Temozolomide Resistance Research Articles

Temozolomide-Promoted MGMT Transcription Contributes to Chemoresistance by Activating the ERK Signalling Pathway in Malignant Melanoma.

Tumour cells possess a multitude of chemoresistance mechanisms, which could plausibly contribute to the ineffectiveness of chemotherapy. O6-methylguanine-DNA methyltransferase (MGMT) is an important effector protein associated with Temozolomide (TMZ) resistance in various tumours. To some extent, the expression level of MGMT determines the sensitivity of cells to TMZ, but the mechanism of its expression regulation has not been fully elucidated. Cultured malignant melanoma cell lines A375 and Sk-MEL28 were employed. A luciferase assay was used to detect the transcriptional activity of the MGMT promoter. Western blotting was used to compare the expression levels of phosphorylated ERK1/2 (P-ERK1/2) after TMZ treatment. Immunofluorescent staining was used to detect TMZ-induced DNA damage protein levels. The sensitivity of melanoma cells to TMZ was detected by MTT assay and animal experiments. The expression of MGMT mRNA was tested by Quantitative real-time PCR (RT-qPCR). Flow cytometry was used to measure the apoptosis of TMZ-treated cells. TMZ enhanced the transcription of MGMT through activating the ERK pathway. ERK inhibitors U0126 and vemurafenib (vMF) inhibited the TMZ induced transcription of MGMT. The expression of MGMT and p-ERK1/2 was closely related in human MM tissues. vMF increased the sensitivity of melanoma (MM) to TMZ invitro and invivo through downregulating MGMT and promoting the TMZ induced DNA damage in MM. TMZ-promoted MGMT transcription contributed to instinctive chemoresistance by activating the ERK signalling pathway in malignant melanoma. Our study indicates that the use of the ERK inhibitor in combination with TMZ could potentially enhance the effectiveness of clinical treatment for malignant melanoma.

Glycosylated Delphinidins Decrease Chemoresistance to Temozolomide by Regulating NF-κB/MGMT Signaling in Glioblastoma

Glioblastoma (GB) is a highly malignant brain tumor with a poor prognosis, with a median survival of only 14.6 months despite aggressive treatments. Resistance to chemotherapy, particularly temozolomide (TMZ), is a significant challenge. The DNA repair enzyme MGMT and glioblastoma stem cells (GSCs) often mediate this resistance. Recent studies highlight the therapeutic potential of natural compounds, particularly delphinidins, found in deep purple berries. Delphinidins are known for their ability to inhibit NF-κB signaling, a critical pathway for GB progression, chemoresistance, and MGMT expression. Our research demonstrates that glycosylated delphinidins have potential adjuvant use in the treatment of GB, offering a promising natural strategy to combat TMZ resistance. Specifically, we observed that delphinidin 3,5 di-glucoside has potent anticancer effects when used alone. Meanwhile, delphinidin 3 glucoside acted in synergy with temozolomide to decrease cell viability, highlighting its potential as an adjuvant. It also exerted a faster and more sustained inhibition of NF-κB, highlighting its potential for long-lasting therapeutic effects. These findings open new avenues for targeted therapies against glioblastoma, particularly to overcome treatment resistance.

Adiponectin facilitates the cell cycle, inhibits cell apoptosis and induces temozolomide resistance in glioblastoma via the Akt/mTOR pathway.

Adiponectin (ADN) regulates DNA synthesis, cell apoptosis and cell cycle to participate in the pathology and progression of glioblastoma. The present study aimed to further explore the effect of ADN on temozolomide (TMZ) resistance in glioblastoma and the underlying mechanism of action. Glioblastoma cell lines (U251 and U87-MG cells) were treated with ADN and TMZ at different concentrations; subsequently, 3.0 µg/ml ADN and 1.0 mM TMZ were selected as the optimal concentrations for the experimental conditions. LY294002 (a PI3K inhibitor) was added to ADN or ADN + TMZ-treated glioblastoma cell lines. Cell growth rate was determined using the Cell Counting Kit-8 assay, the apoptotic rate and cell cycle were evaluated using Annexin V/propidium iodide and cell cycle assays, and p-Akt (Thr308), p-Akt (Ser473), Akt, p-mTOR, c-caspase 3, caspase 3, Bax, cyclin B1 and cyclin D1 expression was determined by western blotting. Adiponectin receptor (ADIPOR) 1 and ADIPOR2 were expressed in glioblastoma cell lines. The glioblastoma cell line growth rate was increased by ADN in a concentration- and time-dependent manner. ADN inhibited glioblastoma cell line apoptosis and facilitated cell cycle. Of note, ADN activated the Akt/mTOR pathway and the addition of LY294002 reversed the effect of ADN, indicating that ADN activated the Akt/mTOR pathway to suppress apoptosis and promote cell cycle in glioblastoma cell lines. Notably, TMZ inhibited glioblastoma cell line growth, promoted apoptosis and increased G2 phase cell cycle arrest. However, the addition of ADN reversed the effect of TMZ in glioblastoma cell lines, disclosing that ADN induced TMZ resistance. Markedly, ADN-mediated TMZ resistance was further attenuated by LY294002, suggesting that ADN activated the Akt/mTOR pathway to induce TMZ resistance in glioblastoma cell lines. In conclusion, ADN activated the Akt/mTOR pathway to facilitate cell cycle, inhibit cell apoptosis and induce TMZ resistance in glioblastoma.

