriptional circuit that plays a role in GSC stemness maintenance, invasion, and tumor growth, particularly in EGFRvIII-driven gliomas. Targeting this axis may offer a new avenue for GBM treatment.

ABSTRACT Background As Coccidioides spp. are endemic in the southwestern United States, awareness of the risk factors for severe disease and dissemination is important to avoid delays in diagnosis and treatment. In addition to immunocompetent patients, individuals of certain ethnic backgrounds, especially African and Filipino ancestry, are at higher risk for dissemination. Diagnostic testing may be complicated by the lack of available invasive specimens for histopathologic examination and culture. Case Summary A 29-year-old African American woman presented with a 6-year history of abdominal bloating and discomfort, which was previously diagnosed as endometriosis and possible irritable bowel syndrome. After multiple visits to primary care, urgent care, and emergency settings, she was referred for outpatient gynecology and gastrointestinal follow-up. Imaging revealed diffuse peritoneal nodules potentially concerning for malignancy, and biopsies revealed Coccidioides spp. spherules on the frozen section. Routine bacterial and fungal cultures from the biopsied nodules grew Coccidioides immitis/posadasii . The patient was placed on fluconazole with rapid symptom resolution and was maintained on fluconazole at a 2-year follow-up. Conclusion The case presented highlights a reasonably common occurrence in endemic regions—the delayed diagnosis of coccidioidomycosis due to the lack of consideration as the etiology of the patient’s symptoms. This patient was at risk for dissemination due to her ethnicity, and while the differential for her presenting symptoms was broad, coccidioidomycosis should have been considered. In immunocompetent patients, like the case presented, serologic testing should be pursued, as severe or disseminated cases typically have positive IgG responses.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with few effective treatments. EGFR alterations, including expression of the truncated variant EGFRvIII, are among the most frequent genomic changes in these tumors. EGFRvIII is known to preferentially signal through STAT5 for oncogenic activation in GBM, yet targeting EGFRvIII has yielded limited clinical success to date. In this study, we employed patient-derived xenograft (PDX) models expressing EGFRvIII to determine the key points of therapeutic vulnerability within the EGFRvIII-STAT5 signaling axis in GBM. Our findings reveal that exogenous expression of paralogs STAT5A and STAT5B augments cell proliferation and that inhibition of STAT5 phosphorylation in vivo improves overall survival in combination with temozolomide (TMZ). STAT5 phosphorylation is independent of JAK1 and JAK2 signaling, instead requiring Src family kinase (SFK) activity. Saracatinib, an SFK inhibitor, attenuates phosphorylation of STAT5 and preferentially sensitizes EGFRvIII+ GBM cells to undergo apoptotic cell death relative to wild-type EGFR. Constitutively active STAT5A or STAT5B mitigates saracatinib sensitivity in EGFRvIII+ cells. In vivo, saracatinib treatment decreased survival in mice bearing EGFR WT tumors compared to the control, yet in EGFRvIII+ tumors, treatment with saracatinib in combination with TMZ preferentially improves survival.

Abstract Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM (50%). One example includes extracellular domain missense mutations like G598V, A289V, and R108K (10-15%). These extracellular domain missense mutations increase ligand binding affinity, inducing a highly active receptor. Earlier reports have shown these EGFR missense mutations exhibit therapeutic resistance; however, the molecular mechanism of these missense mutations in GBM remains unknown. In this study, we show high oligodendrocyte transcription factor 2 (OLIG2) expression in GSCs with EGFR-G598V and EGFR-A289V missense mutations. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Here, we report GSCs with these point mutations display increased self-renewal, growth, invasion, and therapeutic resistance. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. These missense mutation(s) also represent different pathway-based GBM subclassifications, where A289V induces a Neuronal subtype and G598V induces a Glycolytic/Plurimetabolic subtype. We hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients. Citation Format: Sidney Weaver, Aunay Miller, Mylan Blomquist, James McNamara, Keely Orndorff, Chris Sereduk, Jean Kloss, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5444.

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion. Citation Format: Aunay Miller, Costanza Lo Cascio, Mylan Blomquist, James B. McNamara, Keely Orndorff, Matthew Dufault, Christopher Sereduk, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. A novel transcriptional complex of OLIG2-STAT5 mediates glioma stem cell self renewal and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 256.

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion.

Abstract BACKGROUND Treatment of high-grade glioma (HGG) remains a challenge. Several groups have developed ex vivo drug sensitivity assays to evaluate susceptibility of the tumor tissue to specific chemotherapeutics. This tool, in addition to molecular analysis of tumors might provide another layer of confidence in choosing treatments for patients with high-grade gliomas. METHODS 3D PredictTM platform (Kiyatec, Greenville, SC, USA) was tested at three academic medical centers in the United States. Twelve patients with newly diagnosed and recurrent HGGs were included. Tumor spheroids created ex vivo were exposed to twelve pre-selected drugs. Responses were reported as: no response, moderate response and response. We analyzed: demographics, histopathology, stage of disease, success rate and results of the ex vivo assay and clinical implications. RESULTS Majority of patients were women (67%). Median age was 58.5. Histology: GBM-83%, HGGs-17%. Eighty three percent of the samples represented recurrent disease. Sixty percent represented first recurrence while the remaining 40% represented 2nd or 3rd recurrence. Eight out of 12 tissue samples (67%) were successfully tested ex vivo. In six cases (75%), the information was used for immediate clinical decision making. In 2 cases, the decision was deferred until the next progression of the disease. The drugs chosen for treatment based on assay results included: olaparib (the assay was positive for response to rucaparib), temozolomide, lomustine, carboplatin and irinotecan. CONCLUSIONS This platform is easy to use in a standard clinical setting, the success rate for creating viable spheroids for drug sensitivity testing is high and the information obtained can be useful in clinical practice. Addition of other agents to the assay, correlation with molecular testing of the tumor tissue and attempt to assess response to drug combinations should be considered in further development of this technology. Prospective clinical trial is needed to validate this approach for routine clinical use.

Abstract Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM. EGFRvIII (deletion of exons 2-7) is observed in GBM (20-25%) resulting in a constitutively activated EGFR receptor. In addition, extracellular domain missense point mutations, (e.g. G598V, A289V, R108K) are observed in GBM (14.4%). These missense mutations in the EGFR extracellular domain also induces a highly active receptor by enhancing ligand binding. Earlier reports have shown that these EGFR missense point mutations have exhibited therapeutic resistance; however, the molecular mechanism of these missense point mutations in GBM remains unknown. Previous studies in our lab showed that EGFRvIII promotes GSC maintenance and therapeutic resistance via oligodendrocyte transcription factor 2 (OLIG2) signaling. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Increased OLIG2 expression promotes invasion, growth, proliferation, and survival. Here we report an increase in OLIG2 protein expression in GSCs harboring EGFR-G598V or EGFR-A289V mutations, suggesting these mutations may support stemness properties in GBM tumors. GSC expressing these point mutations display increased self-renewal, growth, and survival. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. Hence, we hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients.