The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma.

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

Metabolic response to radiation therapy in cancer.

Tumor metabolism has emerged as a hallmark of cancer and is involved in carcinogenesis and tumor growth. Reprogramming of tumor metabolism is necessary for cancer cells to sustain high proliferation rates and enhanced demands for nutrients. Recent studies suggest that metabolic plasticity in cancer cells can decrease the efficacy of anticancer therapies by enhancing antioxidant defenses and DNA repair mechanisms. Studying radiation-induced metabolic changes will lead to a better understanding of radiation response mechanisms as well as the identification of new therapeutic targets, but there are few robust studies characterizing the metabolic changes induced by radiation therapy in cancer. In this review, we will highlight studies that provide information on the metabolic changes induced by radiation and oxidative stress in cancer cells and the associated underlying mechanisms.

Serum erythropoietin levels, breast cancer and breast cancer-initiating cells.

BACKGROUND: Cancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation. METHODS: Using four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment. RESULTS: Our results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells. CONCLUSIONS: We conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

PURPOSE: Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radiation (IR) on PK-M2-mediated metabolic changes in TNBC cells, and their survival. In addition, we determine the effect of PK-M2 activators on breast cancer stem cells, a radioresistant subpopulation of breast cancer stem cells. METHODS: Glucose uptake, lactate production, and glutamine consumption were assessed. The cellular localization of PK-M2 was evaluated by western blot and confocal microscopy. The small molecule activator of PK-M2, TEPP46, was used to promote its pyruvate kinase function. Finally, effects on cancer stem cell were evaluated via sphere forming capacity. RESULTS: Exposure of TNBC cells to IR increased their glucose uptake and lactate production. As expected, PK-M2 expression levels also increased, especially in the nucleus, although overall pyruvate kinase activity was decreased. PK-M2 nuclear localization was shown to be associated with breast cancer stem cells, and activation of PK-M2 by TEPP46 depleted this population. CONCLUSIONS: Radiotherapy can induce metabolic changes in TNBC cells, and these changes seem to be mediated, at least in part by PK-M2. Importantly, our results show that activators of PK-M2 can deplete breast cancer stem cells in vitro. This study supports the idea of combining PK-M2 activators with radiation to enhance the effect of radiotherapy in resistant cancers, such as TNBC.

Cancer stem cells, cancer cell plasticity and radiation therapy.

Since the first prospective identification of cancer stem cells in solid cancers the cancer stem cell hypothesis has reemerged as a research topic of increasing interest. It postulates that solid cancers are organized hierarchically with a small number of cancer stem cells driving tumor growth, repopulation after injury and metastasis. They give rise to differentiated progeny, which lack these features. The model predicts that for any therapy to provide cure, all cancer stem cells have to be eliminated while the survival of differentiated progeny is less critical. In this review we discuss recent reports challenging the idea of a unidirectional differentiation of cancer cells. These reports provide evidence supporting the idea that non-stem cancer cells exhibit a remarkable degree of plasticity that allows them to re-acquire cancer stem cell traits, especially in the context of radiation therapy. We summarize conditions under which differentiation is reversed and discuss the current knowledge of the underlying mechanisms.

The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells.

Cancer stem cells (CSCs) or tumor-initiating cells, similar to normal tissue stem cells, rely on developmental pathways, such as the Notch pathway, to maintain their stem cell state. One of the regulators of the Notch pathway is Musashi-1, a mRNA-binding protein. Musashi-1 promotes Notch signaling by binding to the mRNA of Numb, the negative regulator of Notch signaling, thus preventing its translation. CSCs have also been shown to downregulate their 26S proteasome activity in several types of solid tumors, thus making them resistant to proteasome-inhibitors used as anticancer agents in the clinic. Interestingly, the Notch pathway can be inhibited by proteasomal degradation of the Notch intracellular domain (Notch-ICD); therefore, downregulation of the 26S proteasome activity can lead to stabilization of Notch-ICD. Here, we present evidence that the downregulation of the 26S proteasome in CSCs constitutes another level of control by which Musashi-1 promotes signaling through the Notch pathway and maintenance of the stem cell phenotype of this subpopulation of cancer cells. We demonstrate that Musashi-1 mediates the downregulation of the 26S proteasome by binding to the mRNA of NF-YA, the transcriptional factor regulating 26S proteasome subunit expression, thus providing an additional route by which the degradation of Notch-ICD is prevented, and Notch signaling is sustained.