Development and experimental validation of dephosphorylation-related biomarkers to assess prognosis and immunotherapeutic response in gliomas.

Gliomas are common aggressive brain tumors with poor prognosis. Dephosphorylation-related biomarkers are in a void in gliomas. This study aims to construct a validated prognostic risk model for dephosphorylation, which will provide new directions for clinical treatment, prognostic assessment, and temozolomide (TMZ) resistance in glioma patients. Screening dephosphorylation-related genes (DRGs) and transcriptome expression data from The Cancer Genome Atlas (TCGA), Molecular signatures database (MSigDB) and constructing risk scoring models. Kaplan-Meier (K-M), nomogram and ROC curve were used to assess the predictive efficacy of the model. Gene set enrichment analysis (GSEA), immune cell infiltration, immunotherapy response and chemotherapeutic drug sensitivity analysis were performed in this study. The correlation between chemotherapeutic drugs and the half maximal inhibitory concentration (IC50) values of 12 DRGs was analyzed. Cell division cycle 25A (CDC25A) and TMZ were screened and verified by experiments. Quantitative Real-Time PCR (qRT-PCR) detection of mRNA expression of 12 genes in human normal glial cells and two glioma cell lines. Transfection techniques overexpressed and knocked down CDC25A. qRT-PCR and Western Blot (WB) were used to detect the mRNA and protein expression levels of CDC25A. Subsequently, verify the effect of CDC25A on TMZ resistance in glioma cells. The model established in this study was able to accurately predict the prognosis of glioma patients. Besides, there were significant differences in GSEA, immune cell infiltration, immunotherapeutic response and chemotherapeutic drug sensitivity analysis between glioma patients in the high and low risk groups. The results of CCK8 experiments showed that overexpression of CDC25A increased the susceptibility of U251 and LN229 cells to TMZ, and knockdown of CDC25A attenuated the susceptibility of U251 and LN229 cells to TMZ.

ADAR1 Promotes the Progression and Temozolomide Resistance of Glioma Through p62-Mediated Selective Autophagy.

Resistance to temozolomide (TMZ) remains is an important cause of treatment failure in patients with glioblastoma multiforme (GBM). ADAR1, as a member of the ADAR family, plays an important role in cancer progression and chemotherapy resistance. However, the mechanism by which ADAR1 regulates GBM progression and TMZ resistance is still unclear. We first constructed stable transfected strains in which ADAR1 was knocked down and overexpressed to investigate the effect of ADAR1 on the first-line glioma chemotherapy drug TMZ. Subsequently, we validated that ADAR1 induces autophagy activation and used autophagy inhibitors to suppress autophagy, demonstrating that ADAR1 enhances TMZ resistance through autophagy. We further knocked down p62 (SQSTM1) based on the overexpression of ADAR1, and the results showed that ADAR1 regulates selective autophagy through the p62 regulation. Finally, we demonstrated through mutations at both edited and nonedited sites that ADAR1 regulates selective autophagy in an edited dependent way. Further analysis showed that in the presence of TMZ, elevated ADAR1 promoted TMZ induced autophagy activation. Further research revealed that ADAR1 enhances TMZ resistance through p62-mediated selective autophagy. Further, ADAR1 regulates selective autophagy in an edited dependent way. Our results indicate a relationship between ADAR1 levels and the response of glioma patients to TMZ treatment. We found that the expression of ADAR1 is upregulated in GBM and is associated with tumor grade and TMZ resistance. Elevated expression of ADAR1 predicts poor prognosis in GBM patients and promotes tumor growth invivo or invitro.

PSMC2 promotes resistance against temozolomide in glioblastoma via suppressing JNK-mediated autophagic cell death.

