Radiation-induced Cell Killing Research Articles

Restoration of Radiosensitivity by Soya Isoflavone Genistein is Accomplished by Facilitating DNA Damage Response in Radioresistant Cervical Cancer in vitro

ABSTRACT Context: Enhancing radiotherapeutic efficacy in tumor cells and sparing the normal tissues are major clinical concerns for the betterment of cancer therapy. Genistein (GEN) being a radiosensitizer ameliorates the effectiveness of radiation-induced cell killing by inducing DNA damage. This molecule is accountable for minimizing radiation-related toxicity and protecting healthy cells. However, the explicit mechanism of action of such molecules needs exploration. Aims: The objective of this study is to investigate the mechanistic action of GEN in cervical cancer cell radiosensitization. Settings and Design: Cervical squamous carcinoma cell SiHa and a radioresistant subline SiHa/RR (developed and isolated from SiHa) were taken for this study. The experiments were performed by pretreating the cells with IC30 dose of GEN, followed by acute irradiation to detect the impact of GEN in imparting radiosensitivity. Subjects and Methods: Optimal dose selection of GEN was performed by MTT assay, and radiosensitizing potency was determined by pretreating the cells with IC30 dose of GEN, followed by challenging with acute incremental doses of radiation. Mechanistic parameters were checked by clonogenic assay, cell cycle analysis, DNA damage estimation, apoptosis, and wound healing-sphere-forming assay. Statistical Analysis Used: Statistical analysis was performed in GraphPad software by performing the Student’s t-test. Results: Results depicted decreased numbers of colonies, increased frequency of DNA damage and apoptotic cells, and suppressed wound healing ability along with restrained sphere-forming ability upon the intervention of cells with GEN before radiation exposure. Such observations implied that GEN pretreatment renders improved radiosensitivity in cervical cancer by increased DNA damage-mediated G2/M arrest with subsequent apoptosis. Conclusions: GEN by inducing DNA damage stimulates radiation-induced cell killing in vitro.

HGG-41. COMBINATORIAL BH3-MIMETIC STRATEGY FOR OVERCOMING RADIORESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND High-grade gliomas (HGGs) are a leading cause of cancer-associated death in children. Radiation therapy (RT) continues to be the only standard-of-care treatment across HGG subtypes, but disease frequently recurs following only a transient response. We have recently identified that HGGs utilize the pro-survival BCL-2 family of proteins to evade cell death after radiation, and we demonstrated that effective inhibition of BCL-2 with the CNS-penetrant drug venetoclax could be utilized to disrupt this resistance and augment radiation-induced cell killing. While this treatment significantly prolonged survival in orthotopic mouse models of pediatric HGG, tumors invariably developed resistance to single-agent venetoclax and recurred. METHODS We profiled tumors from venetoclax-resistant animals for alternate BCL-2 family binding. We then assessed susceptibility to MCL1 inhibition in venetoclax-treated or treatment naïve culture models. Synergy was tested in vitro, and mitochondrial ROS production and induction of apoptosis was assessed using the MCL1-specific agent S63845. Finally, venetoclax and S63845 were tested in combination following fractionated radiotherapy in orthotopic models of DIPG. RESULTS MCL1 is an acquired dependency following treatment with RT + venetoclax. S63845 synergizes with venetoclax following RT to drive mitochondrial ROS production and apoptosis. The combination of venetoclax and S63845 significantly prolongs survival in xenograft models of DIPG. CONCLUSIONS Combinatorial targeting with BH3 mimetics augments radiation-induced cell killing and may open new therapeutic avenues for pediatric HGG.

Morphological Effects and In Vitro Biological Mechanisms of Radiation-Induced Cell Killing by Gold Nanomaterials.