Metabolic differences in breast cancer stem cells and differentiated progeny.

In general, tumor cells display a more glycolytic phenotype compared to the corresponding normal tissue. However, it is becoming increasingly clear that tumors are composed of a heterogeneous population of cells. Breast cancers are organized in a hierarchical manner, with the breast cancer stem cells (BCSCs) at the top of the hierarchy. Here, we investigate the metabolic phenotype of BCSCs and their differentiated progeny. In addition, we determine the effect of radiation on the metabolic state of these two cell populations. Luminal, basal, and claudin-low breast cancer cell lines were propagated as mammospheres enriched in BCSCs. Lactate production, glucose consumption, and ATP content were compared with differentiated cultures. A metabolic flux analyzer was used to determine the oxygen consumption, extracellular acidification rates, maximal mitochondria capacity, and mitochondrial proton leak. The effect of radiation treatment of the metabolic phenotype of each cell population was also determined. BCSCs consume more glucose, produce less lactate, and have higher ATP content compared to their differentiated progeny. BCSCs have higher maximum mitochondrial capacity and mitochondrial proton leak compared to their differentiated progeny. Radiation treatment enhances the higher energetic state of the BCSCs, while decreasing mitochondrial proton leak. Our study indicated that breast cancer cells are heterogeneous in their metabolic phenotypes and BCSCs reside in a distinct metabolic state compared to their differentiated progeny. BCSCs display a reliance on oxidative phosphorylation, while the more differentiated progeny displays a more glycolytic phenotype. Radiation treatment affects the metabolic state of BCSCs. We conclude that interfering with the metabolic requirements of BCSCs may prevent radiation-induced reprogramming of breast cancer cells during radiation therapy, thus improving treatment outcome.

Tumor cells with low proteasome subunit expression predict overall survival in head and neck cancer patients.

BACKGROUND: Experimental and clinical data suggest that solid cancers contain treatment-resistant cancer stem cells that will impair treatment efficacy. The objective of this study was to investigate if head and neck squamous cell carcinoma (HNSCC) also contain cancer stem cells that can be identified by low 26S proteasome activity and if their presence correlates to clinical outcome. METHODS: Human HNSCC cells, engineered to report lack of proteasome activity based on accumulation of a fluorescent fusion protein, were separated based on high (ZsGreen-cODCneg) or low (ZsGreen-cODCpos) proteasome activity. Self-renewal capacity, tumorigenicity and radioresistance were assessed. Proteasome subunit expression was analyzed in tissue microarrays and correlated to survival and locoregional cancer control of 174 patients with HNSCC. RESULTS: HNSCC cells with low proteasome activity showed a significantly higher self-renewal capacity and increased tumorigenicity. Irradiation enriched for ZsGreen-cODCpos cells. The survival probability of 82 patients treated with definitive radio- or chemo-radiotherapy exhibiting weak, intermediate, or strong proteasome subunit expression were 21.2, 28.8 and 43.8 months (p = 0.05), respectively. Locoregional cancer control was comparably affected. CONCLUSIONS: Subpopulations of HNSCC display stem cell features that affect patients' tumor control and survival. Evaluating cancer tissue for expression of the proteasome subunit PSMD1 may help identify patients at risk for relapse.

Folate receptor-ß in activated macrophages: ligand binding and receptor recycling kinetics.

Activated macrophages overexpress a receptor for the vitamin folic acid termed the folate receptor ß (FR-ß). Because conjugation of folate to low molecular weight drugs, genes, liposomes, nanoparticles, and imaging agents has minor effects on FR binding, the vitamin can be exploited to target both therapeutic and imaging agents to activated macrophages without promoting their uptake by other healthy cells. In this paper, we characterize the binding, internalization, and recycling kinetics of FR-ß on activated macrophages in inflamed tissues of rats with adjuvant-induced arthritis. Our results demonstrate that saturation of macrophage FR is achieved at injection doses of ∼150-300 nmol/kg, with more rapidly perfused tissues saturating at lower doses than inflamed appendages. After binding, FR-ß internalizes and recycles back to the cell surface every ∼10-20 min, providing empty receptors for additional folate conjugate uptake. Because the half-life of low molecular weight folate conjugates in the vasculature is usually <1 h, these data suggest that targeting of folate conjugates to activated macrophages in vivo can be maximized by frequent dosing at conjugate concentrations that barely saturate FR (∼150 nmol/kg), thereby minimizing nonspecific binding to receptor-negative tissues and maximizing the probability that unoccupied cell surface receptors will be exposed to folate-drug conjugate.

