THOC1 complexes with SIN3A to regulate R-loops and promote glioblastoma progression.

Glioblastoma (GBM), the most common and aggressive malignant brain tumor in adults, has a median survival of 21 months. To identify drivers of GBM proliferation, we conducted a CRISPR-knockout screen, which revealed THO Complex 1 (THOC1) as a key driver. Knocking down THOC1 significantly reduced GBM cell viability across patient-derived xenograft (PDX) lines, enhancing survival (p<0.01) in primary PDX models. Conversely, overexpressing THOC1 in non-cancerous cells bolstered viability, decreasing survival and causing tumor engraftment in vivo (p<0.01). Further investigation revealed THOC1's robust interaction with SIN3A, a histone deacetylase complex. Histone deacetylation has been previously shown to prevent the buildup of R-loops, structures that form normally during transcription but can be lethal in excess. We found that THOC1-knockdown leads to elevated R-loop levels and reduced histone deacetylation levels. Next, to understand the networks specifically regulated by THOC1-mediated R-loop prevention, we conducted unbiased RNA-sequencing on control and THOC1-knockdown GBM cells. We found that THOC1's role in R-loop prevention primarily affects telomeres, critical regions for cell replication. We further show that THOC1-knockdown results in significantly increased telomeric R-loop levels and shortened telomeres. Ultimately, this study suggests that targeting THOC1 shows promise as a therapeutic strategy to disrupt the delicate R-loop landscape and undermine GBM's replicative potential.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress.

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

B cell-based therapy produces antibodies that inhibit glioblastoma growth.

Glioblastoma (GBM) is a highly aggressive and malignant brain tumor with limited therapeutic options and a poor prognosis. Despite current treatments, the invasive nature of GBM often leads to recurrence. A promising alternative strategy is to harness the potential of the immune system against tumor cells. Our previous data showed that the BVax (B cell-based vaccine) can induce therapeutic responses in preclinical models of GBM. In this study, we aimed to characterize the antigenic reactivity of BVax-derived Abs and evaluate their therapeutic potential. We performed immunoproteomics and functional assays in murine models and samples from patients with GBM. Our investigations revealed that BVax distributed throughout the GBM tumor microenvironment and then differentiated into Ab-producing plasmablasts. Proteomics analyses indicated that the Abs produced by BVax had unique reactivity, predominantly targeting factors associated with cell motility and the extracellular matrix. Crucially, these Abs inhibited critical processes such as GBM cell migration and invasion. These findings provide valuable insights into the therapeutic potential of BVax-derived Abs for patients with GBM, pointing toward a novel direction for GBM immunotherapy.

Genomic analysis of human brain metastases treated with stereotactic radiosurgery reveals unique signature based on treatment failure.

Stereotactic radiosurgery (SRS) has been shown to be efficacious for the treatment of limited brain metastasis (BM); however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis on resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in human, biological evidence of differential effects of SRS across BM's.

ARF4-mediated retrograde trafficking as a driver of chemoresistance in glioblastoma.

BACKGROUND: Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear. METHODS: To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor. RESULTS: Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (P < .01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU-57788, in mice with a recurrent PDX line resulted in prolonged survival (P < .01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. CONCLUSIONS: Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

Metixene is an incomplete autophagy inducer in preclinical models of metastatic cancer and brain metastases.

A paucity of chemotherapeutic options for metastatic brain cancer limits patient survival and portends poor clinical outcomes. Using a CNS small-molecule inhibitor library of 320 agents known to be blood-brain barrier permeable and approved by the FDA, we interrogated breast cancer brain metastasis vulnerabilities to identify an effective agent. Metixene, an antiparkinsonian drug, was identified as a top therapeutic agent that was capable of decreasing cellular viability and inducing cell death across different metastatic breast cancer subtypes. This agent significantly reduced mammary tumor size in orthotopic xenograft assays and improved survival in an intracardiac model of multiorgan site metastases. Metixene further extended survival in mice bearing intracranial xenografts and in an intracarotid mouse model of multiple brain metastases. Functional analysis revealed that metixene induced incomplete autophagy through N-Myc downstream regulated 1 (NDRG1) phosphorylation, thereby leading to caspase-mediated apoptosis in both primary and brain-metastatic cells, regardless of cancer subtype or origin. CRISPR/Cas9 KO of NDRG1 led to autophagy completion and reversal of the metixene apoptotic effect. Metixene is a promising therapeutic agent against metastatic brain cancer, with minimal reported side effects in humans, which merits consideration for clinical translation.

Ribonucleotide reductase regulatory subunit M2 drives glioblastoma TMZ resistance through modulation of dNTP production.

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 (RRM2), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients' tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production.

Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Under the Phase-II Clinical Trial (NCT03398694) Reveals DNA Damage Repair at the Peripheral Tumor Edge.

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

Endoplasmic Reticulum Stress in the Brain Tumor Immune Microenvironment.

