GDNF/GFRA1 signaling contributes to chemo- and radioresistance in glioblastoma.

Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.

Stress granule-mediated sequestration of EGR1 mRNAs correlates with lomustine-induced cell death prevention.

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

BACKGROUND: Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers. OBJECTIVE: To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. METHODS: We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations. RESULTS: TMZ (200 µM) or CCNU alone (5 µM) reduced the irradiated survival fraction (4 Gy) from 60% to 38% (p = 0.0074) and 26% (p = 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4 Gy) more potently to 12% (p < 0.0001). After long-term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p = 0.0006) and TMZ (p = 0.0326) treatment combined with irradiation (4 Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p < 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4 Gy) reduced the cell survival by 88% (p = 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines. CONCLUSIONS: Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.

Abrogation of Cellular Senescence Induced by Temozolomide in Glioblastoma Cells: Search for Senolytics.

A first-line therapeutic for high-grade glioma, notably glioblastoma (GBM), is the DNA methylating drug temozolomide (TMZ). Previously, we showed that TMZ induces not only apoptosis and autophagy, but also cellular senescence (CSEN). We presented the hypothesis that GBM cells may escape from CSEN, giving rise to recurrent tumors. Furthermore, the inflammatory phenotype associated with CSEN may attenuate chemotherapy and drive tumor progression. Therefore, treatments that specifically target senescent cells, i.e., senolytic drugs, may lead to a better outcome of GBM therapy by preventing recurrences and tumor inflammation. Here, we tested Bcl-2 targeting drugs including ABT-737, ABT-263 (navitoclax), several natural substances such as artesunate, fisetin and curcumin as well as lomustine (CCNU) and ionizing radiation (IR) for their senolytic capacity in GBM cells. Additionally, several proteins involved in the DNA damage response (DDR), ATM, ATR, Chk1/2, p53, p21, NF-kB, Rad51, PARP, IAPs and autophagy, a pathway involved in CSEN induction, were tested for their impact in maintaining CSEN. Treatment of GBM cells with a low dose of TMZ for 8-10 days resulted in >80% CSEN, confirming CSEN to be the major trait induced by TMZ. To identify senolytics, we treated the senescent population with the compounds of interest and found that ABT-737, navitoclax, chloroquine, ATMi, ATRi, BV-6, PX-866 and the natural compounds fisetin and artesunate exhibit senolytic activity, inducing death in senescent cells more efficiently than in proliferating cells. Curcumin showed the opposite effect. No specific effect on CSEN cells was observed by inhibition of Chk1/Chk2, p21, NF-kB, Rad51 and PARP. We conclude that these factors neither play a critical role in maintaining TMZ-induced CSEN nor can their inhibitors be considered as senolytics. Since IR and CCNU did not exhibit senolytic activity, radio- and chemotherapy with alkylating drugs is not designed to eliminate TMZ-induced senescent cancer cells.

Viability fingerprint of glioblastoma cell lines: roles of mitotic, proliferative, and epigenetic targets.

Despite the use of multimodal treatment combinations, the prognosis of glioblastoma (GB) is still poor. To prevent rapid tumor recurrence, targeted strategies for the treatment of GB are widely sought. Here, we compared the efficacy of focused modulation of a set of signaling pathways in two GB cell lines, U-251 MG and T98-G, using a panel of thirteen compounds targeting cell cycle progression, proliferation, epigenetic modifications, and DNA repair mechanism. In parallel, we tested combinations of these compounds with temozolomide and lomustine, the standard chemotherapy agents used in GB treatment. Two major trends were found: within individual compounds, the lowest IC50 values were exhibited by the Aurora kinase inhibitors, whereas in the case of mixtures, the addition of DNA methyltransferase 1 inhibitor azacytidine to lomustine proved the most beneficial. The efficacy of cell cycle-targeting compounds was further augmented by combination with radiation therapy using two different treatment regimes. The potency of azacytidine and lomustine mixtures was validated using a unique assay pipeline that utilizes automated imaging and machine learning-based data analysis algorithm for assessment of cell number and DNA damage extent. Based on our results, the combination of azacytidine and lomustine should be tested in GB clinical trials.

Patient-reported outcomes in a phase II randomised study of regorafenib compared with lomustine in patients with relapsed glioblastoma (the REGOMA trial).

BACKGROUND: The REGOMA trial showed that regorafenib significantly improved overall survival in patients with recurrent glioblastoma compared with lomustine. Patients treated with regorafenib experienced a higher occurrence of grade 3-4 drug-related adverse events than those receiving the standard treatment. Because this safety profile was expected, it was considered of great importance to assess the patient point of view regarding the disease and treatment impact on different aspects of life and patient well-being. We here report the final results of the health-related quality of life (HRQoL) assessment, a secondary end-point of the study. This trial is registered with ClinicalTrials.gov, NCT02926222. METHODS: Patient-reported outcomes were assessed, within a prospective, randomised, multicentre, open-label phase II trial, by the European Organisation for Research and Treatment of Cancer core questionnaire and brain module at baseline and every 8-weekly neuroradiological assessment till disease progression. Mixed-effect linear models were fitted for each of the HRQoL domain to examine the change over progression-free time within and between arms. Furthermore, differences were also classified as clinically meaningful changes. To correct for multiple comparisons and avoid type I errors, the level of significance was set at P = 0.01 (2-sided). RESULTS: Of 119 enrolled patients, 56/59 (95%) patients and 58/60 (97%) patients treated with regorafenib and lomustime completed questionnaires at baseline, respectively. No significant differences were observed in any generic or cancer-specific domain during treatment in both arms, or between the two arms, except for the appetite loss and diarrhoea scales which were significantly worse in patients treated with regorafenib. The rate of patients with a clinically meaningful worsening for appetite loss, diarrhoea and for any other domain was not statistically different between the two arms. CONCLUSIONS: Regorafenib did not negatively affect HRQoL in patients with recurrent glioblastoma. These data combined with the survival benefit shown in the REGOMA trial support the use of regorafenib as a treatment option for these patients.

