AbstractPurpose: Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM. Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro. Results: This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells. Conclusions: Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas arising from Schwann cells and characterized by marked cellular and molecular heterogeneity. Although bulk multi-omic studies have provided valuable insights into MPNST biology, recent advances in single-cell profiling have deepened our understanding of the tumor microenvironment and molecular mechanisms underlying malignant transformation. Single cell analyses have revealed distinct Schwann cell-like, malignant neural crest-like, immune, and stromal cellular subpopulations within MPNSTs and their precursor lesions. Comparative profiling of MPNSTs, neurofibromas, and atypical neurofibromatous neoplasms of uncertain biologic potential, suggest that MPNST progression involves Schwann cell dedifferentiation into a more primitive, stem-like state. In this review, we summarize key discoveries from single-cell characterization studies, and discuss how these findings illuminate MPNST tumorigenesis, cellular plasticity, and potential therapeutic vulnerabilities.

Supplemental Figure 1. Gene-set enrichment analysis (GSEA). Based on the list of top 200 differentially expressed genes with HK2 knockdown in the two cell lines (U87 and GSC30), a GSEA using the molecular signature database identified oncogenic signatures downregulated (A) or upregulated (B) with HK2 knockdown.

Supplemental Figure 2. Ketoconazole and Posaconazole inhibit GBM growth in a U87 xenograft model.

Background Adolescent and young adult (AYA) patients remain underrepresented in neuro-oncology research. Despite being the second most common primary brain tumor in this population, meningiomas have not been studied using age-specific molecular analyses. DNA methylation-based classification and prognostic tools have transformed meningioma care. This study aimed to evaluate the performance of these tools across age groups.Methods We analyzed 1,568 meningiomas with DNA methylation and clinical data, including 18 pediatric patients (<15 years), 195 AYA patients (15-39 years), and 1,355 adult patients (>39 years). Pediatric and AYA (P/AYA) tumors were combined and compared with adult tumors. The performance of established molecular classifiers and recurrence predictors, as well as differences in chromosomal copy number alterations were compared across age groups.Results While histologic grading was comparable between cohorts, P/AYA tumors displayed significantly fewer aggressive molecular groups and lower frequencies of chromosomal arm losses, including 1p, 6q, and 14q. The adult-trained recurrence predictor failed in the P/AYA population (AUC 0.57), despite similar score distributions. Retraining the model on an age-specific cohort using an identical analytic framework improved performance (AUC 0.79) and enabled effective stratification of progression-free survival (P = 0.00054). Importantly, 1p loss retained prognostic significance within the P/AYA group, supporting its clinical utility.Conclusions Molecular tools developed in adult-dominant cohorts do not generalize to younger patients due to both biological divergence and exclusion from model development. These findings underscore the need for age-specific molecular frameworks and highlight the imperative of including P/AYA populations in precision neuro-oncology research to ensure lifespan-equitable care.