Gold nanomaterials have been shown to augment radiation therapy both in vitro and in vivo. However, studies on these materials are mostly phenomenological due to nanoparticle heterogeneity and the complexity of biological systems. Even accurate quantification of the particle dose still results in bulk average biases; the effect on individual cells is not measured but rather the effect on the overall population. To perform quantitative nanobiology, we coated glass coverslips uniformly at varying densities with Au nanoparticle preparations with different morphologies (45 nm cages, 25 nm spheres, and 30 nm rods). Consequently, the effect of a specific number of particles per unit area in contact with breast cancer cells growing on the coated surfaces was ascertained. Gold nanocages showed the highest degree of radiosensitization on a per particle basis, followed by gold nanospheres and gold nanorods, respectively. All three materials showed little cytotoxic effect at 0 Gy, but clonogenic survival decreased proportionally with the radiation dose and particle coverage density. A similar trend was seen in vivo in the combined treatment antitumor response in 4T1 tumor-bearing animals. The presence of gold affected the type and quantity of reactive oxygen species generated, specifically superoxide and hydroxyl radicals, and the concentration of nanocages correlated with the development of more numerous double-stranded DNA breaks and increased protein oxidation as measured by carbonylation. This work demonstrates the dependence on morphology and concentration of radiation enhancement by gold nanomaterials and may lead to a novel method to differentiate intra- and extracellular functionalities of gold nanomedicine treatment strategies. It further provides insights that can guide the rational development of gold nanomaterial-based radiosensitizers for clinical use.

An artificial intelligence-based model for cell killing prediction: development, validation and explainability analysis of the ANAKIN model

The present work develops ANAKIN: an Artificial iNtelligence bAsed model for (radiation-induced) cell KIlliNg prediction. ANAKIN is trained and tested over 513 cell survival experiments with different types of radiation contained in the publicly available PIDE database. We show how ANAKIN accurately predicts several relevant biological endpoints over a wide broad range on ion beams and for a high number of cell-lines. We compare the prediction of ANAKIN to the only two radiobiological models for Relative Biological Effectiveness prediction used in clinics, that is the Microdosimetric Kinetic Model and the Local Effect Model (LEM version III), showing how ANAKIN has higher accuracy over the all considered cell survival fractions. At last, via modern techniques of Explainable Artificial Intelligence (XAI), we show how ANAKIN predictions can be understood and explained, highlighting how ANAKIN is in fact able to reproduce relevant well-known biological patterns, such as the overkilling effect.

Ultrasound-Stimulated Microbubbles Enhance Radiation-Induced Cell Killing.

Recent in vivo studies using ultrasound-stimulated microbubbles as a localized radiosensitizer have had impressive results. While in vitro studies have also obtained similar results using human umbilical vein endothelial cells (HUVEC), studies using other cell lines have had varying results. This study was aimed at investigating any increases in radiation-induced cell killing in vitro using two carcinoma lines not previously investigated before (metastatic follicular thyroid carcinoma cells [FTC-238] and non-small cell lung carcinoma cells [NCI-H727]), in addition to HUVEC. Cells were treated using a combination of 1.6% (v/v) microbubbles, ∼90 s of 2-MHz ultrasound (mechanical index=0.8) and 0-6 Gy of kilovolt or MV X-rays. Cell viability assays obtained 72 h post-treatment were normalized to untreated controls, and analysis of variance was used to determine statistical significance. All cells treated with combined ultrasound-stimulated microbubbles and radiation exhibited decreased normalized survival, with statistically significant effects observed for the NCI-H727 cells. No statistically significant differences in effects were observed using kV compared with MV radiation. Further studies using increased microbubble concentrations may be required to achieve statistically significant results for the FTC-238 and HUVEC lines.

