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

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<0.01) 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, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. CONCLUSION: 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.

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.

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.

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.

Chemotherapeutic Stress Induces Transdifferentiation of Glioblastoma Cells to Endothelial Cells and Promotes Vascular Mimicry.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor affecting adults, with a median survival of approximately 21 months. One key factor underlying the limited efficacy of current treatment modalities is the remarkable plasticity exhibited by GBM cells, which allows them to effectively adapt to changes induced by anticancer therapeutics. Moreover, GBM tumors are highly vascularized with aberrant vessels that complicate the delivery of antitumor agents. Recent research has demonstrated that GBM cells have the ability to transdifferentiate into endothelial cells (ECs), illustrating that GBM cells may use plasticity in concert with vascularization leading to the creation of tumor-derived blood vessels. The mechanism behind this transdifferentiation, however, remains unclear. Here, we show that treatment with temozolomide (TMZ) chemotherapy induces time-dependent expression of markers for glioma stem cells (GSCs) and immature and mature ECs. In addition, GBM tumors growing as orthotopic xenografts in nude mice showed increased expression of GSC and EC markers after TMZ treatment. Ex vivo FACS analysis showed the presence of immature and mature EC populations. Furthermore, immunofluorescence analysis revealed increased tumor-derived vessels in TMZ-recurrent tumors. Overall, this study identifies chemotherapeutic stress as a new driver of transdifferentiation of tumor cells to endothelial cells and highlights cellular plasticity as a key player in therapeutic resistance and tumor recurrence.

Activation of Dopamine Receptor 2 Prompts Transcriptomic and Metabolic Plasticity in Glioblastoma.

Glioblastoma (GBM) is one of the most aggressive and lethal tumor types. Evidence continues to accrue indicating that the complex relationship between GBM and the brain microenvironment contributes to this malignant phenotype. However, the interaction between GBM and neurotransmitters, signaling molecules involved in neuronal communication, remains incompletely understood. Here we examined, using human patient-derived xenograft lines, how the monoamine dopamine influences GBM cells. We demonstrate that GBM cells express dopamine receptor 2 (DRD2), with elevated expression in the glioma-initiating cell (GIC) population. Stimulation of DRD2 caused a neuron-like hyperpolarization exclusively in GICs. In addition, long-term activation of DRD2 heightened the sphere-forming capacity of GBM cells, as well as tumor engraftment efficiency in both male and female mice. Mechanistic investigation revealed that DRD2 signaling activates the hypoxia response and functionally alters metabolism. Finally, we found that GBM cells synthesize and secrete dopamine themselves, suggesting a potential autocrine mechanism. These results identify dopamine signaling as a potential therapeutic target in GBM and further highlight neurotransmitters as a key feature of the pro-tumor microenvironment.SIGNIFICANCE STATEMENT This work offers critical insight into the role of the neurotransmitter dopamine in the progression of GBM. We show that dopamine induces specific changes in the state of tumor cells, augmenting their growth and shifting them to a more stem-cell like state. Further, our data illustrate that dopamine can alter the metabolic behavior of GBM cells, increasing glycolysis. Finally, this work demonstrates that GBM cells, including tumor samples from patients, can synthesize and secrete dopamine, suggesting an autocrine signaling process underlying these results. These results describe a novel connection between neurotransmitters and brain cancer, further highlighting the critical influence of the brain milieu on GBM.