Glioblastoma (GBM) is the most common and lethal primary brain tumor, with a median survival of 15 months. Standard therapies often fail due to intra-tumoral heterogeneity and resistance mechanisms, largely driven by hyperactive RTK/RAS/MEK/ERK signaling. Capicua (CIC), a tumor suppressor and HMG box transcription factor, represses oncogenic ETV1/4/5 but is degraded by aberrant ERK signaling in GBM, enabling tumor progression. CIC forms a co-repressor complex with Yin Yang 1 (YY1), a transcription factor that can act as a repressor or activator depending on its binding partner. This complex is essential for suppressing oncogenic transcription however it is destabilized in GBM. p90 ribosomal S6 kinase (p90RSK), a downstream ERK effector, has been implicated in tumor progression and therapy resistance. We hypothesize that p90RSK contributes to MEK inhibitor resistance by disrupting the CIC/YY1 complex, sustaining oncogenic transcription. Western blotting, qPCR, immunoprecipitation, and chromatin immunoprecipitation (ChIP-qPCR) were used to assess p90RSK’s effect on CIC/YY1 and its regulation of oncogenic targets. Cell viability assays and xenograft models evaluated the therapeutic potential of combined MEK and p90RSK inhibition. MEK inhibition failed to restore CIC protein levels and did not suppress p90RSK activation, suggesting an alternative resistance mechanism. p90RSK destabilized the CIC/YY1 complex, leading to derepression of ETV1/4/5. Dual MEK and p90RSK inhibition restored CIC function, suppressed oncogenic transcription, and reduced tumor growth. Targeting p90RSK overcomes MEK inhibitor resistance by restoring CIC/YY1 function, highlights a promising therapeutic strategy for GBM.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a median survival of just 15 months following diagnosis. Standard therapies remain largely ineffective due to pronounced intra-tumoral heterogeneity and rapid development of resistance mechanisms, underscoring the need for novel therapeutic approaches. A hallmark of GBM is hyperactivation of the RTK/RAS/MEK/ERK signaling pathway, which drives tumor growth and progression. One critical downstream effector is Capicua (CIC), a tumor suppressor and HMG box transcription factor. Normally, CIC represses oncogenic transcription factors such as ETV1, ETV4, and ETV5. In GBM, sustained ERK activity leads to CIC degradation, lifting this repression and promoting oncogene expression. While this mechanism is well characterized, our data indicates that CIC degradation persists even when ERK is pharmacologically inhibited, suggesting alternative regulatory mechanisms. A phospho-kinase array identified p90 ribosomal S6 kinase (p90RSK), a downstream ERK effector, as significantly upregulated following ERK inhibition. Notably, p90RSK has been implicated in regulating CIC function and contributing to resistance in other malignancies. Additionally, CIC forms a co-repressor complex with Yin Yang 1 (YY1), a multifunctional transcription factor. The stability of this complex is essential for repressing oncogenic programs; however, hyperactive RTK signaling may destabilize it, potentially via p90RSK-mediated mechanisms. Biochemical, molecular, and in vivo assays, including Western blotting, qPCR, immunoprecipitation, ChIP-qPCR, cell viability assays, and xenograft models, were used to assess p90RSK’s role in regulating CIC/YY1 function and GBM growth. MEK inhibition alone failed to restore CIC levels or suppress p90RSK activation. p90RSK interacted with and destabilized the CIC/YY1 complex, derepressing ETV1/4/5. Dual MEK and p90RSK inhibition restored CIC function, suppressed oncogenic transcription, reduced GBM stem cell viability, and inhibited tumor growth. p90RSK decreases GBM sensitivity to MEK inhibitors by destabilizing the CIC/YY1 complex and sustaining oncogenic transcription. Dual inhibition restores CIC function, suppresses oncogenic programs, and enhances therapeutic efficacy in GBM.

CIC-rearranged sarcoma (CRS) is a rare disease driven by a specific fusion protein involving the CIC gene. The occurrence in the brain is 3% in all CRS patients, and these tumors frequently metastasize to the brain. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. Preliminary data show that CIC-DUX4/NUTM1 fusions activate JAK and its downstream effector STAT1/3. We hypothesize that JAK/STAT1/3 signaling cooperates with CIC-fusions to drive CIC-sarcomas by inducing ETV1/4/5 expression. Patient-derived CRS cell lines showed elevated levels of JAK1/STAT1/3 activation compared to fusion-negative sarcoma lines. Inhibition of JAK1 using Ruxolitinib and Solicitinib reduced STAT1/3 phosphorylation, downregulated ETV1/4/5 expression at both mRNA and protein levels, and diminished ETV5 promoter activity, cell proliferation, and tumorigenicity. Although the mechanism by which CIC-fusions activate oncogenic targets is still under investigation, histone acetylation appears to play a central role. STAT1/3 interacts with p300/CBP to enhance transcription, and STAT1 is necessary for p300 acetyltransferase activity. Ruxolitinib significantly reduced histone acetylation at ETV1/4/5 promoters in hMSC cells expressing CIC-DUX4/NUTM1, as well as in CRS cell lines. Unlike the p300 inhibitor C646, which causes global hypoacetylation, Ruxolitinib’s effects were promoter-specific, indicating its potential as a more targeted and less toxic therapeutic option. Importantly, we found that STAT1/3 binds to ETV1/4/5 promoters only in the presence of CIC-fusions. Luciferase assays confirmed that STAT1/3 alone cannot activate ETV5 transcription without CIC-fusions, revealing a novel cooperative mechanism. In vivo, Ruxolitinib treatment of CRS xenografts led to significant reductions in tumor volume, STAT1/3 activation, and ETV1/4/5 expression. These findings support JAK1/STAT1/3 inhibition as a promising therapeutic strategy for CRS and warrant further preclinical investigation.