Immunotherapy has emerged as a powerful strategy for halting cancer progression. However, primary malignancies affecting the brain have been exempt to this success. Indeed, brain tumors continue to portend severe morbidity and remain a globally lethal disease. Extensive efforts have been directed at understanding how tumor cells survive and propagate within the unique microenvironment of the central nervous system (CNS). Cancer genetic aberrations and metabolic abnormalities provoke a state of persistent endoplasmic reticulum (ER) stress that in turn promotes tumor growth, invasion, therapeutic resistance, and the dynamic reprogramming of the infiltrating immune cells. Consequently, targeting ER stress is a potential therapeutic approach. In this work, we provide an overview of how ER stress response is advantageous to brain tumor development, discuss the significance of ER stress in governing antitumor immunity, and put forth therapeutic strategies of regulating ER stress to augment the effect of immunotherapy for primary CNS tumors.

Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma.

PURPOSE: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. EXPERIMENTAL DESIGN: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. RESULTS: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. CONCLUSIONS: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

Circadian Regulator CLOCK Drives Immunosuppression in Glioblastoma.

The symbiotic interactions between cancer stem cells and the tumor microenvironment (TME) are critical for tumor progression. However, the molecular mechanism underlying this symbiosis in glioblastoma (GBM) remains enigmatic. Here, we show that circadian locomotor output cycles kaput (CLOCK) and its heterodimeric partner brain and muscle ARNT-like 1 (BMAL1) in glioma stem cells (GSC) drive immunosuppression in GBM. Integrated analyses of the data from transcriptome profiling, single-cell RNA sequencing, and TCGA datasets, coupled with functional studies, identified legumain (LGMN) as a direct transcriptional target of the CLOCK-BMAL1 complex in GSCs. Moreover, CLOCK-directed olfactomedin-like 3 (OLFML3) upregulates LGMN in GSCs via hypoxia-inducible factor 1-alpha (HIF1α) signaling. Consequently, LGMN promotes microglial infiltration into the GBM TME via upregulating CD162 and polarizes infiltrating microglia toward an immune-suppressive phenotype. In GBM mouse models, inhibition of the CLOCK-OLFML3-HIF1α-LGMN-CD162 axis reduces intratumoral immune-suppressive microglia, increases CD8+ T-cell infiltration, activation, and cytotoxicity, and synergizes with anti-programmed cell death protein 1 (anti-PD-1 therapy). In human GBM, the CLOCK-regulated LGMN signaling correlates positively with microglial abundance and poor prognosis. Together, these findings uncover the CLOCK-OLFML3-HIF1α-LGMN axis as a molecular switch that controls microglial biology and immunosuppression, thus revealing potential new therapeutic targets for patients with GBM.

Checkpoint: Inspecting the barriers in glioblastoma immunotherapies.

Despite an aggressive standard of care involving radiation therapy, temozolomide-based chemotherapy, and surgical resection, glioblastoma multiforme (GBM) continues to exhibit very high recurrence and mortality rates partly due to the highly plastic and heterogenous nature of the tumor. In recent years, activation of the immune system has emerged as a promising strategy in cancer therapies. However, despite recent successes in other fields, immunotherapeutic approaches continue to encounter challenges in GBM. In this review, we first discuss immunotherapies targeting the most well-studied immune checkpoint proteins, CTLA-4 and PD-1, followed by discussions on therapies targeting immune-stimulatory molecules and secreted metabolic enzymes. Finally, we address the major challenges with immunotherapy in GBM and the potential for combination and neoadjuvant immunotherapies to tip the scales in the fight against glioblastoma.

Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer.

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood-brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Spelling Out CICs: A Multi-Organ Examination of the Contributions of Cancer Initiating Cells' Role in Tumor Progression.

Tumor invasion and metastasis remain the leading causes of mortality for patients with cancer despite current treatment strategies. In some cancer types, recurrence is considered inevitable due to the lack of effective anti-metastatic therapies. Recent studies across many cancer types demonstrate a close relationship between cancer-initiating cells (CICs) and metastasis, as well as general cancer progression. First, this review describes CICs' contribution to cancer progression. Then we discuss our recent understanding of mechanisms through which CICs promote tumor invasion and metastasis by examining the role of CICs in each stage. Finally, we examine the current understanding of CICs' contribution to therapeutic resistance and recent developments in CIC-targeting drugs. We believe this understanding is key to advancing anti-CIC clinical therapeutics.

Neural stem cell delivery of an oncolytic adenovirus in newly diagnosed malignant glioma: a first-in-human, phase 1, dose-escalation trial.

