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The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α–helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Extended Data relating to Main Figure 3/AKT-driven progression of ALDH1A1+ cells in glioblastoma. A, Flow cytometry histograms showing ALDH1A1 expression in the paired treatment-naive vs. clinical relapse patient samples quantified in main Fig. 3A. Isotype controls in gray. B, Flow cytometry histograms of ALDH1A1 derived from treatment-naive and paired experimental relapse (TMZ→eR) BN46 cells. Data quantification in Supplementary Fig. S2B. C, Cartoon illustrating course of neurosphere experiments, quantified in main Fig. 3B and in Supplementary Fig. S3D, applying treatment-naive and paired experimental and clinical relapse patient cells. Right: Exemplary qRT-PCR data presenting knockdown efficacy of the siRNA approach in respective cells, normalized to siNT control. Mean of duplicates ± SD. D, Source data for Fig. 3B, and for Supplementary Fig. S3E. Dotplots show percent neurosphere-forming cells estimated from neurospheres at 12 days after seeding. Paired patient cell analysis, evaluating 1° and 2° neurospheres of indicated knockdown (siALDH1A1) and respective control (siNT) cell samples. Data shown as mean ± SD. E, Neurosphere assay, similar to main Fig. 3B on paired treatment-naive vs. experimental relapse (TMZ→eR) BN46 cells. Source data in Supplementary Fig. S3D. Data as mean ± SD. F, Protein patterns, similar to Fig. 3D. on paired cells from 2 additional discovery cohort samples. G, Flow cytometry histograms depicting pAKT(Ser473) expression in the paired treatment-naive vs. clinical relapse patient samples quantified in main Fig. 3E. Isotype controls in gray. H, Flow cytometry histograms of pAKT(Ser473) and, right, quantification of data derived from treatment-naive and paired experimental relapse (TMZ→eR) BN46 cells. Data represent mean fluorescence intensities (MFI), ± SD, normalized to isotype control (gray). I, Representative source data for Fig. 3F. Flow cytometry profiles of ALDH1A1/pAKT(Ser473)-labeled, paired treatment-naive vs. clinical relapse cell samples. J, Flow cytometry profiles of of ALDH1A1/pAKT(Ser473)-labeled, paired treatment-naive vs. experimental relapse conditions of BN46 cells (in vitro exposure to TMZ (TMZ→eR) or irradiation (RT→eR)).

Purpose:Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy.Experimental Design:To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts.Results:We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy.Conclusions:Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.

Supplementary Table S1. Discovery cohort of vital cells; Supplementary Table S2. Validation cohort/FFPE tissue samples; Supplementary Table S3. Differentially regulated genes; Supplementary Table S4. Selected gene variants compared between the paired treatment-naive vs. relapse cell samples.

Purpose: Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy. Experimental Design: To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts. Results: We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy. Conclusions: Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in humans and remains to have a fatal prognosis. Recent studies in animal models and human ALS patients indicate that increased reactive oxygen species (ROS) play an important role in the pathogenesis. Considering previous studies revealing the influence of ROS on mitochondrial physiology, our attention was focused on mitochondria in the murine ALS model, wobbler mouse. The aim of this study was to investigate morphological differences between wild-type and wobbler mitochondria with aid of superresolution structured illumination fluorescence microscopy, TEM, and TEM tomography. To get an insight into mitochondrial dynamics, expression studies of corresponding proteins were performed. Here, we found significantly smaller and degenerated mitochondria in wobbler motor neurons at a stable stage of the disease. Our data suggest a ROS-regulated, Ox-CaMKII-dependent Drp1 activation leading to disrupted fission-fusion balance, resulting in fragmented mitochondria. These changes are associated with numerous impairments, resulting in an overall self-reinforcing decline of motor neurons. In summary, our study provides common pathomechanisms with other ALS models and human ALS cases confirming mitochondria and related dysfunctions as a therapeutic target for the treatment of ALS.