Radiation-induced Pancreatic Cancer Cell Killing by Simultaneous Inhibition of NF-kB and Warburg Metabolism

Radiation-induced Pancreatic Cancer Cell Killing by Simultaneous Inhibition of NF-kB and Warburg Metabolism

Radiation-induced Pancreatic Cancer Cell Killing by Simultaneous Inhibition of NF-kB and Warburg Metabolism

Radiation-induced Pancreatic Cancer Cell Killing by Simultaneous Inhibition of NF-kB and Warburg Metabolism

Modeling the impact of spatial oxygen heterogeneity on radiolytic oxygen depletion during FLASH radiotherapy

Purpose. It has been postulated that the delivery of radiotherapy at ultra-high dose rates (‘FLASH’) reduces normal tissue toxicities by depleting them of oxygen. The fraction of normal tissue and cancer cells surviving radiotherapy depends on dose and oxygen levels in an exponential manner and even a very small fraction of tissue at low oxygen levels can determine radiotherapy response. To quantify the differential impact of FLASH radiotherapy on normal and tumour tissues, the spatial heterogeneity of oxygenation in tissue should thus be accounted for. Methods. The effect of FLASH on radiation-induced normal and tumour tissue cell killing was studied by simulating oxygen diffusion, metabolism, and radiolytic oxygen depletion (ROD) over domains with simulated capillary architectures. To study the impact of heterogeneity, two architectural models were used: (1) randomly distributed capillaries and (2) capillaries forming a regular square lattice array. The resulting oxygen partial pressure distribution histograms were used to simulate normal and tumour tissue cell survival using the linear quadratic model of cell survival, modified to incorporate oxygen-enhancement ratio effects. The ratio (‘dose modifying factors’) of conventional low-dose-rate dose and FLASH dose at iso-cell survival was computed and compared with empirical iso-toxicity dose ratios. Results. Tumour cell survival was found to be increased by FLASH as compared to conventional radiotherapy, with a 0–1 order of magnitude increase for expected levels of tumour hypoxia, depending on the relative magnitudes of ROD and tissue oxygen metabolism. Interestingly, for the random capillary model, the impact of FLASH on well-oxygenated (normal) tissues was found to be much greater, with an estimated increase in cell survival by up to 10 orders of magnitude, even though reductions in mean tissue partial pressure were modest, less than ∼7 mmHg for the parameter values studied. The dose modifying factor for normal tissues was found to lie in the range 1.2–1.7 for a representative value of normal tissue oxygen metabolic rate, consistent with preclinical iso-toxicity results. Conclusions. The presence of very small nearly hypoxic regions in otherwise well-perfused normal tissues with high mean oxygen levels resulted in a greater proportional sparing of normal tissue than tumour cells during FLASH irradiation, possibly explaining empirical normal tissue sparing and iso-tumour control results.

CD81 Enhances Radioresistance of Glioblastoma by Promoting Nuclear Translocation of Rad51.

Simple SummaryCD81 is highly expressed in glioblastoma (GBM) as a transmembrane protein. The functional study demonstrated that CD81 contributed to radioresistance of GBM. Further evidence showed that CD81 expression was closely related to DNA damage response and homologous recombination repair (HRR) was responsible for the CD81 mediated radioresistance. Particularly, nuclear membrane protein CD81 assisted the nuclear transport of Rad51, a key protein involved in HRR process after irradiation. Overall, CD81 may be utility as a predictive biomarker and therapeutic target of radioresistant GBM.Glioblastoma (GBM) is the most common type of primary tumor in central nervous system in adult with a 5-year survival rate of ≤5%. Despite of recent advances in tumor radiotherapy, the prognosis of GBM remains to be dismal due to radioresistance. In this study, we identified CD81 as a potential biomarker of GBM radioresistance with the analysis of upregulated genes in human glioma radioresistant cell lines U251R and T98G in comparison with U251 cells. In vitro and in vivo experiments demonstrated that suppressing CD81 by siRNA/shRNA enhanced radiation-induced cell killing and DNA damage of γ-H2AX formation, and delayed tumor xenograft growth of GBM. Mechanistically, we found that knockdown of CD81 significantly decreased radiation-induced expression of nuclear Rad51, a key protein involved in homologous recombination repair (HRR) of DNA, suggesting that CD81 is essential for DNA damage response. Meanwhile, when the cells were treated with B02, a Rad51 inhibitor, silencing CD81 would not sensitize GBM cells to radiation, which further illustrates that Rad51 acts as an effector protein of CD81 in tumor radioresistance. Dual immunofluorescence staining of CD81 and Rad51 illustrated that nuclear membrane CD81 contributed to the nuclear transport of Rad51 after irradiation. In conclusion, we demonstrated for the first time that CD81 not only played a vital role in DNA repair through regulating Rad51 nuclear transport, but also might serve as a potential target of GBM radiotherapy.

