Mechanism Of Temozolomide Resistance Research Articles

Abstract 1996: Novel mechanism of temozolomide resistance in glioblastoma

Abstract Intrinsic expression of PD-1 has been discovered in different types of tumurs, exhibiting either oncogenic or tumor-suppressing function. In glioblastoma (GBM), intrinsic PD-1 has been found to play a role in promoting the proliferation and self-renewal of brain tumor-initiating cells through nuclear factor κB without involving PD-L1 ligation, with unknown factors activating this signalling pathway. Meanwhile, TMZ resistance significantly contributes to treatment failure and an extremely poor prognosis for GBM. This study investigated whether intrinsic PD-1 is also expressed in differentiated human GBM cell lines and its role in GBM proliferation and temozolomide (TMZ) resistance. In vitro studies demonstrated the presence of PD-1 in both U87 and U251 TMZ-sensitive and -resistant cell lines, with a higher expression on TMZ-resistant cell lines. PDCD1 knockdown led to decreased GBM progression, including cell proliferation, colony formation and cell migration, in TMZ-resistant U87 and U251 cell lines using cell viability assays. It also resensitizes these cell lines to TMZ. Overexpression of PDCD1 in the PDCD1-knockdown cell lines increases their proliferation and restores their resistance to TMZ. Treatment of U87 and U251 TMZ-resistant cell lines with TMZ upregulated PD-1 expression. An orthotopic xenograft mouse model is further deployed by injecting previously cultured U87 and U251 cell lines into the mouse brains. A smaller tumour size is observed in the model with PDCD1 knockdown model than the one without PDCD1 knockdown when treated with TMZ. This study reveals that intrinsic PD-1 expression contributes to TMZ resistance in GBM and TMZ may be a factor activating the PD-L1-independent PD-1 signalling. PD-1 and its downstream signalling pathway, such as SHP-2 and IKK, may be potential therapeutic targets for GBM patients with TMZ resistance. Co-administration of TMZ and agents inhibiting the intrinsic PD-1 signalling pathway can potentially produce synergistic therapeutic effects. The higher expression of PD-1 on TMZ-resistant GBM than on TMZ-sensitive GBM also suggests that the intrinsic PD-1 level can be a predictor of TMZ outcome. Citation Format: Yuet Yi Charmaine Hung, Mei Yee Karrie Kiang, Ka Kit Gilberto Leung. Novel mechanism of temozolomide resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1996.

RUNX1 (RUNX family transcription factor 1), a target of microRNA miR-128-3p, promotes temozolomide resistance in glioblastoma multiform by upregulating multidrug resistance-associated protein 1 (MRP1)

ABSTRACT Glioblastoma multiform (GBM) is the most frequent type of malignant brain tumor with a poor prognosis. After optimal surgery, radiotherapy plus temozolomide (TMZ) is the standard treatment for GBM patients. However, the development of TMZ resistance limits its efficacy in GBM management. Runt Related Transcription Factor 1 (RUNX1) and microRNAs have been implicated in drug resistance of TMZ in GBM. In this study, we revealed the underlying mechanism of TMZ resistance and identified miR-128-3p/RUNX1 axis as a novel target for TMZ resistance in GBM. RUNX1 expression was significantly upregulated in GBM tissues as compared to normal tissues, and its expression was even higher in recurrent GBM tissues and TMZ-resistant GBM cells. RUNX1 depletion inhibited the viability, proliferation, migration, invasion and TMZ resistance of GBM cells, which could be rescued by RUNX1 overexpression. We further identified miR-128-3p as a tumor-suppressor whose overexpression restored the sensitivity of TMZ in GBM cells. miR-128-3p negatively regulated RUNX1 and subsequently downregulated multidrug resistance-associated protein 1 (MRP1). Together, the present study indicates that RUNX1 confers TMZ resistance in GBM by upregulating MRP1, which is negatively regulated by miR-128-3p. Targeting miR-128-3p/RUNX1/MRP1 axis provides a potential strategy to overcome TMZ resistance in GBM.