Radiotherapy (RT) is applied in 45–60% of all cancer patients either alone or in multimodal therapy concepts comprising surgery, RT and chemotherapy. However, despite technical innovations approximately only 50% are cured, highlight a high medical need for innovation in RT practice. RT is a multidisciplinary treatment involving medicine and physics, but has always been successful in integrating emerging novel concepts from cancer and radiation biology for improving therapy outcome. Currently, substantial improvements are expected from integration of precision medicine approaches into RT concepts.Altered metabolism is an important feature of cancer cells and a driving force for malignant progression. Proper metabolic processes are essential to maintain and drive all energy-demanding cellular processes, e.g. repair of DNA double-strand breaks (DSBs). Consequently, metabolic bottlenecks might allow therapeutic intervention in cancer patients.Increasing evidence now indicates that oncogenic activation of metabolic enzymes, oncogenic activities of mutated metabolic enzymes, or adverse conditions in the tumor microenvironment can result in abnormal production of metabolites promoting cancer progression, e.g. 2-hyroxyglutarate (2-HG), succinate and fumarate, respectively. Interestingly, these so-called “oncometabolites” not only modulate cell signaling but also impact the response of cancer cells to chemotherapy and RT, presumably by epigenetic modulation of DNA repair.Here we aimed to introduce the biological basis of oncometabolite production and of their actions on epigenetic regulation of DNA repair. Furthermore, the review will highlight innovative therapeutic opportunities arising from the interaction of oncometabolites with DNA repair regulation for specifically enhancing the therapeutic effects of genotoxic treatments including RT in cancer patients.

Introduction Treatment of patients with metastatic melanoma is hampered by drug-resistance and often requires combination with radiotherapy as last-resort option. However, also after radiotherapy, clinical relapses are common. Methods & results Our preclinical models indicated a higher rate of tumour relapse when melanoma cells were first treated with BRAFV600E inhibition (BRAFi) followed by radiotherapy as compared to the reverse sequence. Accordingly, retrospective follow-up data from 65 stage-IV melanoma patients with irradiated melanoma brain metastases confirmed a shortened duration of local response of mitogen-activated protein kinase (MAPK)-inhibitor–pretreated compared with MAPK-inhibitor–naive intracranial metastases. On the molecular level, we identified JARID1B/KDM5B as a cellular marker for cross-resistance between BRAFi and radiotherapy. JARID1Bhigh cells appeared more frequently under upfront BRAFi as compared with upfront radiation. JARID1B favours cell survival by transcriptional regulation of genes controlling cell cycle, DNA repair and cell death. Conclusion The level of cross-resistance between combined MAPK inhibition and radiotherapy is dependent on the treatment sequence. JARID1B may represent a novel therapy-overarching resistance marker.

High tumor heterogeneity and increased therapy resistance acquired in a hypoxic tumor microenvironment remain major obstacles to successful radiotherapy. Others and we have shown that adaptation of cancer cells to cycling severe hypoxia and intermittent reoxygenation stress (chronic-cycling hypoxia) increases cellular antioxidant capacity thereby supporting resistance to chemotherapy and radiotherapy. Here we explored the involvement of antioxidant-associated mitochondrial transport-systems for maintenance of redox-homeostasis in adaptation to chronic-cycling hypoxia and associated radioresistance. Genetic or pharmacological inhibition of the mitochondrial dicarboxylate carrier (SLC25A10) or the oxoglutarate-carrier (SLC25A11) increased the cytotoxic effects of ionizing radiation (IR). But only targeting of SLC25A10 was effective in overcoming chronic-cycling hypoxia-induced enhanced death resistance in vitro and in vivo by disturbing increased antioxidant capacity. Furthermore, in silico analysis revealed that overexpression of SLC25A10 but not SLC25A11 is associated with reduced overall survival in lung- and breast-cancer patients. Our study reveals a role of SLC25A10 in supporting both, redox- and energy-homeostasis, ensuring radioresistance of cancer cells with tolerance to chronic-cycling hypoxia thereby proposing a novel strategy to overcome a mechanism of hypoxia-induced therapy resistance with potential clinical relevance regarding decreased patient survival.