Nucleoside Analogs Radiosensitize G0 Cells by Activating DNA End Resection and Alternative End-Joining

Alternative end-joining (alt-EJ) is a DNA end resection-dependent, error-prone pathway utilized by vertebrate cells to repair DNA double-strand breaks (DSBs), but its engagement is linked to chromosomal translocations and genomic instability. Here, we report that when proliferating cells are exposed to ionizing radiation, treatment with nucleoside analogs (NAs) causes strong radiosensitization by increasing engagement of alt-EJ, while at the same time suppressing homologous recombination (HR) in S- and G2phase cells. This NA-mediated pathway shift may reflect a passive compensatory engagement of alt-EJ following HR suppression that is specific for S- and G2-phase cells, and/or the direct activation of alt-EJ throughout the cell cycle. To distinguish between these possibilities, we utilize here a cell culture model that exploits genetic and cell cycle-dependent inactivation of DSB repair pathways, to exclusively study alt-EJ and its modulation by NAs in murine and human cell lines. To this end, we allow LIG4-/--deficient cells to accumulate in G1/G0 phase by transfer to serum-deprived media and obtain cells deficient in c-NHEJ owing to the genetic LIG4 knockout, deficient in HR owing to the absence of S- or G2-phase cells, and compromised in their ability to carry out alt-EJ owing to their accumulation in G0. We find that in these cells irradiation and treatment with the NA, β-arabinofuranosyladenine (araA), and to a lesser degree with other NAs, promptly activates suppressed alt-EJ that now functions at levels approximating those of c-NHEJ in wild-type cells. Results at high dose (20 Gy) generated using pulsed-field gel electrophoresis (PFGE) are corroborated by results at low dose (1 Gy) generated by scoring 53BP1 foci. Strikingly, araA treatment activates a normally undetectable DNA-end-resection at DSBs, which requires ATR activity, but proceeds unimpeded after CtIP knockdown. Treatment with araA increases the formation of chromosomal aberrations and enhances radiation-induced cell killing. The results support direct stimulation of resection by NAs and alt-EJ as a mechanism of their documented radiosensitizing potential. We propose that this stimulation also occurs in repair-proficient cells and that it occurs throughout the cell cycle. It may therefore be harnessed to develop protocols combining NAs with radiation to treat human cancer.

ATM Kinase Inhibition Preferentially Sensitises PTEN-Deficient Prostate Tumour Cells to Ionising Radiation.

Simple SummaryProstate cancer is the most frequently diagnosed cancer in men. Despite the importance of radical radiotherapy for the management of this disease, recurrence remains a challenge. PTEN is a tumour suppressor that is frequently inactivated in advanced prostate cancer and has been associated with relapse following radiotherapy. The present study shows that the role of PTEN in response to ionizing radiation is complex. Furthermore, it demonstrates that in the absence of PTEN, an increased response to combined treatment using radiotherapy and the ATM inhibitor KU-60019 can be observed. Our findings provide a strong rationale for evaluating loss of PTEN in prostate cancer as a therapeutic target for ATM inhibitor in combination with radiotherapy in the clinical setting.Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8–18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

Role of Inhibition of DNA Double-Strand Break Repair and Autophagy in Pancreatic Cancer Cytotoxicity and Radiation-Induced Cell Killing