HOX (homeobox) genes are virtually absent in healthy adult brains but are detected in malignant brain tumors, particularly gliomas. In 2021, the World Health Organization (WHO) classified adult-type diffuse gliomas into three distinct categories: astrocytomas (isocitrate dehydrogenase [IDH]-mutated), oligodendrogliomas (IDH-mutated and 1p/19q-deleted), and glioblastomas, IDH-wildtype (GBM). GBM is the most common and aggressive primary malignant tumor of the Central Nervous System (CNS), characterized by its high recurrence rate and rapid growth. Dysregulation of HOX genes is a well-established phenomenon in both solid and liquid malignancies, playing crucial roles in various fundamental characteristics of cancer, including GBM. In recent years, HOX genes have gained recognition not only as key regulators of tumor progression but also as potential biomarkers for predicting disease outcomes and as promising therapeutic targets for GBM. This review compiles the latest research on HOX genes in GBM, encompassing studies published before and after the 2021 WHO classification of CNS tumors. Our goal is to provide a comprehensive overview of key findings on the role of HOX gene clusters, which are groups of genes involved in regulating the development of the body plan along the anterior–posterior axis, in GBM initiation, progression, prognosis, and treatment response.

The receptor tyrosine kinase (RTK)/Ras/Raf/MEK/ERK signaling pathway is one of the most tumorigenic pathways in cancer, with its hyperactivation strongly linked to the aggressive nature of glioblastoma (GBM). Although extensive research has focused on developing therapeutics targeting this pathway, clinical success remains elusive due to the emergence of resistance mechanisms. This review investigates how inhibition of the RTK/Ras/Raf/MEK/ERK pathway alters transcription factors, contributing to acquired resistance mechanisms in GBM. It also highlights the critical role of transcription factor dysregulation in therapeutic resistance. Findings from key studies on the RTK/Ras/Raf/MEK/ERK pathway in GBM were synthesized to explore the role of transcription factor dysregulation in resistance to targeted therapies, radiation, and chemotherapy. The review highlights that transcription factors undergo significant dysregulation following RTK/Ras/Raf/MEK/ERK pathway inhibition, contributing to therapeutic resistance. Transcription factors are promising targets for overcoming treatment resistance in GBM, with cotreatment strategies combining RTK/Ras/Raf/MEK/ERK pathway inhibitors and transcription factor-targeted therapies presenting a novel approach. Despite the challenges of targeting complex structures and interactions, advancements in drug development and precision technologies hold great potential. Continued research is essential to refine these strategies and improve outcomes for GBM and other aggressive cancers.