Introduction: Balancing redox-homeostasis and energy metabolism allows cancer cells exposed to chemotherapy or radiotherapy to ensure DNA repair and survival. Our work revealed that adaptive changes in antioxidant defense and cancer cell metabolism in adverse environments enhance cancer cell radioresistance and create exploitable therapeutic metabolic dependencies for overcoming adaptive radioresistance1-4. Here, we aimed to decipher the mechanisms behind enhances lethality of ionizing radiation (IR) upon SLC25A1-inhibition (SLC25A1i) beyond disturbance of mitochondrial metabolism. Therefore, we explored a potential impact of SLC25A1i on the suggested accumulation of D-2-hydroxyglutarate (D2HG) and the repair of IR-induced lethal DNA lesions with a focus on homologous recombination (HR) repair. We hypothesized that metabolic disturbance of HR by SLC25A1i might offer opportunities for context-dependent lethality approaches. Methods: We used genetic (SLC25A1 RNAi) or pharmacologic SLC25A1i (small molecule SLC25A1 inhibitor CTPi2) to determine the effects of SLC25A1i alone or in combination with IR on 2HG-accumulation, cellular function, radiosensitivity and DNA repair kinetics in lung (A549, NCI-H460) and glioblastoma (U87-MG, T98G) cell lines. Isogenic MEF cell lines with defects in HR repair or non-homologous end joining (NHEJ) DSB repair were used to explore potential differences in the radiosensitizing effects of SLC25A1i in cells with defects in specific DSB repair pathways. Finally, we analyzed the potential of SLC25A1i to induce context-dependent lethality of IR with clinically relevant inhibitors of poly(ADP-ribose)-polymerase (PARP) or of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in cancer cells. Results: Genetic and pharmacologic SLC25A1i increased D2HG-levels, and sensitized lung cancer and glioblastoma cells to the cytotoxic action of IR. Radiosensitization was abrogated in MEFs with a HR defect. Accumulation of D2HG was associated with increased DNA damage and delayed resolution of IR-induced gH2AX and Rad51 foci. Combining SLC25A1i with PARP- or DNA-PKcs-inhibitors further delayed DNA repair kinetics of IR-induced DNA damage and enhanced the radiation-induced eradication of clonogenic tumor cells. Conclusion: The metabolic effects of SLC25A1i involve disturbance of energy metabolism and inhibition of HR execution. SLC25A1i increases the efficacy of IR in cancer cells but not in MEFs with a defect in HR. SLC25A1i might offer opportunities for the metabolic induction of HRness and thus increased lethality of radiotherapy in cells with defects in end-joining pathways or in combination with clinically relevant DSB repair inhibitors in cancer cells without or weak HR defects. 1 Matschke et al., Antioxid Redox Signal 2016,25:89-107, 2 Matschke et al., Radiat Oncol 2016,11(1):75, 3 Hlouschek et al., Cancer Lett. 2018 Dec 28;439:24-38, 4 Hlouschek et al., Front Oncol. 2018 May 25;8:170 Supported by grants of the DFG (GRK1739/2, MA 8970/1-1) and Mildred Scheel-Stiftung (70112711). Citation Format: Christian Kalthoff, Kexu Xiang, Corinna Muench, Julian Hlouschek, Verena Jendrossek, Johann Matschke. Inhibition of the citrate carrier SLC25A1 affects repair of radiation-induced DNA damage, increases radiosensitivity, and enhances lethality of IR in combination with DNA repair defects or DSB inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-022.