Low survival rates of pancreatic ductal adenocarcinoma (PDAC) are attributed to its detection at advanced stage and high chemotherapy and radiation resistance. Previous work in the lab has shown that resistance to radiation therapy can be decreased through simultaneous inhibition of the anti-apoptotic NF-kB cell survival signaling pathway by dimethyl-amino-parthenolide (DMAPT) and inhibition of Warburg cancer metabolism by dichloroacetate (DCA). We sought to determine the mechanism by which dual treatment with DMAPT and DCA increases cytotoxicity and radiation-induced cell killing in pancreatic cancer. Established pancreatic cancer cell lines, MIA-PaCa-2, Panc-1, and AsPc-1 cells were plated in T-25 flasks. For single fraction experiments, cells were treated with no drugs or both DMAPT and DCA at 24 and 48 hours after plating and irradiated with 0-6Gy with 160 kVp X-rays. To assess split dose repair, experimental groups were 5Gy, 2 x 2.5Gy, 5Gy with DMAPT and DCA, and 2x2.5Gy with DMAPT and DCA. Colonies were counted after 10 days of growth, and plating efficiency and survival fractions were calculated. Western blots of autophagy specific ATGs, LC-3a/b, and P62- SQSTM1 proteins were also determined. To assess radiation DNA damage response (DDR), cells were grown on chambered slides, treated with no drugs or dual DMAPT and DCA, irradiated at 2Gy, and fixed at 0hr, 1hr, and 24hrs after irradiation. After labeling with Gamma-H2Ax antibody, 53BP1 antibody, and DAPI, cells were imaged on a Leica Sp8 confocal microscope. Foci quantification was performed using ImageJ software. Forty-eight hours of DMAPT and DCA dual treatment significantly reduced plating efficiency of MIA-PaCa-2 cells from 0.406±0.092 (40.6%) to 0.253±.0.10 (25.3%), of Panc-1 cells from 0.528±0.11 (52.8%) to 0.0755±0.044 (7.55%), and of AsPc-1 cells from 0.294±0.16 (29.4%) to 0.0300±0.031 (3.0%). Cell death involves alterations of autophagy regulatory proteins. Treatment of cells with two 2.5Gy fractions given four hours apart with DMAPT and DCA dual treatment significantly reduced cell survival fraction compared to split-dose fractionated cells without drug treatment in MIA-PaCA-2 (0.224±0.098 to 0.0716±0.064), Panc-1(0.260±0.040 to 0.0130±0.012), and AsPc-1 cells (0.121±0.011 to 0.0370±0.026). Furthermore, for all three cell lines, persistence of gamma-H2Ax foci was seen at 24 hours post 2Gy irradiation in DMAPT and DCA dual treated cells compared to 2 Gy irradiated only cells, where foci levels returned to baseline levels. Dual DMAPT and DCA treatment of pancreatic cancer decreases plating efficiency, inhibits autophagy, inhibits split dose repair, and results in persistence of gamma-H2Ax foci at 24 hours post irradiation. We conclude that dual DMAPT and DCA treatment of pancreatic cancer increases radiation-induced cell killing through inhibition of DNA double-strand break repair.

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

Mathematical Modeling of Preclinical Alpha-Emitter Radiopharmaceutical Therapy.

Preclinical studies, in vivo, and in vitro studies, in combination with mathematical modeling can help optimize and guide the design of clinical trials. The design and optimization of alpha-particle emitter radiopharmaceutical therapy (αRPT) is especially important as αRPT has the potential for high efficacy but also high toxicity. We have developed a mathematical model that may be used to identify trial design parameters that will have the greatest impact on outcome. The model combines Gompertzian tumor growth with antibody-mediated pharmacokinetics and radiation-induced cell killing. It was validated using preclinical experimental data of antibody-mediated 213Bi and 225Ac delivery in a metastatic transgenic breast cancer model. In modeling simulations, tumor cell doubling time, administered antibody, antibody specific-activity, and antigen-site density most impacted median survival. The model was also used to investigate treatment fractionation. Depending upon the time-interval between injections, increasing the number of injections increased survival time. For example, two administrations of 200 nCi, 225Ac-labeled antibody, separated by 30 days, resulted in a simulated 31% increase in median survival over a single 400 nCi administration. If the time interval was 7 days or less, however, there was no improvement in survival; a one-day interval between injections led to a 10% reduction in median survival. Further model development and validation including the incorporation of normal tissue toxicity is necessary to properly balance efficacy with toxicity. The current model is, however, useful in helping understand preclinical results and in guiding preclinical and clinical trial design towards approaches that have the greatest likelihood of success. SIGNIFICANCE: Modeling is used to optimize αRPT.