Temozolomide is universally used to treat glioblastoma due to its unique ability to cross the blood-brain barrier and inhibit tumor growth through DNA alkylation. However, over time, the inevitable emergence of resistance to temozolomide impedes successful treatment of this cancer. As a result, there is an urgent need to identify new therapeutic targets to improve treatment outcomes for this malignancy. In this work, acquired temozolomide-resistant glioblastoma cell lines LN18 (LN18-TR) and T98G (T98G-TR) exhibited stronger aggressiveness and lower endoplasmic reticulum (ER) stress than their parental cells.. Besides, temozolomide resistance was associated with elevated proteasome activity that suppressed ER stress, which was restored upon inhibition of the proteasome with MG132. Specifically, our study revealed that the 19S proteasomal regulatory subunit PSMC2, which was overexpressed in adapted temozolomide-resistant glioblastoma cells, reduced pro-death autophagy and decreased temozolomide sensitivity in parental cells when overexpressed. While autophagy increased in parental cells following temozolomide treatment, it was not elevated in temozolomide-resistant glioblastoma cells. Genetic suppression of PSMC2 triggered the JNK signalling pathway causing phosphorylation of BCL2, allowing Beclin1 to be released from the BCL2-Beclin1 complex. This boosted autophagosome nucleation, increased pro-death autophagy, and restored apoptosis in temozolomide-resistant glioblastoma cells. Finally, targeting PSMC2 provided a unique method for interrupting autophagy-mediated ER stress maintenance and temozolomide resistance in glioblastoma.

A novel nuclear RNA HSD52 scaffolding NONO/SFPQ complex modulates DNA damage repair to facilitate temozolomide resistance.

Temozolomide (TMZ) is used in the treatment of glioblastoma (GBM). However, the primary obstacle remains the emergence of TMZ chemotherapy resistance. NONO and SFPQ are multifunctional nuclear proteins involved in genome stability and gene regulation. However, the specific role of NONO and SFPQ in TMZ resistance of GBM remains to be explored. RIP-chip and RNA microarray of TMZ-resistant and parental cells were performed for the gain of HSD52. The effects of HSD52 on TMZ resistance were investigated through in vitro assays, intracranial xenograft and GBM organoid models. The underlying mechanisms were explored by DNA methylation chip, RIP, RNA pulldown assays, among others. GBM clinical samples were rolled in to investigate the clinical significance of HSD52. We identified a novel non-coding RNA, HSD52, that was highly expressed in TMZ-resistant GBM and facilitated the interaction between NONO and SFPQ. H3 ubiquitination attenuation and reduced DNMT1 recruitment increased HSD52 transcription via DNA hypo-methylation. HSD52 formed an RNA duplex with UFL1 mRNA, thereby promoting NONO/SFPQ complex binding to UFL1 mRNA and enhancing its stability, and then contributed to TMZ resistance through activating ATM signaling pathway. In vivo xenograft and GBM organoid models showed significant repression in tumor growth after HSD52 knockout with TMZ treatment. In GBM clinical samples, HSD52 was responsible for the malignant progression and TMZ resistance. Our results revealed that HSD52 could serve as a promising therapeutic target to overcome TMZ resistance, improving the clinical efficacy of TMZ chemotherapy in GBM.

Comprehensive whole-genome sequencing reveals origins of mutational signatures associated with aging, mismatch repair deficiency and temozolomide chemotherapy.

In a comprehensive study to decipher the multi-layered response to the chemotherapeutic agent temozolomide (TMZ), we analyzed 427 genomes and determined mutational patterns in a collection of ∼40 isogenic DNA repair-deficient human TK6 lymphoblast cell lines. We first demonstrate that the spontaneous mutational background is very similar to the aging-associated mutational signature SBS40 and mainly caused by polymerase zeta-mediated translesion synthesis (TLS). MSH2-/- mismatch repair (MMR) knockout in conjunction with additional repair deficiencies uncovers cryptic mutational patterns. We next report how distinct mutational signatures are induced by TMZ upon sequential inactivation of DNA repair pathways, mirroring the acquisition of chemotherapy resistance by glioblastomas. The most toxic adduct induced by TMZ, O6-meG, is directly repaired by the O6-methylguanine-DNA methyltransferase (MGMT). In MGMT-/- cells, MMR leads to cell death and limits mutagenesis. MMR deficiency results in TMZ resistance, allowing the accumulation of ∼105 C>T substitutions corresponding to signature SBS11. Under these conditions, N3-methyladenine (3-meA), processed by base excision repair (BER), limits cell survival. Without BER, 3-meA is read through via error-prone TLS, causing T>A substitutions but not affecting survival. Blocking BER after abasic site formation results in large deletions and TMZ hypersensitization. Our findings reveal potential vulnerabilities of TMZ-resistant tumors.

Higher isoform of hnRNPA1 confer Temozolomide resistance in U87MG & LN229 glioma cells.

