DIRAS2 Contributes to Radiation Resistance of Renal Cell Carcinoma Via Autophagy Induction and MKK4-JNK1 Pathway Activation

Radiation resistance has been regarded as a main obstacle to improve the definitive treatment of renal cell carcinoma (RCC), of which clear cell RCC (ccRCC) is the most common histological type. However, the molecular mechanism underlying the radiation resistance remains largely unclear. In this study, we investigated the effect of DIRAS2 on the response to ionizing radiation (IR) in human ccRCC cells. Here, we found the expression level of DIRAS2 was significantly upregulated in human ccRCC tissues using the Oncomine platform and the Cancer Genome Atlas (TCGA) database, which was further validated by immunohistochemistry. Overexpression of DIRAS2 promoted radiation resistance of ccRCC cells based on clonogenic survival assay and enhanced the levels of radiation induced-autophagy. Moreover, inhibition of autophagy by chloroquine (CQ) pre-treatment largely eliminated the effect of DIRAS2 overexpression on radiation-resistance. Finally, molecular mechanism investigation showed that overexpression of DIRAS2 upregulated the activity of mitogen-activated protein kinase (MAPK) kinase 4 (MKK4)- c-Jun NH2-terminal kinase 1 (JNK1)-Bcl-2 pathway in response to IR. Taken together, these results indicate that DIRAS2 may confer radiation resistance on human RCC via autophagy induction through MKK4-JNK1-Bcl-2 signaling pathway.

Microdosimetric characterization of a clinical proton therapy beam: comparison between simulated lineal energy distributions in spherical water targets and experimental measurements with a silicon detector

Objective Treatment planning based on computer simulations were proposed to account for the increase in the relative biological effectiveness (RBE) of proton radiotherapy beams near to the edges of the irradiated volume. Since silicon detectors could be used to validate the results of these simulations, it is important to explore the limitations of this comparison. Approach Microdosimetric measurements with a MicroPlus Bridge V2 silicon detector (thickness = 10 µm) were performed along the Bragg peak of a clinical proton beam. The lineal energy distributions, the dose mean values, and the RBE calculated with a biological weighting function were compared with simulations with PHITS (microdosimetric target = 1 µm water sphere), and published clonogenic survival in vitro RBE data for the V79 cell line. The effect of the silicon-to-water conversion was also investigated by comparing three different methodologies (conversion based on a single value, novel bin-to-bin conversions based on SRIM and PSTAR). Main results Mainly due to differences in the microdosimetric targets, the experimental dose-mean lineal energy and RBE values at the distal edge were respectively up to 53% and 28% lower than the simulated ones. Furthermore, the methodology chosen for the silicon-to-water conversion was proven to affect the dose mean lineal energy and the RBE10 up to 32% and 11% respectively. The best methodology to compensate for this underestimation was the bin-to-bin silicon-to-water conversion based on PSTAR. Significance This work represents the first comparison between PHITS-simulated lineal energy distributions in water targets and corresponding experimental spectra measured with silicon detectors. Furthermore, the effect of the silicon-to-water conversion on the RBE was explored for the first time. The proposed methodology based on the PSTAR bin-to-bin conversion appears to provide superior results with respect to commonly used single scaling factors and is recommended for future studies.

A Combination of Cabozantinib and Radiation Does Not Lead to an Improved Growth Control of Tumors in a Preclinical 4T1 Breast Cancer Model

The tyrosine kinase inhibitor Cabozantinib has been applied in clinical studies in combination with radiotherapy. We investigated the effect of such combination on triple-negative 4T1 cells as a metastatic breast cancer model in vitro and in vivo upon inoculation in BALB/c mice. In vitro assays indicated a potential for improved effects using the combination. Both Cabozantinib (2.5 µM) and 10 Gy of 250 kV x-rays were able to cease the growth of 4T1 cells as revealed by growth curves. In a clonogenic survival assay, the effect of Cabozantinib added on the effects of irradiation and the effectiveness of inhibiting the clonogenic survival was found to be 2 (RBE10). Additionally, cell death measurements of apoptosis plus necrosis revealed a synergistic effect when combining irradiation with Cabozantinib. Surprisingly, however, in vivo tumor growth kinetics showed no additional effect in growth control when irradiation was used together with Cabozantinib. Since both ionizing radiation and Cabozantinib are acknowledged to feature immunogenic effects, we additionally investigated the effect of the treatments on lung metastases. No difference to the control groups was found here, neither for irradiation nor Cabozantinib alone nor in combination. Yet, upon analysis of the mice’ livers, CD11b-positive cells, indicating immune suppressive myeloid derived suppressor cells were found diminished following treatment with Cabozantinib. In conclusion, despite promising in vitro controls of the combination of Cabozantinib and irradiation, tumor growth control was not increased by the combination, which was true also for the occurrence of lung metastases.

DNA repair inhibitors sensitize cells differently to high and low LET radiation

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6–1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G2/M arrest.