Modeling the radiation-induced cell death in a therapeutic proton beam using thermoluminescent detectors and radiation transport simulations

Changes in the relative biological effectiveness (RBE) of the radiation-induced cell killing of human salivary glands (HSG) were assessed along the Bragg peak of a 60 MeV clinical proton beam by means of coupling biophysical models with the results of Monte Carlo radiation transport simulations and experimental measurements with luminescent detectors. The fluence- and dose-mean unrestricted proton LET were determined along the Bragg peak using a recently developed methodology based on the combination of the response of 7LiF:Mg,Ti (MTS-7) and 7LiF:Mg,Cu,P (MCP-7) thermoluminescent detectors. The experimentally assessed LET values were compared with the results of radiation transport simulations using the Monte Carlo code PHITS, showing a good agreement. The cell survival probabilities and RBE were then calculated using the linear-quadratic model with the linear term derived using a phenomenological LET-based model (Carabe A et al 2012 Phys. Med. Biol. 57 1159) in combination with the experimentally-assessed or PHITS-simulated dose mean proton LET values. To the same aim, PHITS simulated microdosimetric spectra were used as input to the modified microdosimetric kinetic model (modified MKM, (Kase et al 2006 Radiat. Res. 166 629–38)). The RBE values calculated with the three aforementioned approaches were compared and found to be in very good agreement between each other, proving that by using dedicated pairs of thermoluminescent detectors it is possible to determine ionization density quantities of therapeutic proton beams which can be applied to predict the local value of the RBE.

Inhibition of P65 and PDK2 Increases Cytotoxicity and Radiation Sensitivity of Pancreatic Cancer Cells

Background: Pancreatic ductal adenocarcinoma (PDAC) is currently the 4th leading cause of cancer death in the US because of late detection and resistance to chemotherapy and radiation. This tumor resistance is partially due to high activity of the transcription factor, NF-κB which promotes cell survival and pyruvate dehydrogenase kinase 2 (PDK2) which regulates aerobic glycolysis (Warburg Metabolism), cell proliferation, and inhibition of apoptosis. We have shown that simultaneous treatment with the chemical NF-κB inhibitor DMAPT and the Warburg inhibitor DCA is cytotoxic and enhances radiation-induced cell death in human PDAC cells. We have also shown that double gene knockdown of the P65 (RELA) subunit of NF-κB and PDK2 by siRNA increased cytotoxicity and radiation sensitivity of pancreatic cancer cells but to a lesser extent than DMAPT and DCA treatment. Experimental Design: Western blots suggested the siRNA did not completely suppress p65 and PDKS protein levels. To test the hypothesis that more complete inhibition of NF-κB and PDK2 is required to increase cytotoxicity in PDAC cells, we transfected the Mia PaCa-2 cell line with appropriate siRNA and exposed the cells to the chemical inhibitors DMAPT and DCA. The cells were plated and followed by irradiation with either 0, 2, 4, or 6 Gy of 160 kVp X-rays Results: Compared to previous experiments that utilized siRNA knockdown alone, the combination of dual drugs and dual siRNA shows an increase in both cytotoxicity and radiation-induced cell killing (p< 0.001, t test) Conclusion: The data suggest that a more definitive approach such as utilizing CRISPR-Cas9 may be required to ensure complete gene knockdown of p65 and PDK2 to elucidate the mechanism(s) by which simultaneous inhibition of NF-kB and PDK2 inhibition enhance cytotoxicity and radiation-induced cell killing in pancreatic cancer.