Gliblastoma is a malignant brain tumor; despite available treatment modalities, the tumor reoccurrence rate persist in the currently prescribed Temozolomide chemotherapy. Study aimed to study the inquisitive role of RNA binding splice factor protein hnRNPA1 in promoting glioma resistance against Temozolomide drug and therapeutic insights. In this study two non-expressing O6-methylguanine-DNA methyltransferase (MGMT) glioma cell lines U87MG & LN229. U87MG cells were grown in Temozolomide from 50μM upto 400μM & LN229 cells grown upto 200μM, till then both these cells acquired Temozolomide resistance. Both of these cells were grown & maintained continously in its highest dose of Temozolomide (TMZ). Splice factor protein SF2/ASF1 was functionally correlated with abundance of hnRNPA1 protein in Temozolomide (TMZ) resistant cells using its specific siRNA transfection approach, in detrmining SF2/ASF1 mediated hnRNPA1 splicing and Temozolomide resistant reversal. U87MG TMZ resistance, results an increase in the expression of pre mRNA-splicing factor SF2/ASF1, Heterogeneous Ribonucleoprotein A1 (hnRNPA1) and O6-methylguanine-DNA methyltransferase (MGMT) protein. MGMT expression was not observed in LN229 TMZ resistant cells. Further, mRNA sequencing of hnRNPA1 confirmed the exclusive abundance of its higher isoform in TMZ- resistant cells along with increase in SF2/ASF1 expression. Knocking down of SF2/ASF1 using its specific siRNA reverted the higher isoform of hnRNPA1 isoform Var2 to its lower isoform hnRNPA1 Var1 in U87 TMZ resistant cells, reveals hnRNPA1 alternative higher isoform abundance is SF2/ASF1 splice factor dependent. Additionally, selective knock down of hnRNPA1 higher isoform Var2 in TMZ resistant U87MG & LN229 promotes apoptosis, was further specfically enhanced on Wortmannin (PI3Kinase inhibitor) treatment. Targeting higher isoform Var2 of hnRNPA1 specifically induces chemosensitization in MGMT expressed Temozolomide resistant U87MG as well as in MGMT non-expressed LN229 TMZ resistant cells.

Microglia induce an interferon-stimulated gene expression profile in glioblastoma and increase glioblastoma resistance to temozolomide.

Glioblastoma is the most malignant primary brain tumour. Even with standard treatment comprising surgery followed by radiation and concomitant temozolomide (TMZ) chemotherapy, glioblastoma remains incurable. Almost all patients with glioblastoma relapse owing to various intrinsic and extrinsic resistance mechanisms of the tumour cells. Glioblastomas are densely infiltrated with tumour-associated microglia and macrophages (TAMs). These immune cells affect the tumour cells in experimental studies and are associated with poor patient survival in clinical studies. The aim of the study was to investigate the impact of microglia on glioblastoma chemo-resistance. We co-cultured patient-derived glioblastoma spheroids with microglia at different TMZ concentrations and analysed cell death. In addition, we used RNA sequencing to explore differentially expressed genes after co-culture. Immunostaining was used for validation. Co-culture experiments showed that microglia significantly increased TMZ resistance in glioblastoma cells. RNA sequencing revealed upregulation of a clear interferon-stimulated gene (ISG) expression signature in the glioblastoma cells after co-culture with microglia, including genes such as IFI6, IFI27, BST2, MX1 and STAT1. This ISG expression signature is linked to STAT1 signalling, which was confirmed by immunostaining. The ISG expression profile observed in glioblastoma cells with enhanced TMZ resistance corresponded to the interferon-related DNA damage resistance signature (IRDS) described in different solid cancers. Here, we show that the IRDS signature, linked to chemo-resistance in other cancers, can be induced in glioblastoma by microglia. ISG genes and the microglia inducing the ISG expression could be promising novel therapeutic targets in glioblastoma.

BRD4 Degradation Enhanced Glioma Sensitivity to Temozolomide by Regulating Notch1 via Glu-Modified GSH-Responsive Nanoparticles.

