Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Background: Despite aggressive treatment regimens comprising surgery and radiochemotherapy, glioblastoma (GBM) remains a cancer entity with very poor prognosis. The development of novel, combined modality approaches necessitates adequate preclinical model systems and therapy regimens that closely reflect the clinical situation. So far, image-guided, fractionated radiotherapy of orthotopic GBM models represents a major limitation in this regard. Methods: GL261 mouse GBM cells were inoculated into the right hemispheres of C57BL/6 mice. Tumor growth was monitored by contrast-enhanced conebeam CT (CBCT) scans. When reaching an average volume of approximately 7 mm 3 , GBM tumors were irradiated with daily fractions of 2 Gy up to a cumulative dose of 20 Gy in different beam collimation settings. For treatment planning and tumor volume follow-up, contrast-enhanced CBCT scans were performed twice per week. Daily repositioning of animals was achieved by alignment of bony structures in native CBCT scans. When showing neurological symptoms, mice were sacrificed by cardiac perfusion. Brains, livers, and kidneys were processed into histologic sections. Potential toxic effects of contrast agent administration were assessed by measurement of liver enzyme and creatinine serum levels and by histologic examination. Results: Tumors were successfully visualized by contrast-enhanced CBCT scans with a detection limit of approximately 2 mm 3 , and treatment planning could be performed. For daily repositioning of the animals, alignment of bony structures in native CT scans was well feasible. Fractionated irradiation caused a significant delay in tumor growth translating into significantly prolonged survival in clear dependence of the beam collimation setting and margin size. Brain sections revealed tumors of similar appearance and volume on the day of euthanasia. Importantly, the repeated contrast agent injections were well tolerated, as liver enzyme and creatinine serum levels were only subclinically elevated, and liver and kidney sections displayed normal histomorphology.

Background Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. Methods We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. Results Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4—two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. Conclusions Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

PD-L1 expression on tumor cells as a potential predictive biomarker for patients with unresectable stage III non-small cell lung cancer treated with chemoradiotherapy followed by durvalumab maintenance treatment. Transl Cancer Res 2023.

Background Concurrent platinum-based chemoradiotherapy (CRT) followed by durvalumab maintenance treatment represents the new standard of care in unresectable stage III non-small cell lung cancer (NSCLC). In this prospective hypothesis-generating single-center study, we aim to identify a framework of prognostic and predictive biomarkers by longitudinal characterization of tumor- and patient (host)-related parameters over all phases of multimodal treatment. Methods This study will enroll 40 patients (≥18 years, Eastern Cooperative Oncology Group performance status (ECOG PS) 0–2, with a diagnosis of PD-L1 positive (≥1%), inoperable stage III NSCLC) with an indication for CRT followed by maintenance treatment with durvalumab according to European Medicines Agency (EMA) approval. Comprehensive analysis will include peripheral blood cellular and humoral immunophenotyping and circulating tumor DNA as well as gut/saliva microbiota analyses. Additional morphological analysis with 18 F-FDG-PET/computed tomography (CT) before, 6 weeks, 6 and 12 months after the end of CRT is included. Statistical analysis using multiple testing will be used to examine the impact of different parameters on progression-free survival (PFS) and overall survival (OS) as well as tumor response and response duration. Discussion This protocol describes the methodology of a comprehensive biomarker study in order to identify a framework of prognostic and predictive markers for unresectable stage III NSCLC in a real-world setting. Trial Registration ClinicalTrials.gov identifier (NCT05027165), data registered on August 2021.

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Purpose In patients with unresectable stage III non-small-cell lung cancer (NSCLC), durvalumab maintenance treatment after chemoradiotherapy (CRT) significantly improves survival. So far, however, metabolic changes of tumoral lesions and secondary lymphoid organs under durvalumab are unknown. Hence, we assessed changes on [18F]FDG PET/CT in comparison to patients undergoing CRT alone. Methods Forty-three patients with [18F]FDG PET/CT both before and after standard CRT for unresectable stage III NSCLC were included, in 16/43 patients durvalumab maintenance treatment was initiated (CRT-IO) prior to the second PET/CT. Uptake of tumor sites and secondary lymphoid organs was compared between CRT and CRT-IO. Also, readers were blinded for durvalumab administration and reviewed scans for findings suspicious for immunotherapy-related adverse events (irAE). Results Initial uptake characteristics were comparable. However, under durvalumab, diverging metabolic patterns were noted: There was a significantly higher reduction of tumoral uptake intensity in CRT-IO compared to CRT, e.g. median decrease of SUVmax –70.0% vs. –24.8%, p = 0.009. In contrast, the spleen uptake increased in CRT-IO while it dropped in CRT (median + 12.5% vs. –4.4%, p = 0.029). Overall survival was significantly longer in CRT-IO compared to CRT with few events (progression/death) noted in CRT-IO. Findings suggestive of irAE were present on PET/CT more often in CRT-IO (12/16) compared to CRT (8/27 patients), p = 0.005. Conclusion Durvalumab maintenance treatment after CRT leads to diverging tumoral metabolic changes, but also increases splenic metabolism and leads to a higher proportion of findings suggestive of irAE compared to patients without durvalumab. Due to significantly prolonged survival with durvalumab, survival analysis will be substantiated in correlation to metabolic changes as soon as more clinical events are present.

Despite intensive basic scientific, translational, and clinical efforts in the last decades, glioblastoma remains a devastating disease with a highly dismal prognosis. Apart from the implementation of temozolomide into the clinical routine, novel treatment approaches have largely failed, emphasizing the need for systematic examination of glioblastoma therapy resistance in order to identify major drivers and thus, potential vulnerabilities for therapeutic intervention. Recently, we provided proof-of-concept for the systematic identification of combined modality radiochemotherapy treatment vulnerabilities via integration of clonogenic survival data upon radio(chemo)therapy with low-density transcriptomic profiling data in a panel of established human glioblastoma cell lines. Here, we expand this approach to multiple molecular levels, including genomic copy number, spectral karyotyping, DNA methylation, and transcriptome data. Correlation of transcriptome data with inherent therapy resistance on the single gene level yielded several candidates that were so far underappreciated in this context and for which clinically approved drugs are readily available, such as the androgen receptor (AR). Gene set enrichment analyses confirmed these results, and identified additional gene sets, including reactive oxygen species detoxification, mammalian target of rapamycin complex 1 (MTORC1) signaling, and ferroptosis/autophagy-related regulatory circuits to be associated with inherent therapy resistance in glioblastoma cells. To identify pharmacologically accessible genes within those gene sets, leading edge analyses were performed yielding candidates with functions in thioredoxin/peroxiredoxin metabolism, glutathione synthesis, chaperoning of proteins, prolyl hydroxylation, proteasome function, and DNA synthesis/repair. Our study thus confirms previously nominated targets for mechanism-based multi-modal glioblastoma therapy, provides proof-of-concept for this workflow of multi-level data integration, and identifies novel candidates for which pharmacological inhibitors are readily available and whose targeting in combination with radio(chemo)therapy deserves further examination. In addition, our study also reveals that the presented workflow requires mRNA expression data, rather than genomic copy number or DNA methylation data, since no stringent correlation between these data levels could be observed. Finally, the data sets generated in the present study, including functional and multi-level molecular data of commonly used glioblastoma cell lines, represent a valuable toolbox for other researchers in the field of glioblastoma therapy resistance.