Cancer resistance to chemotherapy, radiotherapy and molecular-targeted agents is a major obstacle to successful cancer therapy. Herein, aberrant activation of the phosphatidyl-inositol-3-kinase (PI3K)/protein kinase B (Akt) pathway is one of the most frequently deregulated pathways in cancer cells and has been associated with multiple aspects of therapy resistance. These include, for example, survival under stress conditions, apoptosis resistance, activation of the cellular response to DNA damage and repair of radiation-induced or chemotherapy-induced DNA damage, particularly DNA double strand breaks (DSB). One further important, yet not much investigated aspect of Akt-dependent signaling is the regulation of cell metabolism. In fact, many Akt target proteins are part of or involved in the regulation of metabolic pathways. Furthermore, recent studies revealed the importance of certain metabolites for protection against therapy-induced cell stress and the repair of therapy-induced DNA damage. Thus far, the likely interaction between deregulated activation of Akt, altered cancer metabolism and therapy resistance is not yet well understood. The present review describes the documented interactions between Akt, its target proteins and cancer cell metabolism, focusing on antioxidant defense and DSB repair. Furthermore, the review highlights potential connections between deregulated Akt, cancer cell metabolism and therapy resistance of cancer cells through altered DSB repair and discusses potential resulting therapeutic implications.

Summary Cancer bioenergetics fuel processes necessary to maintain viability and growth under stress conditions. We hypothesized that cancer metabolism supports the repair of radiation-induced DNA double-stranded breaks (DSBs). We combined the systematic collection of metabolic and radiobiological data from a panel of irradiated cancer cell lines with mathematical modeling and identified a common metabolic response with impact on the DSB repair kinetics, including a mitochondrial shutdown followed by compensatory glycolysis and resumption of mitochondrial function. Combining ionizing radiation (IR) with inhibitors of the compensatory glycolysis or mitochondrial respiratory chain slowed mitochondrial recovery and DNA repair kinetics, offering an opportunity for therapeutic intervention. Mathematical modeling allowed us to generate new hypotheses on general and individual mechanisms of the radiation response with relevance to DNA repair and on metabolic vulnerabilities induced by cancer radiotherapy. These discoveries will guide future mechanistic studies for the discovery of metabolic targets for overcoming intrinsic or therapy-induced radioresistance., Graphical abstract, Highlights • Mathematical modeling revealed common early mitochondrial shutdown upon irradiation • Mitochondrial shutdown correlates to oxidizing effects of ionizing radiation • Compensatory glycolysis fuels DNA repair during mitochondrial shutdown • Oncogenic mutations impact mitochondrial recovery and DNA repair kinetics, Mathematical biosciences; Cancer systems biology; Cancer

Flexibility and reprogramming of cancer metabolism supports cancer progression and therapy resistance. In previous work we described opportunities for overcoming environment-induced resistance to ionizing radiation (IR) by pharmacologic inhibition of metabolic processes [1,2,3]. Here, we hypothesized that certain aspects of cancer metabolism will support the repair of radiation-induced DNA double-strand breaks (DSBs) and that the identification of pathways critical to the repair of radiation-induced DNA damage and cell survival will allow to enhance sensitivity of cancer cells to IR by interfering with such metabolic liabilities. Yet little is known about metabolic aspects of the cancer cell response to IR. We therefore systematically screened a panel of cancer cell lines with distinct genetic backgrounds for time-dependent changes in their acute metabolic response to IR and recorded the kinetics of IR-induced DSB in parallel. Mathematical modelling of the obtained metabolic and radiobiological data revealed a common metabolic response of irradiated cancer cells with impact on the kinetics of DSB repair. These were characterized by a rapid but transient downregulation of glycolysis and mitochondrial respiration, and gradual reactivation of glycolysis prior to mitochondrial recovery, presumably to compensate for disturbed metabolic functions of impaired mitochondria. Application of fractionated IR doses retarded mitochondrial recovery, highlighting the inhibitory effects of IR on mitochondrial functions. Furthermore, combining IR with inhibitors of compensatory glycolysis slowed mitochondrial recovery and DNA repair kinetics, supporting the functional relevance of compensatory glycolysis for DNA repair. Mathematical modeling of the metabolic response to IR allowed us to generate new hypotheses on the metabolic requirements of irradiated cancer cells and potential associated metabolic vulnerabilities. Further mechanistic studies will reveal the role of oncogenic drivers and co-mutations in metabolic enzymes for metabolic flexibility and metabolic liabilities of irradiated cancer cells in support of personalized approaches for combinatorial treatments. [1] Matschke J, et al., Antioxid Redox Signal 2016; 25: 89-107. [2] Hlouschek J et al., Front Oncol 2018; 8: 170. [3] Hlouschek J et al., Cancer Lett 2018; 439: 24-38. Supported by grants of the DFG (GRK1739/2 to V.J. and MA 8970/1-1 to J.M.), the Deutsche Krebshilfe/Mildred Scheel-Stiftung (70112711 to V.J.). Citation Format: Adam Krysztofiak, Klaudia A. Szymonowicz, Julian Hlouschek, Christoph Waterkamp, Kexu Xiang, Daniel Hoffmann, Verena Jendrossek, Johann Matschke. Modeling common aspects of the metabolic response of cancer cells to ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3065.