Temozolomide (TMZ) serves as the principal chemotherapeutic agent for glioma; nonetheless, its therapeutic efficacy is compromised by the rapid emergence of drug resistance, the inadequate targeting of glioma cells, and significant systemic toxicity. ARV-825 may play a role in modulating drug resistance by degrading the BRD4 protein, thereby exerting anti-glioma effects. Therefore, to surmount TMZ resistance and achieve efficient and specific drug delivery, a dual-targeted glutathione (GSH)-responsive nanoparticle system (T+A@Glu-NP) is designed and synthesized for the co-delivery of ARV-825 and TMZ. As anticipated, T+A@Glu-NPs significantly enhanced penetration of the blood-brain barrier (BBB), facilitated drug uptake by glioma cells, and exhibited efficient accumulation in brain tissue. Additionally, T+A@Glu-NPs exhibited augmented efficacy against glioma both in vitro and in vivo through the induction of apoptosis, inhibition of proliferation, and cell cycle arrest. Furthermore, mechanistic exploration revealed that T+A@Glu-NPs degraded the BRD4 protein, leading to the downregulation of Notch1 gene transcription and the inhibition of the Notch1 signaling pathway, thereby augmenting the therapeutic efficacy of glioma chemotherapy. Taken together, the findings suggest that T+A@Glu-NPs represents a novel and promising therapeutic strategy for glioma chemotherapy.

DDDR-16. SORTING TO ENRICH FOR CHEMOTHERAPEUTIC-RESISTANT GLIOMA CELLS BASED ON UNIQUE MEMBRANE PROPERTIES

Abstract Glioblastoma, astrocytoma, and oligodendroglioma comprise a class of aggressive and deadly brain tumors called diffuse gliomas. Around 20,000 glioma cases are diagnosed yearly in the US alone. The current standard chemotherapy treatment, temozolomide (TMZ), is insufficient as the 5-year survival rate of glioblastoma patients is as low as 8%. This is due to the presence of TMZ-resistant cells within tumors that lead to chemoresistance and tumor recurrence. Thus, studying the molecular properties of TMZ-resistant cells would enable more effective means of targeting them and thus improve patient outcomes. We leveraged dielectrophoresis (DEP), a phenomenon by which non-uniform electric fields induce differential movement of cells, as a novel label-free technique to sort glioma cells based on their membrane electrophysiological properties. We were able to successfully use DEP to sort cells from glioblastoma and oligodendroglioma established cell lines, which yielded fractions that differed in membrane capacitance, the membrane’s ability to store charge. Interestingly, the DEP-sorted cell fractions also exhibited significant differences in TMZ resistance, indicating that we were able to use DEP to enrich for TMZ-resistant cells. Further, the differences in TMZ resistance were coupled with their membrane capacitance differences. Because we previously found that changes in membrane capacitance were linked to differences in glycosylation, we utilized lectin analysis to assess glycan expression of DEP-sorted oligodendroglioma cells. Our data show that TMZ-resistant oligodendroglioma cells enriched from DEP sorting had reduced levels of mannose glycans compared to less TMZ-resistant fractions. Collectively, our novel findings demonstrate (a) TMZ-resistant glioma cells can be enriched by DEP, and (b) TMZ resistance is correlated with membrane capacitance and cell surface glycosylation. These findings could enable further characterization of TMZ-resistant cells to better understand molecular mechanisms of TMZ resistance and the development of more effective treatment strategies for gliomas through targeting membrane properties such as glycosylation.

TMIC-86. THE ROLE AND THERAPEUTIC RESEARCH OF NETS IN PROMOTING TMZ RESISTANCE IN GLIOBLASTOMA

Abstract OBJECTIVE This study aims to investigate the issue of resistance to temozolomide (TMZ) treatment in high-grade glioblastoma (GBM), specifically the association between TMZ resistance, the tumor microenvironment (TME), and neutrophil activity. The research hypothesis posits that changes in neutrophils induced by TMZ treatment, particularly the formation of neutrophil extracellular traps (NETs), may lead to TMZ resistance in GBM cells. METHODS The study utilized clinical data analysis and animal model research to evaluate changes in neutrophils during TMZ treatment and their impact on treatment efficacy. Proteomics analysis and high-throughput sequencing were employed to investigate the effect of TMZ on cytokine secretion by GBM cells and how these cytokines stimulate the release of NETs. Additionally, gene ontology (GO) analysis and bioinformatics methods were used to study how NETs promote the epithelial-mesenchymal transition (EMT) process and TMZ resistance by activating specific signaling pathways such as Jak-Stat3. RESULTS The study found that TMZ treatment led to increased neutrophil infiltration, which was associated with poor prognosis in GBM patients. TMZ treatment altered the levels of cytokines secreted by GBM cells, particularly IL-6, promoting the release of NETs by neutrophils. The formation of NETs was closely related to the activation of Stat3 signaling and the promotion of the EMT process in tumor cells, thereby enhancing TMZ resistance. Magnesium-based micromotors were found to improve TMZ efficacy by scavenging ROS production in neutrophils, inhibiting the release of NETs. CONCLUSION This study confirms the association between TMZ resistance and neutrophil activity, particularly the role of NETs in promoting EMT and resistance. These findings provide new insights into the treatment of GBM and offer a theoretical basis for the development of therapeutic strategies targeting NETs, which may improve the response of glioblastoma to TMZ treatment by inhibiting ROS production. The development of this novel therapeutic approach offers new treatment options for postoperative chemotherapy in GBM.