MDM2 inhibitor AMG-232 and radiation therapy in treating patients with soft tissue sarcoma with wild-type TP53: A phase IB study (NRG-DT001).

TPS11076 Background: Over-expression of mdm2 is a major block to p53 activation in soft tissue sarcoma (STS). AMG-232 specifically inhibits human MDM2-p53 interactions in vitro and in vivo, thereby activating p53. Resistance mechanisms to MDM2 inhibition by AMG-232 is through accumulation of MDM2 and MDMX. However, DNA damaging agents, such as radiotherapy (RT), promote MDMX degradation and lead to sustained p53 activation, which is critically important for radiation-induced tumor cell killing in STS. Indeed, preclinical studies reveal that AMG-232 synergizes with RT in vitro and in vivo when treating STS with wild type (WT) P53 gene . Since a majority of STS harbor WT TP53 gene and RT is part of the standard of care in treating STS; it is the next rational step to test the safety and efficacy of combination of AMG-232 and RT followed by surgery. In this clinical trial, we hypothesize that preoperative AMG-232 plus RT is safe and well tolerated in 2 separate cohorts: (A) extremity and body wall; (B) abdomen/pelvis/retroperitoneum. Methods: A key feature of this trial is to incorporate TP53 NGS sequencing result as one of the eligibility criteria. Adult patients with pathologically proven grade 2-3 STS with size ≥ 5 cm are eligible to enroll if there is a planned definitive surgical resection of the primary tumor. There must be sufficient tissue to submit to central laboratory for NGS sequencing of the TP53 gene, which is an integral biomarker. Only those with tumors harboring WT p53 gene will be allowed to continue protocol treatment. The primary objective is to evaluate the safety and tolerability of AMG-232 in combination with standard-dose RT in STS in two separate cohort; as well as to determine the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of AMG-232 in combination with RT. We define DLT as grade 4-5 toxicities attributable to AMG-232 for up to 4 weeks after the completion of combination treatment (prior to surgery) as well as any AMG-232 related grade 3 toxicity that should lead to > 2 weeks treatment delay or ≥ two dose reductions. There are 3 dose levels for AMG-232: dose level 1 is 120 mg daily for 3 days per week; dose level 2 is 120 mg daily 4 days per week and dose level 5 is 120 mg daily 5 days a week. If patients did not tolerate dose level 1, AMG-232 will be de-escalated to a lower level, which is 120 mg daily 2 days a week. The trial was open since 11/2017; 3 patients have been enrolled to cohort A and 2 patients in cohort B at dose level 1. The first patient has a tumor with WT p53 gene and has completed protocol treatment safely. Clinical trial information: NCT03217266.

Adrenergic Receptor Signaling Regulates the Response of Tumors to Ionizing Radiation.

While ionizing radiation is a major form of cancer therapy, radioresistance remains a therapeutic obstacle. We have previously shown that the mandated housing temperature for laboratory mice (∼22°C) induces mild, but chronic, cold stress resulting in increased circulating norepinephrine, which binds to, and triggers activation of, beta-adrenergic receptors (β-AR) on tumor and immune cells. This adrenergic signaling increases tumor cell intrinsic resistance to chemotherapy and suppression of the anti-tumor immune response. These findings led us to hypothesize that adrenergic stress signaling increases radioresistance in tumor cells in addition to suppressing T-cell-mediated anti-tumor immunity, thus suppressing the overall sensitivity of tumors to radiation. We used three strategies to test the effect of adrenergic signaling on responsiveness to radiation. For one strategy, mice implanted with CT26 murine colon adenocarcinoma were housed at either 22°C or at thermoneutrality (30°C), which reduces physiological adrenergic stress. For a second strategy, we used a β-AR antagonist ("beta blocker") to block adrenergic signaling in mice housed at 22°C. In either case, tumors were then irradiated with a single 6 Gy dose and the response was compared to mice whose adrenergic stress signaling was not reduced. For the third strategy, we used an in vitro approach in which several different tumor cell lines were treated with a β-AR agonist and irradiated, and cell survival was then assessed by clonogenic assay. Overall, we found that adrenergic stress significantly impaired the anti-tumor efficacy of radiation by inducing tumor cell resistance to radiation-induced cell killing and by suppression of anti-tumor immunity. Treatment using beta blockers is a promising strategy for increasing the anti-tumor efficacy of radiotherapy.