Oxygen is essential to the human life and life of all aerobic organisms. The complete oxidation of nutrients for the biological energy supply is one of the most important prerequisites for the formation of higher life forms. However, cells that benefit from oxidative respiration also suffer from reactive oxygen species because they adapted to oxygen as an energy source. Healthy cells balance the formation and elimination of reactive oxygen species thereby creating and keeping reactive oxygen species-homeostasis. When the concentration of free radicals exceeds a critical level and homeostasis is disturbed, oxidative stress occurs leading to damage of multiple cellular molecules and compartments. Therefore, oxidative stress plays an important role in the physiology and pathology of various diseases. Often, the antioxidant protection system becomes pathologically unbalanced in the genesis of several diseases, leading to functional losses of the organism, as in the case of amyotrophic lateral sclerosis, or cells develop metabolic mechanisms to use this system as protection against external influences, such as in the case of glioblastoma cells. Either way, understanding the underlying deregulated mechanisms of the oxidative protection system would allow the development of novel treatment strategies for various diseases. Thus, regardless of the direction in which the reactive oxygen species-homeostasis disequilibrate, the focus should be on the oxidative protection system.

Poster-Abstract

Introduction Hypoxia-mediated resistance of solid tumours to ionising radiation is a major obstacle to successful radiotherapy. We showed previously that chronic cycling hypoxia drives the evolution of anoxia/reoxygenation-tolerant (ART) cancer cells with increased resistance to ionising radiation. Radiation resistance of ART cancer cells was associated with complex metabolic reprogramming, Matschke et al., Antioxid Redox Signal 201625:89–107; Matschke et al., Radiat Oncol 201611(1:75). Aim of the present study was to gain a more comprehensive understanding of the metabolic adaptation of cancer cells and to systematically explore opportunities for targeted pharmacologic intervention based on their suspected specific metabolic needs upon irradiation. Material and methods We compared gene expression profiles of ART and control cancer cells by microarray analysis and validated genes of interest by qRT-PCR. We used LC-MS high-throughput metabolomics, metabolic flux analyses, nutrient deprivation and drugs interfering with metabolism to characterise the cellular metabolic state without/with irradiation. Results and discussions Our microarray data indicated changes in major metabolic pathways after chronic cycling hypoxia selection. Furthermore, tolerance to severe hypoxia was associated with the formation of enlarged mitochondria in ART NCI-H460 cells. The analysis of metabolic alterations in irradiated cancer cells by LC-MS high-throughput metabolome analysis demonstrated a high and time-dependent need of irradiated cancer cells in central metabolism. Targeting of induced metabolic alterations disturbed redox homeostasis, altered mitochondrial metabolism and sensitised cancer cells to ionising radiation. Conclusion Specific metabolic requirements under stress conditions such as severe hypoxia or irradiation render cancer cells vulnerable to metabolic inhibitors alone and in combination with ionising radiation in a context and cell type-dependent manner. Supported by grants of the DFG (GRK1739/2) and the Deutsche Krebshilfe (1 10 355 and 70112711).