DUSP6 regulates radio-sensitivity in glioblastoma by modulating the recruitment of p-DNAPKcs at DNA double-strand breaks

Glioblastoma (GBM) has poor median survival due to its resistance to chemo-radiotherapy regimen, resulting in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is up-regulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies (n=11) compared to primary biopsies (n=11). Importantly, although reported as cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse population of GBM cell lines generated from in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radio-sensitized primary and importantly, relapse GBM cells by inhibiting the recruitment of p-DNAPKcs, subsequently down-regulating the recruitment of γH2AX and 53BP1. This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

Expressing MLH1 in HCT116 cells increases cellular resistance to radiation by activating the PRKAC

Mut L homolog-1 (MLH1) is a key DNA mismatch repair protein which participates in the sensitivity to DNA damaging agents. However, its role in the radiosensitivity of tumor cells is less well characterized. In this study, we investigated the role of MLH1 in cellular responses to ionizing radiation (IR) and explored the signaling molecules involved. The isogenic pair of MLH1 proficient (MLH1+) and deficient (MLH1–) human colorectal cancer HCT116 cells was exposed to IR for 24 h at the dose of 3 cGy. The clonogenic survival was examined by the colony formation assay. Cell cycle distribution was analyzed with flow cytometry. Changes in the protein level of MLH1, DNA damage marker γH2AX, and protein kinase A catalytic subunit (PRKAC), a common target for anti-tumor drugs, were examined with Western blotting. The results showed that the HCT116 (MLH1+) cells demonstrated increased radio-resistance with increased S population, decreased G2 population, a low level of γH2AX, a reduced ratio of phosphorylated PRKACαβ to total PRKAC, and an elevated level of total PRKAC and phosphorylated PRKACβII following IR compared with the HCT116 (MLH1–) cells. Importantly, silencing PRKAC in HCT116 (MLH1+) cells increased the cellular radiosensitivity. In conclusion, MLH1 may increase cellular resistance to IR by activating PRKAC. Our finding is the first to demonstrate the important role of PRKAC in MLH1-mediated radiosensitivity, suggesting that PRKAC has potential as a biomarker and a therapeutic target for increasing radio-sensitization.

Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases a subpopulation of the treated cells can exhibit a sublethal response, termed minority MOMP. In this phenomenon, the affected cells survive, despite a low level of caspase activation and a subsequent limited activation of the endonuclease CAD (DFFB). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. To discover genes affecting MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells. Among these were genes related to mitochondrial dynamics and mitophagy, which participate in the mitochondrial quality control (MQC) system. To test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

EXTH-48. NOVEL COMBINATION THERAPY IN PRECLINICAL GLIOMA MODELS USING THE CELL CYCLE STABILIZING COMPOUND ARGYRIN F AND CHECKPOINT INHIBITION

Glioblastoma are incurable aggressive tumors and remain a therapeutic challenge. Glioblastoma frequently harbor alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. Here, we aimed to investigate the anti-glioma activity of the cell cycle-stabilizing compound Argyrin F and its potential treatment-induced vulnerabilities to exploit possibilities for novel combination therapies. We investigated cell viability, clonogenic survival, cell cycle status and immunoblots of human and murine glioma cells treated with Argyrin F. Moreover, we established an ex vivo glioma model using residual freshly resected tissue from patients, i.e. patient-derived microtumors (PDMs). Additionally, we extracted autologous tumor infiltrating lymphocytes (TILs) to perform co-culturing experiments. We performed mass spectrometry-based immunopeptidomics and used the orthotopic syngeneic SMA560/VM/Dk glioma mouse model. Argyrin F displayed anti-glioma efficacy in glioma cell lines in vitro and in PDM models ex vivo. Moreover, Argyrin F treatment induced cell cycle arrest, reduced clonogenic survival in vitro and prolonged survival in vivo. Argyrin F-treated SMA560 glioma displayed 4.6-fold more glioma-infiltrating CD8+ T cells. We discovered a distinctive treatment-induced immunopeptidome. Combination of Argyrin F plus PD-1 antibody increased cellular toxicity in PDM/TILs co-cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. We conclude that our experimental data suggest a novel combination of Argyrin F plus PD-1 blockade and its clinical translation.

CBIO-24. NF2 LOSS-OF-FUNCTION COOPERATES WITH HYPOXIA TO DRIVE RADIATION RESISTANCE IN GRADE 2 MENINGIOMAS

World health organization grade 2 meningiomas (G2M) are associated with an aggressive natural history characterized by frequent recurrence, resistance to radiation therapy (RT), and poor survival. We and others have shown that intratumoral necrosis is an independent predictor of disease progression in G2Ms treated with RT, suggesting that necrosis is a histopathological biomarker of radiation resistance. However, the mechanisms of radiation resistance in G2M remain unclear and treatment of radioresistant G2M is challenging. We performed genetic sequencing and histopathological analysis of 121 G2Ms and found that NF2 mutations are associated with intratumoral necrosis. To further investigate the relationship between NF2 loss-of-function, necrosis, and radiation resistance, we developed an in vitro model system using the IOMM-Lee meningioma cell line. In clonogenic survival assays, we found that NF2 knockdown under normoxia had little to no effect on radiation resistance compared to control. However, in the setting of hypoxia, NF2 knockdown significantly increased radiation resistance by maintaining cellular proliferation without impacting cell death. To understand the molecular basis of NF2 loss-of-function on radiation resistance, we performed bulk RNA sequencing of cells in our in vitro hypoxia/RT model and 10 patient G2Ms (5 necrotic and 5 non-necrotic tumors). GO and KEGG pathway analysis of the in vitro dataset revealed that NF2 deficiency triggers upregulation of pathways involved in DNA damage repair and cellular proliferation. Integration of differentially expressed genes between the in vitro NF2 loss-of-function model and necrotic patient tumors identified shared upregulation of S100A4, which promotes cell cycle progression, and downregulation of WNK2, a cell proliferation inhibitor. Together, these results suggest NF2 loss-of-function in the setting of hypoxia confers radiation resistance through a transcriptional program that promotes proliferation and DNA repair.