EXTH-65. EXPLORING ANTIHISTAMINES TO TREAT GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and lethal primary malignant brain tumor with a 5-year survival of 6.8%. Interestingly, people with allergies, atopy, or asthma are less likely to develop GBM. A recent study recognized that high histamine receptor 1 (HRH1) expression is inversely related to overall and progression-free survival. METHODS We used a bioinformatics tool, iLINCS, which, after analyzing 99 GBM and 38 healthy samples, identified 36 drugs that can reverse GBM signatures. Based on the concordance score and blood-brain barrier permeability, we selected brompheniramine (Brom), a first-generation HRH1 antagonist, as a potential candidate. HRH1 expression analysis was done on human and mouse GBM cell lines by immunoblotting. The effect of Brom alone or in combination with temozolomide (TMZ) was investigated on proliferation, DNA damage and apoptosis in human (U118 and U251) and three syngeneic (EGFRvIII, p16-/- & GFAP Cre, PTEN+/-, p53R172H+/-, EGFRvIII & GFAP Cre, PTEN-/-, p53R172H-/- & GFAP Cre) cell lines. GBM orthograft mouse models were used to test the efficacy of combination therapy. RESULTS HRH1 is highly expressed in human and syngeneic GBM cells. This expression is further increased in TMZ-resistant cells. Interestingly, the addition of histamine, induced TMZ resistance in GBM cells. TMZ with Brom decreases proliferation in mouse and human GBM cells, and Brom is non-toxic to non-transformed cells. Combination treatment increases DNA damage and decreases the DNA repair mechanism, possibly by downregulating the expression of O6methylguanine-DNA methyltransferase (MGMT). In the GBM orthograft mouse model, the combination therapy reduced tumor volume and significantly increased survival compared to single-agent treatment. CONCLUSIONS Brom is non-toxic; Brom, in combination with TMZ, enhanced the anti-tumor efficacy of TMZ both in in vitro and in vivo GBM models. Further studies in spontaneous mouse models are planned.

EXTH-31. TARGETINGMIR-19B/PPP2R5E REGULATORY AXIS TO MAXIMISE ALKYLATING AGENT EFFICACY: A NEW AVENUE TO COUNTERACT TEMOZOLOMIDE RESISTANCE IN RECURRENT GLIOBLASTOMAS

Abstract Glioblastoma, IDH-wildtype (GBM), notorious for its inevitable recurrence despite aggressive multi-modal standard of care, presents a pressing need for novel treatment paradigms. The extensive DNA damage caused by the frontline DNA alkylating agent, temozolomide (TMZ) is counteracted by intricate or acquired survival mechanisms. Through functional microRNA screens we identified miR-19b-overexpressing clones possessing extra fitness in presence of escalating doses of TMZ. Our study uncovers a previously unrecognized TMZ resistance mechanism governed by miR-19b. Notably, miR-19b attenuation intensified DNA damage response and CHEK1 and CHEK2 phosphorylation, instigating persistent DNA lesions and consequently priming GBM cells for heightened TMZ cytotoxicity. Mechanistically, we showed that miR-19b exerts its function by targeting PPP2R5E, a subunit of protein phosphatase PP2A, frequently downregulated in GBM. We uncovered that the elevated DNA damage response in the miR-19b-attenuated cells was a consequence of enhanced nuclear and mitochondrial ROS production. This in turn, induced ROS-mediated senescence, and an intriguing susceptibility to ferroptosis, possibly due to increased ROS-mediated lipid peroxidation. Of note, all these phenotypes were reversed in GBM cells with concomitant miR-19b and PPP2R5E attenuation, demonstrating that miR-19b exerts its role in TMZ resistance by targeting PPP2R5E. In line with this finding, an orthotopic human-derived glioblastoma stem cell xenograft model confirmed that PPP2R5E attenuation not only decreases TMZ cytotoxicity in the brain, but also at the spinal metastatic location. Consistently, treating cells with the PP2A-activating drug FTY720 or knocking down endogenous PP2A-inhibiting proteins mirrored the effects of miR-19b attenuation in enhancing TMZ cytotoxicity. This intricate interplay between miR-19b and PPP2R5E underscores a novel strategy to modulate the TMZ response, highlighting promising results in our pre-clinical models. In conclusion, our findings advocate for the therapeutic targeting of the miR-19b/PPP2R5E regulatory axis as a potential novel strategy in the pursuit of effective therapeutic interventions for the formidable recurrent GBM tumours.