Tumor hypoxia is a major biological factor causing poor patient outcome. Evidence is increasing that improved protection against reactive oxygen species (ROS) participates in therapy resistance of chronically hypoxic cancer cells. We aimed at characterizing the relevance of improved ROS defense for radiation resistance of cancer cells with tolerance to cycling anoxia/re-oxygenation stress ("anoxia-tolerant") and at designing rational treatment strategies for overcoming the resulting therapy resistance by targeting the underlying mechanisms identified in an in vitro model.We demonstrate that chronic exposure of NCH-H460 lung adenocarcinoma, DU145 prostate cancer, and T98G glioblastoma cells to cycling anoxia/re-oxygenation stress induced upregulation of the aspartate-aminotransferase glutamic-oxaloacetic transaminase (GOT1), particularly in RAS-driven anoxia-tolerant NCI-H460 cells. Altered glutamine utilization of the anoxia-tolerant cancer cells contributed to the observed decrease in cellular ROS levels, the increase in cellular glutathione levels, and improved cell survival on ROS-inducing treatments, including exposure to ionizing radiation. Importantly, targeting glutamine-dependent antioxidant capacity or glutathione metabolism allowed us to hit anoxia-tolerant cancer cells and to overcome their increased resistance to radiation-induced cell death. Targeting glutathione metabolism by Piperlongumine also improved the radiation response of anoxia-tolerant NCI-H460 cells in vivo.Improved antioxidant capacity downstream of up-regulated GOT1-expression is a characteristic of anoxia-tolerant cancer cells and is predictive for a specific vulnerability to inhibition of glutamine utilization or glutathione metabolism, respectively.Unraveling the molecular alterations underlying improved ROS defense of anoxia-tolerant cancer cells allows the design of rational strategies for overcoming radiation resistance caused by tumor cell heterogeneity in hypoxic tumors. Antioxid. Redox Signal. 25, 89-107.

Introduction Melanoma progression is often associated with the growth of anatomically critical metastases that require immediate therapy response, e.g. in the brain. In such situations, systemic drugs are often sequentially combined with radiotherapy. However, there is a lack of clinical and preclinical studies that systematically examine the potential effects of therapy-timing on treatment efficacy and radio-cross-resistance in melanoma. Material and methods We established an experimental platform comprising cell culture and mouse xenograft models that allows predicting the outcome of different BRAF V600E inhibitor-radiotherapy sequences. Retrospectively collected clinical follow up data of 65 stage IV melanoma patients with brain metastases were analysed for their intracranial response after radiotherapy depending on the sequence of co-administered MAPK inhibition. Results and discussions Our models indicated a higher rate of tumour relapse when melanoma cells were first treated with BRAF V600E inhibition followed by radiotherapy as compared to the reverse sequence. We identified the H3K4 demethylase JARID1B/KDM5B as a cellular marker for cross-resistance between BRAF V600E inhibition and radiotherapy. JARID1B high cells appeared more frequently in vitro and in vivo under upfront BRAF V600E inhibition as compared to upfront radiation. Accordingly, retrospective follow-up data from irradiated melanoma brain metastases confirmed a shortened duration of response of MAPK inhibitor-pretreated compared to MAPK inhibitor-naive metastases. Mechanistically, JARID1B favours cell survival by transcriptional regulation of genes controlling cell cycle, DNA repair, and cell death. Conclusion Our preclinical results and the clinical follow up of patients support a higher rate of tumour relapse when melanomas are treated with upfront MAPK inhibition as compared to upfront radiation. JARID1B may represent a novel therapy-overarching resistance marker that could guide future therapy sequencing.