The Rolipram-Perillyl Alcohol Conjugate (NEO214) Is A Mediator of Cell Death through the Death Receptor Pathway.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Treatment with temozolomide, standard of care for gliomas, usually results in drug resistance and tumor recurrence. Therefore, there is a great need for drugs that target GBM. NEO214 was generated by covalently linking rolipram to perillyl alcohol (POH) via a carbamate bond to form the rolipram-perillyl alcohol conjugate. We show here that NEO214 is effective against both temozolomide-sensitive and temozolomide-resistant glioma cells. Furthermore, NEO214 is effective for different mechanisms of temozolomide resistance: overexpression of MGMT (O6-methylguanine methyl-transferase); deficiency in specific mismatch repair proteins; and overexpression of base excision repair (BER) proteins. NEO214-induced cytotoxicity involves apoptosis triggered by endoplasmic reticulum (ER) stress, as well as activating the Death Receptor 5 (DR5)/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) pathway. In vitro studies show that glioma cells treated with NEO214 express DR5 and exhibit cell death in the presence of recombinant TRAIL, a growth factor constitutively produced by astrocytes. Our in vitro 3D coculture data show that induction of DR5 in glioma cells with NEO214 and TRAIL cause tumor cell death very effectively and specifically for glioma cells. In vivo studies show that NEO214 has antitumor efficacy in orthotropic syngeneic rodent tumor models. Furthermore, NEO214 has therapeutic potential especially for brain tumors because this drug can cross the blood-brain barrier (BBB), and is effective in the TRAIL-rich astrocyte microenvironment. NEO214 is a strong candidate for use in the treatment of GBMs.

Effect of microRNA-20a on the temozolomide resistance of glioma cells

Objective To examine the expression of microRNA (miRNA, miR)-20a in the glioma cells with temozolomide (TMZ) resistance, and explore the potential molecular mechanisms of TMZ resistance. Methods TMZ resistant glioma cell line, called U251/TMZ cell line, was constructed by the stepwise revulsion with TMZ. The drug resistance and cell viability were detected by cell counting kit-8 (CCK-8) assay. The expression of miR-20a was detected by real-time quantitative polymerase chain reaction (Real-time PCR). The phosphorylated level of mammalian target of rapamycin (mTOR) signal was defined by Western blotting. The level of miR-20a expression in U251/TMZ cells was downregulated by miR-20a inhibitor transfection using lipidosome. Results As cmpared with U251 cells, the expression of miR-20a and the phosphorylated level of mTOR signal were significantly increased in U251/TMZ cells (2.28±0.18 vs. 1.03±0.05, P<0.05). When the expression level of miR-20a in U251/TMZ cells was downregulated by miR-20a inhibitor, the expression of miR-20a and the phosphorylated level of mTOR signal were decreased, and cell viability was decreased in U251/TMZ cells with 50 μmol/L TMZ existing (P<0.01). Conclusion The expression of miR-20a might be contributed to TMZ resistance of glioma cells, which may be related to mTOR signaling way. Key words: Glioma; MicroRNA-20a; Mammalian target of rapamycin; Temozolomide

Abstract CT168: Phase I/II study of dianhydrogalactitol (VAL-083) with radiation therapy in patients with newly diagnosed glioblastoma, MGMT-unmethylated