CIC-rearranged sarcoma (CRS) is a rare and aggressive malignancy driven by CIC fusion proteins, primarily CIC-DUX4, with a propensity for brain metastases. Other fusion partners, such as CIC-NUTM1, have been identified in a subset of pediatric primitive neuroectodermal tumors. Native CIC functions as a transcriptional repressor of genes downstream of the RTK/Ras/ERK pathway, including ETV1/4/5, which play a key role in CRS oncogenesis. Interestingly, CIC fusions convert a transcriptional repressor into an activator, though the precise oncogenic mechanisms remain unclear. Our data reveal that CIC fusions activate the JAK1/STAT1/3 pathway, leading to increased mRNA and protein expression of ETV1/4/5. Patient-derived CRS cell lines (NCC-SCC-89/C) exhibit high JAK1/STAT1/3 activation compared to fusion-negative sarcoma lines. JAK1 inhibition with Solcitinib or Ruxolitinib suppresses STAT1/3 phosphorylation, ETV1/4/5 expression, cell proliferation, and tumorigenicity. DUX4’s role in histone acetylation and STAT1’s interaction with the p300/CBP complex suggest a transcriptional activation mechanism. We demonstrate that CIC fusions enhance STAT1/3 binding to ETV1/4/5 promoters, while JAK1 inhibition reduces histone acetylation and suppresses transcription. In vivo, Ruxolitinib significantly reduced tumor volume, STAT1/3 activation, and ETV1/4/5 expression in CRS xenografts. Additionally, Ruxolitinib combined with doxorubicin exhibited synergistic effects, further reducing tumor volume. In conclusion, we identify JAK1/STAT1/3 as a key oncogenic driver in CRS, promoting histone acetylation and ETV1/4/5 upregulation, leading to increased cellular proliferation. These findings support JAK1/STAT1/3 inhibition as a promising therapeutic strategy for CRS and warrant further preclinical investigation.

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical GBM such as necrosis, high levels of mitosis and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.

Abstract Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. Purpose & Hypothesis: To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway. METHODS: Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation. RESULTS: Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation. CONCLUSIONS: The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.

Abstract CIC-rearranged sarcoma (CRS) is an rare disease driven by a specific fusion protein involving the CIC gene. Occurrence in the brain is 3% in all CRS patients. The native CIC protein is a transcriptional repressor of the (RTK)/Ras/ERK signaling pathway, which is one of the most tumorigenic pathways in cancer. The most common rearrangement is with the double homeobox 4 (DUX4) transcription factor (CIC-DUX4), and others, such as CIC-NUTM1 fusions, have been identified in a subset of pediatric primitive neuroectodermal tumors. However, the molecular mechanisms by which CIC-fusions drive CRS remain unknown. Preliminary data shows that CIC-DUX4/NUTM1 fusions activate JAK and its downstream effector STAT1/3. We hypothesize that the JAK/STAT1/3 signal transduction pathway cooperates with CIC-fusions to induce the expression of oncogenic transcription factors ETV1/4/5 and drive these sarcomas. We show high levels of JAK1/STAT1/3 activation in patient-derived CRS cell lines (NCC-SCC-89/C) as compared to other sarcoma cell lines without the fusion. JAK1 inhibition using Solicitinib and Ruxolitinib reduced phosphorylation of STAT1/3 as well as protein and mRNA expression of ETV1/4/5, promoter activity of ETV5, cell proliferation and tumorgenicity. Interestingly DUX4 is involved in histone acetylation and STAT1 has been shown to activate p300/CBP complex which lead us to evaluating the role of STAT1 in the gene activation CRS. We elucidate mechanistically that the fusion proteins increase binding of STAT1/3 at the promoters of ETV 1/4/5. Importantly, JAK1 inhibition effectively reduces histone acetylation induced by CIC-fusions at the promoters of ETV1/4/5. To evaluate the pre-clinical effect of targeting the JAK1/STAT1/3 pathway, A CRS cell line (NCC-SCC-89/C) were grafted into NSG mice. Ruxolitinib treatment significantly reduced tumor volume, STAT activation, and ETV1/4/5. In conclusion, we show that the JAK1/STAT1/3 pathway plays a critical role in cooperating with CIC-fusions to drive CRS, providing insight into potential therapeutic avenues for these aggressive tumors.

PDF file - 609K, Dasatinib, but not imatinib, markedly inhibits JMML colony formation in the absence or presence of GM-CSF

PDF file - 4783K, Dasatinib inhibits growth of primary JMML cells from patients with CBL, NF1, NRAS, or PTPN11 mutation in a dose dependent manner