Increased infiltration of CD8 T cells in recurrent glioblastoma patients is a useful biomarker for assessing the response to combined bevacizumab and lomustine therapy.

PURPOSE: Treatment options for recurrent glioblastoma (rGBM) remain scarce, which may be due to the limited understanding of its molecular characteristics. METHODS: Based on gene expression profiling, the infiltration scores of 26 immune cell types were calculated using gene set variation analysis. The differences between rGBM and other cancer subtypes were estimated to characterize the specific immune characteristics of rGBM, and the prognostic value of immune cells in rGBM was estimated using univariate and multivariate Cox analysis. Subgroup analyses and Kaplan-Meier analyses were performed to identify whether CD8 T-cell infiltration could be useful in selecting treatment options for rGBM patients. RESULTS: We found that rGBM patients were associated with enrichment of activated CD8 T cells, and high CD8 T-cell infiltration was associated with superior overall survival. Patients exhibiting high CD8 T-cell infiltration who received treatment with bevacizumab and lomustine combination therapy experienced a significant benefit in overall survival and progression-free survival, whereas patients with low CD8 T-cell infiltration did not experience such a benefit. CD8 T cells remained an independent prognostic factor in multivariate analyses (cohort 1: hazard ratio [HR] = 0.546, 95% confidence interval [CI]: 0.316-0.945, P = 0.031; cohort 3: HR = 0.615, 95% CI: 0.387-0.978, P = 0.040) after adjusting for clinicopathological and molecular factors. CONCLUSIONS: Activated CD8 T-cells is a promising biomarker for predicting overall survival in rGBM patients and could be used for assisting treatment selection.

Electrochemical behaviour of anticancer drug lomustine and in situ evaluation of its interaction with DNA.

Electrochemical techniques were used to investigate the behavior of lomustine (CCNU) and its degradation in aqueous solution at a glassy carbon electrode (GCE). The in situ interaction of CCNU and chemically degraded CCNU (cdCCNU) with dsDNA was then investigated in dsDNA incubated solutions, using dsDNA electrochemical biosensors and comet assays. CCNU undergoes electrochemical reduction in two irreversible, diffusion-controlled, and pH-dependent redox processes, each with transfer of two electrons and one proton. At pH ≥ 10.1, the peak potential for the two processes was essentially pH-independent and involved only one electron. A mechanism was proposed for the reduction of CCNU in a neutral medium. In addition, it was found that CCNU underwent spontaneous degradation during incubation in aqueous solution, without the formation of electroactive degradation products. The degradation process was faster in basic media. Moreover, this pro-drug interacted with the DNA. Its metabolite(s) initially caused condensation of the double helix chains, followed by the unwinding of these chains. In addition, free guanine (Gua) was released from the dsDNA and oxidative damage to the DNA by the CCNU metabolite(s) was evidenced from the detection of 8-oxoGua and 2,8-oxoAde. These results were confirmed by the poly(dA)- and poly(dG)-polyhomonucleotide biosensors, which revealed the oxidative damage caused to both bases (guanine and adenine) of the dsDNA by the CCNU metabolite(s). The comet assay indicated breaks in the single strand DNA, complementing the results of the studies using differential pulse voltammetry. Conformational changes of dsDNA caused by CCNU and cdCCNU were confirmed using comet assays.

A Retrospective Study of Multi-agent Chemotherapy including either Cyclophosphamide or Lomustine as Initial Therapy for Canine High-grade T-cell Lymphoma (2011-2017).

Multi-agent chemotherapy (vincristine, epirubicin and prednisolone) including either cyclophosphamide (CEOP) or lomustine (LEOP) was given as first-line chemotherapy to treatment-naïve canine lymphoma patients with measurable, high grade T-cell lymphoma (HGTCL). All patients responded to either CEOP or LEOP. Toxicity was typical of multi-agent chemotherapy protocols and 25% of dogs receiving lomustine exhibited mild-to-moderate ALT elevation and 29% grade 3 or 4 neutropenia. Median progression-free survival (100 versus 269 days) and overall survival (155 versus 327 days) were significantly higher in patients receiving LEOP compared to CEOP. Overall survival was improved for patients receiving LEOP compared to those receiving CEOP followed by lomustine-based rescue therapy. The results of this retrospective study support further evaluation of lomustine as part of first-line, multi-agent therapy for patients with HGTCL.