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard-of-care includes surgery followed by concomitant chemo-radiation and adjuvant temozolomide (TMZ). Unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT), a validated biomarker for TMZ-resistance, is strongly correlated with TMZ-resistance. In addition, defective DNA mismatch repair (MMR) has been identified as a secondary mechanism of TMZ resistance. MGMT-unmethylated tumors represent a majority of newly diagnosed GBM patients demonstrating significantly inferior progression free and overall survival compared to MGMT-methylated GBM patients. VAL-083 is a first-in-class bi-functional DNA-targeting agent that has shown activity against GBM in NCI-sponsored clinical trials both as a single agent and in combination with radiotherapy. VAL-083 rapidly induces interstrand DNA cross-links at N7-guanine, leading to persistent DNA double-strand breaks and cell death in GBM cell lines and GBM cancer stem cells (CSCs). VAL-083's unique cytotoxic mechanism circumvents MGMT-mediated chemoresistance and maintains cytotoxic activity in cancer cells deficient in MMR. Furthermore, VAL-083 acts as a radiosensitizer in GBM CSCs and non-CSCs, in vitro. We completed a 3:3 dose-escalation trial of VAL-083 in temozolomide- and bevacizumab-refractory rGBM. 40mg/m2/day given on days 1, 2, and 3 of a 21-day cycle was well-tolerated with 6% grade 3 or 4 thrombocytopenia. This dose was selected for further clinical development in a pivotal Phase 3 study in bevacizumab refractory rGBM. The present trial is an ongoing open-label, biomarker-driven, Phase 1/2 study to evaluate the tolerability and efficacy of VAL-083 in combination with radiotherapy in newly diagnosed MGMT-unmethylated GBM patients. A treatment regimen, consisting of a 6-week induction period of VAL-083 and concurrent radiation (2 Gy given daily, 5 days/week) followed by up to 24 weeks of adjuvant maintenance therapy with single-agent VAL-083, is being evaluated as an alternative to standard-of-care in MGMT-unmethylated GBM. The study is being conducted in two parts: 1) a dose-escalation part (20, 30, and 40 mg/m2/day IV infusion on days 1, 2, and 3 of a 21-day cycle) in up to 10 patients, to assess safety and activity of VAL-083 when administered concurrently with radiation therapy; 2) an expansion phase, in which VAL-083 will be studied in up to 20 additional patients at the determined well-tolerated dose. Tumor response will be assessed by MRI, according to RANO criteria. Efficacy endpoints include progression-free survival (PFS) and overall survival (OS). Additional endpoints include safety evaluations and pharmacokinetic assessments of plasma and CSF samples (when available). Enrollment and safety data update will be provided at the meeting. Clinicaltrials.gov identifier: NCT03050736. Citation Format: Zhong-ping Chen, Chengcheng Guo, Jeffrey Bacha, Anne Steino, John Langlands, Claire Kwan, Sarath Kanekal, Richard Schwartz, Lorena Lopez, Dennis M. Brown. Phase I/II study of dianhydrogalactitol (VAL-083) with radiation therapy in patients with newly diagnosed glioblastoma, MGMT-unmethylated [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT168.

Alterations in Cell Motility, Proliferation, and Metabolism in Novel Models of Acquired Temozolomide Resistant Glioblastoma

Glioblastoma (GBM) is an aggressive and incurable tumor of the brain with limited treatment options. Current first-line standard of care is the DNA alkylating agent temozolomide (TMZ), but this treatment strategy adds only ~4 months to median survival due to the rapid development of resistance. While some mechanisms of TMZ resistance have been identified, they are not fully understood. There are few effective strategies to manage therapy resistant GBM, and we lack diverse preclinical models of acquired TMZ resistance in which to test therapeutic strategies on TMZ resistant GBM. In this study, we create and characterize two new GBM cell lines resistant to TMZ in vitro, based on the 8MGBA and 42MGBA cell lines. Analysis of the TMZ resistant (TMZres) variants in conjunction with their parental, sensitive cell lines shows that acquisition of TMZ resistance is accompanied by broad phenotypic changes, including increased proliferation, migration, chromosomal aberrations, and secretion of cytosolic lipids. Importantly, each TMZ resistant model captures a different facet of the “go” (8MGBA-TMZres) or “grow” (42MGBA-TMZres) hypothesis of GBM behavior. These in vitro model systems will be important additions to the available tools for investigators seeking to define molecular mechanisms of acquired TMZ resistance.