BACKGROUND: Malignant glioma is the most common and lethal primary brain tumour, with dismal survival rates and no effective treatment. We examined the safety and activity of NSC-CRAd-S-pk7, an engineered oncolytic adenovirus delivered by neural stem cells (NSCs), in patients with newly diagnosed high-grade glioma. METHODS: This was a first-in-human, open-label, phase 1, dose-escalation trial done to determine the maximal tolerated dose of NSC-CRAd-S-pk7, following a 3 + 3 design. Patients with newly diagnosed, histologically confirmed, high-grade gliomas (WHO grade III or IV) were recruited. After neurosurgical resection, NSC-CRAd-S-pk7 was injected into the walls of the resection cavity. The first patient cohort received a dose starting at 6·25 × 1010 viral particles administered by 5·00 × 107 NSCs, the second cohort a dose of 1·25 × 1011 viral particles administered by 1·00 × 108 NSCs, and the third cohort a dose of 1·875 × 1011 viral particles administered by 1·50 × 108 NSCs. No further dose escalation was planned. Within 10-14 days, treatment with temozolomide and radiotherapy was initiated. Primary endpoints were safety and toxicity profile and the maximum tolerated dose for a future phase 2 trial. All analyses were done in all patients who were included in the trial and received the study treatment and were not excluded from the study. Recruitment is complete and the trial is finished. The trial is registered with ClinicalTrials.gov, NCT03072134. FINDINGS: Between April 24, 2017, and Nov 13, 2019, 12 patients with newly diagnosed, malignant gliomas were recruited and included in the safety analysis. Histopathological evaluation identified 11 (92%) of 12 patients with glioblastoma and one (8%) of 12 patients with anaplastic astrocytoma. The median follow-up was 18 months (IQR 14-22). One patient receiving 1·50 × 108 NSCs loading 1·875 × 1011 viral particles developed viral meningitis (grade 3) due to the inadvertent injection of NSC-CRAd-S-pk7 into the lateral ventricle. Otherwise, treatment was safe as no formal dose-limiting toxicity was reached, so 1·50 × 108 NSCs loading 1·875 × 1011 viral particles was recommended as a phase 2 trial dose. There were no treatment-related deaths. The median progression-free survival was 9·1 months (95% CI 8·5-not reached) and median overall survival was 18·4 months (15·7-not reached). INTERPRETATION: NSC-CRAd-S-pk7 treatment was feasible and safe. Our immunological and histopathological findings support continued investigation of NSC-CRAd-S-pk7 in a phase 2/3 clinical trial. FUNDING: US National Institutes of Health.

De novo purine biosynthesis is a major driver of chemoresistance in glioblastoma.

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

Molecular Classification of Gliomas is Associated with Seizure Control: A Retrospective Analysis.

Classically, histologic grading of gliomas has been used to predict seizure association, with low-grade gliomas associated with an increased incidence of seizures compared to high-grade gliomas. In 2016, WHO reclassified gliomas based on histology and molecular characteristics. We sought to determine whether molecular classification of gliomas is associated with preoperative seizure presentation and/or post-operative seizure control across multiple glioma subtypes. All gliomas operated at our institution from 2007 to 2017 were identified based on ICD 9 and 10 billing codes and were retrospectively assessed for molecular classification of the IDH1 mutation, and 1p/19q codeletion. Logistic regression models were performed to assess associations of seizures at presentation as well as post-operative seizures with IDH status and the new WHO integrated classification. Our study included 376 patients: 82 IDH mutant and 294 IDH wildtype. The presence of IDH mutation was associated with seizures at presentation [OR 3.135 (1.818-5.404), p < 0.001]. IDH-mutant glioblastomas presented with seizures less often than other IDH-mutant glioma subtypes grade II and III [OR 0.104 (0.032-0.340), p < 0.001]. IDH-mutant tumors were associated with worse post-operative seizure outcomes, demonstrated by Engel Class [OR 2.666 (1.592-4.464), p < 0.001]. IDH mutation in gliomas is associated with an increased risk of seizure development and worse post-operative seizure control, in all grades except for GBM.

LNX1 Modulates Notch1 Signaling to Promote Expansion of the Glioma Stem Cell Population during Temozolomide Therapy in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a 100% recurrence rate and 21-month median survival. Our lab and others have shown that GBM contains a subpopulation of glioma stem cells (GSCs) that expand during chemotherapy and may contribute to therapeutic resistance and recurrence in GBM. To investigate the mechanism behind this expansion, we applied gene set expression analysis (GSEA) to patient-derived xenograft (PDX) cells in response to temozolomide (TMZ), the most commonly used chemotherapy against GBM. Results showed significant enrichment of cancer stem cell and cell cycle pathways (False Discovery Rate (FDR) < 0.25). The ligand of numb protein 1 (LNX1), a known regulator of Notch signaling by targeting negative regulator Numb, is strongly upregulated after TMZ therapy (p < 0.0001) and is negatively correlated with survival of GBM patients. LNX1 is also upregulated after TMZ therapy in multiple PDX lines with concomitant downregulations in Numb and upregulations in intracellular Notch1 (NICD). Overexpression of LNX1 results in Notch1 signaling activation and increased GSC populations. In contrast, knocking down LNX1 reverses these changes, causing a significant downregulation of NICD, reduction in stemness after TMZ therapy, and resulting in more prolonged median survival in a mouse model. Based on this, we propose that during anti-GBM chemotherapy, LNX1-regulated Notch1 signaling promotes stemness and contributes to therapeutic resistance.