Determining if Double Gene Knockdown of P65 and PDK2 Increases Cytotoxicity and Radiation Sensitivity of Pancreatic Cancer Cells

Background: Pancreatic ductal adenocarcinoma (PDA) is currently the 4th leading cause of cancer death in the US because of late detection and resistance to chemotherapy and radiation. This tumor resistance is partially due to high activity of the transcription factor, NF-[Symbol]B which promotes cell survival and pyruvate dehydrogenase kinase 2 (PDK2) which regulates aerobic glycolysis (Warburg Metabolism), cell proliferation, and inhibition of apoptosis. We have shown that simultaneous treatment with the chemical NF-B inhibitor DMAPT and the Warburg inhibitor DCA is cytotoxic and enhances radiation-induced cell death in human PDA cells. Experimental Design: We hypothesize that double gene knockdown of the P65 subunit of NF-[Symbol]B and PDK2 by siRNA will lead to increased cytotoxicity and radiation sensitivity. The PDA cell line (Mia PaCa-2) was utilized and was transfected with appropriate siRNAs. The cells were plated and followed by irradiation with either 0, 2, 4, or 6 Gy of 160 kVp X-rays. Results: Analysis of Western blots shows the successful silencing of P65, but the knockdown of PDK2 are pending. The differences in plating efficiency were not found to be statistically significant between the treatment groups. The differences in survival fraction were not found to be statistically significant at 2, 4, or 6 Gy, but there is a trend for enhancement at 6 Gy. Conclusion: Preliminary data indicate there was no increase in cytotoxicity or radiation-induced cell killing after treatment with SiP65, SiPDK2, or both. However, to date we have no evidence that siPDK2 is working and further experiments are underway.

Superoxide Dismutase Mimetic GC4419 Enhances the Oxidation of Pharmacological Ascorbate and Its Anticancer Effects in an H₂O₂-Dependent Manner.

Lung cancer, together with head and neck cancer, accounts for more than one-fourth of cancer deaths worldwide. New, non-toxic therapeutic approaches are needed. High-dose IV vitamin C (aka, pharmacological ascorbate; P-AscH−) represents a promising adjuvant to radiochemotherapy that exerts its anti-cancer effects via metal-catalyzed oxidation to form H2O2. Mn(III)-porphyrins possessing superoxide dismutase (SOD) mimetic activity have been shown to increase the rate of oxidation of AscH−, enhancing the anti-tumor effects of AscH− in several cancer types. The current study demonstrates that the Mn(II)-containing pentaazamacrocyclic selective SOD mimetic GC4419 may serve as an AscH−/O2•− oxidoreductase as evidenced by the increased rate of oxygen consumption, steady-state concentrations of ascorbate radical, and H2O2 production in complete cell culture media. GC4419, but not CuZnSOD, was shown to significantly enhance the toxicity of AscH− in H1299, SCC25, SQ20B, and Cal27 cancer cell lines. This enhanced cancer cell killing was dependent upon the catalytic activity of the SOD mimetic and the generation of H2O2, as determined using conditional overexpression of catalase in H1299T cells. GC4419 combined with AscH− was also capable of enhancing radiation-induced cancer cell killing. Currently, AscH− and GC4419 are each being tested separately in clinical trials in combination with radiation therapy. Data presented here support the hypothesis that the combination of GC4419 and AscH− may provide an effective means by which to further enhance radiation therapy responses.