Connexin 43 Inhibition Sensitizes Chemoresistant Glioblastoma Cells to Temozolomide.

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ-blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM, and perhaps other TMZ-resistant cancers.

Progression of O⁶-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research.

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O(6)-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.

Resveratrol reverses temozolomide resistance by downregulation of MGMT in T98G glioblastoma cells by the NF-κB-dependent pathway

Glioblastoma multiforme (GBM) is the most common intracranial tumor, with a dismal prognosis. Although temozolomide (TMZ)-based chemotherapy following neurosurgery has been proven to be effective, not all patients benefit clinically because of TMZ resistance. Given that protein expression of O(6)-methylguanine-DNA-methyltransferase (MGMT) is the most important determinant of TMZ resistance, great efforts have been made to suppress it by regulating MGMT-related transcription factors. The study presented here demonstrates that resveratrol, a natural polyphenol, is able to reverse TMZ resistance of glioblastoma T98G cells which have relatively high MGMT activity. The data showed that combination treatment with TMZ and resveratrol resulted in an enhanced antitumor potential of TMZ, decreased the 50% inhibiting concentration (IC50) of TMZ and increased the induction of apoptosis in TMZ-resistant T98G cells. Hoechst 33258 staining revealed increased apoptotic morphology, such as chromatin aggregation and nuclear and cytoplasmic condensation, in cells receiving combination treatment. Western blot analysis manifested a significant decreased intracellular content and nuclear translocation of NF-κB and increased cleavage of caspase-3 in cells exposed to combination treatment, compared to those in cells treated with TMZ alone. In addition, recombinant expression of NF-κB subunit p65 remarkably promoted nuclear translocation of NF-κB and abolished the TMZ-resistance reversal induced by combination treatment, suggesting an underlying NF-κB-dependent mechanism. Our study improved the knowledge on the mechanism of TMZ resistance and suggested a novel strategy for TMZ-based chemotherapy in glioblastoma patients.

Phase I Pharmacokinetic and Pharmacodynamic Study of Temozolomide in Pediatric Patients With Refractory or Recurrent Leukemia: A Children's Oncology Group Study

To determine the tolerability, pharmacokinetics, and mechanisms of temozolomide resistance in children with relapsed or refractory leukemia. Cohorts of three to six patients received 200 or 260 mg/m2/d of temozolomide by mouth daily for 5 days every 28 days. Toxicities, clinical response, and pharmacokinetics were evaluated. Pretreatment leukemia cell O6-methylguanine-DNA methyltransferase (MGMT) activity, tumor and plasma MGMT promoter methylation, and microsatellite instability (MSI) were examined in 14 of 16 study patients and in tissue bank samples from children with acute leukemia not treated with temozolomide (MGMT, n = 67; MSI, n = 65). Sixteen patients (nine female, seven male; acute lymphoblastic leukemia [ALL], n = 8; acute myeloid leukemia [AML], n = 8), median age 11 years (range, 1 to 19 years), received either 200 mg/m2/d (nine enrolled, three assessable for toxicity) or 260 mg/m2/d (seven enrolled, three assessable for toxicity) of temozolomide. Temozolomide was well tolerated and no dose-limiting toxicities occurred. The mean clearance of temozolomide was 107 mL/min/m2, with a volume of distribution of 20 L/m2 and half-life of 109 minutes. MGMT activity in leukemia cells was quite variable and was highest in patients with relapsed ALL. Only one patient had MSI. Two patients had a partial response. Both of these patients had no detectable MGMT activity; both also had methylated MGMT promoters and were MSI stable. Temozolomide was well tolerated at doses as high as 260 mg/m2/d for 5 days in children with relapsed or refractory leukemia. Increased MGMT activity may account for the temozolomide resistance in children with relapsed leukemia. Leukemia cell MGMT activity was higher in pediatric ALL than AML (P < .0001).