DDDR-01. TUMOR ASSOCIATED MACROPHAGES VIS-À-VIS TMZ RESISTANCE IN PTEN-DEFICIENT GLIOMA

Abstract Drug resistance is one of the most important obstacles during chemotherapy. Currently temozolomide (TMZ) is still first line chemotherapy agent for gliomas, while drug resistance is one of the main reasons for chemotherapy failure. It has been reported that tumor-associated macrophages (TAMs) could be related to drug resistance, and thus we have investigated the association between TAMs and TMZ response in gliomas. Firstly, our study have found that PTEN-deficient glioma can specifically induce a large number of M2 phenotype TAMs, which can attenuate the TMZ-mediated the killing effect on glioma cells. Further search for the possible mechanism, we have found TAMs may secrete cytokine IL-6 in PTEN-deficient gliomas, which in turn mediate TMZ resistance. We also revealed that IL-6 highly secreted by TAMs can enhance the stemness of glioma cells through activation of the JAK-STAT3 signaling pathway in PTEN-deficient glioma cells. Secondly, have found valproic acid (VPA) can promote the transformation of TAMs from M2 to M1, reduce its IL-6 secretion, and in turn enhancing sensitivity of TMZ in PTEN-deficient gliomas. Our study revealed that TMZ resistance may modulated through soluble factors released by TAMs. Conventional anti-epilepsy drug- VPA may enhance TMZ sensitivity in PTEN-deficient gliomas, and worth to further clinical investigation. Keywords: PTEN-deficient gliomas; tumor-associated macrophages; valproic acid; temozolomide resistance

DDDR-10. MYELOID CELLS COORDINATELY INDUCE GLIOMA CELL-INTRINSIC AND -EXTRINSIC PATHWAYS FOR CHEMORESISTANCE VIA GP130 SIGNALING

Abstract The DNA damage response (DDR) and the blood tumor barrier (BTB) restrict chemotherapeutic success for primary brain tumors like glioblastoma (GBM). Coherently, GBM almost invariably relapse with fatal outcome. We here show that interaction of GBM and myeloid cells simultaneously induces chemoresistance on the genetic and the vascular level by activating GP130 receptor signaling, which can be addressed therapeutically. We performed transcriptomic and immunohistochemical screens with human brain material, pharmacological experiments with a humanized organotypic GBM model, proteomics and cell-based assays and observed that nanomolar concentrations of the signaling peptide Humanin promoted TMZ resistance through DDR activation. GBM mouse models recapitulating intratumoral Humanin-release showed accelerated BTB formation. Genetic analysis of pericyte to endothelial cell signaling in transgenic mice revealed increased GP130 activation in Humanin releasing tumors as compared to controls. GP130 blockade attenuated both DDR activity and BTB formation resulting in improved preclinical chemotherapeutic efficacy. Altogether, we describe an overarching mechanism for TMZ resistance and outline a translatable strategy with predictive markers to improve chemotherapy for GBM.

BIOM-64. EXPLOITING CELL-FREE DNA TO DISSECT MECHANISMS OF RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA CELLS

Abstract Temozolomide (TMZ) is a cytotoxic alkylating agent that serves as a cornerstone in glioblastoma (GBM) treatment, yet is notorious for eliciting hypermutation in recurrent tumors that have developed drug resistance by downregulating mismatch repair (MMR). We reasoned that cell-free DNA (cfDNA), a non-invasive measure for detecting tumor-derived biomarkers, could be used to temporally examine the mechanism of resistance to TMZ. We therefore designed an in-vitro experiment using the patient-derived GBM43 cell line. Over five days, batches of cells were treated with either TMZ or vehicle. Cell counts and cfDNA were collected every 24 hours. We observed an associated increase in both dead cell counts and cfDNA yield in treated cells compared to untreated cells. In TMZ-treated samples, cfDNA showed a periodic fragmentation pattern consistent with internucleosomal cleavage and TMZ-induced apoptosis. Intriguingly, higher numbers of variants were found in the cfDNA of untreated samples when compared to treated cfDNA, possibly reflecting TMZ-induced selection of resistant subclones in treated samples. This was further supported by the finding that the average variant allele frequency (VAF) increased over time in TMZ-treated samples, consistent with purifying selection. Conversely, cfDNA from vehicle-treated samples demonstrated a decrease in the average VAF over time, consistent with clonal diversification. Although we did not observe an enrichment of the TMZ-associated mutational signature SBS11, we did find higher levels of defective MMR mutation signature SBS44 in treated samples, potentially indicating the selection of subclones with defective MMR mechanisms. Overall, our preliminary findings show that cfDNA can be used to resolve mechanisms of TMZ resistance over time. We are addressing open questions by performing longer treatment times, testing other patient-derived cell lines, and performing murine xenotransplantation experiments to test TMZ-associated hypermutation in vivo. Our work will contribute to our understanding of TMZ resistance, and importantly push the development of cfDNA-based biomarkers for measuring TMZ-sensitivity in patients.

DNAR-14. MODULATING POPULATIONAL VARIANCE OF METHYL-GUANINE METHYL TRANSFERASE (MGMT) EXPRESSION THROUGH MICRORNA DEGRADATION: A NOVEL MECHANISM OF TEMOZOLOMIDE RESISTANCE

Abstract O6 methyl-guanine methyltransferase (MGMT) restores alkylated DNA to its undamaged form in a stochiometric manner to prevent the cytotoxic effects of temozolomide (TMZ), a DNA alkylating agent used as a standard-of-care treatment for glioblastomas. We previously demonstrated that he microRNA, miR-181d, post-transcriptionally repressed MGMT expression. Here, we show that treatment of glioblastoma cells with TMZ induced a feed-forward cascade resulting in an ataxia telangiectasia and Rad3 (ATR) and polyribonucleotide nucleotidyl transferase 1 (PNPT1)-dependent degradation of miR-181d. Single-cell analyses revealed such miR-181d degradation induced an: 1) increased mean level of MGMT expression and 2) widened the variance in MGMT expression in the cell population. While the former is known to confer increased population fitness to temozolomide, the importance of the latter is unknown. By mixing cultures derived from isogenic glioblastoma subclones with distinct MGMT expression levels, we generated glioblastoma cell populations with comparable mean MGMT expression levels while differing in the populational variance in MGMT expression (classified as “wide” or “narrow”). Despite comparable mean levels of MGMT expression, the glioblastoma population with wide variance exhibited increased temozolomide resistance relative to the population with the narrow variance, in vitro and in vivo. Transfection of miR-181d into the wide-variance population narrows the variance of MGMT expression and restores TMZ sensitivity to levels comparable to that observed in miR-181d transfected narrow-variance populations. These findings suggest that TMZ-induced miR-181d degradation enhanced TMZ resistance through modulation of both the mean and variance of MGMT expression, mechanisms which may be extrapolated to microRNA biology in general.

DDEL-02. PRECLINICAL EFFICACY OF IL13RΑ2-TARGETING POLYPEPTIDE-DRUG CONJUGATE (SELF-DEPOTTM ONCOPDC) IN GLIOBLASTOMA AND DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are currently incurable cancers. Systemic chemotherapy shows limited efficacy because of the intact blood-brain barrier. Convection-enhanced delivery (CED) offers promise by concentrating high doses of chemotherapeutics directly within these tumors. However, drugs administered via CED often face challenges in confirming precise target delivery and are quickly cleared by increased interstitial fluid flow and drug efflux transporters. This highlights the urgent need for innovative drugs that demonstrate target specificity and prolonged retention capabilities. Intrinsically disordered polypeptides (IDPs), derived from tropoelastin, self-assemble into water-insoluble structures with extended retention properties triggered by body temperature. IL13Rα2, differentially amplified in U87MG GBM and SF8628 DIPG cells, serves as a target for therapeutic intervention. An IL13Rα2-targeting IDP (XM161) was developed by incorporating IL13Rα2 binding sequences into the IDP domains. XM161 demonstrated high selectivity in binding to IL13Rα2, with Kd values of 1,786 nM for IL13Rα1 and 12.8 nM for IL13Rα2. Conjugation of XM161 with SN38, a potent topoisomerase I inhibitor, resulted in XM161-SN38, which remained in a dissolved state up to 50°C but formed insoluble aggregates above 27°C in the presence of cerebrospinal fluid. XM161-SN38 exhibited strong cytotoxicity against U87MG and SF8628 cells, with IC50 values of 14.2 nM and 0.48 nM, respectively. Importantly, XM161-SN38 demonstrated efficacy in overcoming Temozolomide resistance in T98G GBM cells, with IC50 values of 3.35 nM compared to 10.87 nM for unconjugated SN38. In orthotopic xenograft models established in BALB/cSlc-nu/nu male mice using U87MG-Luc2 cells, three doses (3 x 0.95 μg SN38 equivalents) of XM161-SN38 delivered via CED were well tolerated and significantly extended median survival to over 65 days, providing a notable survival benefit compared to untreated controls (42 days) and Topotecan-treated group (42.5 days). These findings validate the feasibility and therapeutic potential of XM161-SN38 for GBM treatment. Ongoing studies are underway to investigate the safety and pharmacokinetics of XM161-SN38 in tumor-naive mouse brains.