Metabolic state of glioma stem cells and nontumorigenic cells.

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs), and it is thought that tumor regrowth originates from GSCs, thus rendering GSCs an attractive target for novel treatment approaches. Cancer cells rely more on glycolysis than on oxidative phosphorylation for glucose metabolism, a phenomenon used in 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography imaging of solid cancers, and targeting metabolic pathways in cancer cells has become a topic of considerable interest. However, if GSCs are indeed important for tumor control, knowledge of the metabolic state of GSCs is needed. We hypothesized that the metabolism of GSCs differs from that of their progeny. Using a unique imaging system for GSCs, we assessed the oxygen consumption rate, extracellular acidification rate, intracellular ATP levels, glucose uptake, lactate production, PKM1 and PKM2 expression, radiation sensitivity, and cell cycle duration of GSCs and their progeny in a panel of glioma cell lines. We found GSCs and progenitor cells to be less glycolytic than differentiated glioma cells. GSCs consumed less glucose and produced less lactate while maintaining higher ATP levels than their differentiated progeny. Compared with differentiated cells, GSCs were radioresistant, and this correlated with a higher mitochondrial reserve capacity. Glioma cells expressed both isoforms of pyruvate kinase, and inhibition of either glycolysis or oxidative phosphorylation had minimal effect on energy production in GSCs and progenitor cells. We conclude that GSCs rely mainly on oxidative phosphorylation. However, if challenged, they can use additional metabolic pathways. Therefore, targeting glycolysis in glioma may spare GSCs.

Targeted elimination of breast cancer cells with low proteasome activity is sufficient for tumor regression.

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs), able to regrow a tumor after sublethal treatment while their progeny lack this feature. Furthermore, BCSCs are highly resistant to conventional anticancer treatments. According to the cancer stem cell hypothesis, all cancer stem cells in a tumor have to be eliminated to achieve cancer cure. In this study we tested if targeted elimination of BCSCs leads to tumor regression. Specific targeting of BCSCs was achieved via a unique imaging and targeting system that relies on their low proteasome activity. In our system breast cancer cells stably express a fluorescent fusion protein, thymidine kinase-ZsGreen-cODC, which is readily degraded after translation in cells with normal 26S proteasome activity. However, cells with low proteasome activity accumulate this fluorescent fusion protein, thus allowing for their identification, tracking, and specific elimination. Here, we show that the activity of the 26S proteasome was significantly down-regulated in MCF-7, T47D, and MDA-MB-231 cultures enriched for BCSCs. Treatment with ganciclovir resulted in abrogation of sphere formation in vitro, and tumor regression in vivo, thus demonstrating that targeted elimination of BCSCs leads to loss of self-renewal in vitro and tumor regression in vivo. We conclude that specific targeting of BCSCs could be a useful strategy to improve treatment outcome.

Radiation-induced reprogramming of breast cancer cells.

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs) able to regrow a tumor while their progeny lack this ability. Recently, several groups reported enrichment for BCSCs when breast cancers were subjected to classic anticancer treatment. However, the underlying mechanisms leading to this enrichment are incompletely understood. Using non-BCSCs sorted from patient samples, we found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs (iBCSCs). iBCSCs showed increased mammosphere formation, increased tumorigenicity, and expressed the same stemness-related genes as BCSCs from nonirradiated samples. Reprogramming occurred in a polyploid subpopulation of cells, coincided with re-expression of the transcription factors Oct4, sex determining region Y-box 2, Nanog, and Klf4, and could be partially prevented by Notch inhibition. We conclude that radiation may induce a BCSC phenotype in differentiated breast cancer cells and that this mechanism contributes to increased BCSC numbers seen after classic anticancer treatment.

Folate-linked drugs for the treatment of cancer and inflammatory diseases.

Folic acid, also known as vitamin B9 (Fig. 9.1), is an essential co-enzyme in one-carbon metabolism pathways, including the biosynthesis of nucleotides (i.e. purines, thymidine) and several amino acids. In general, two functionally different systems mediate the cellular uptake of folate: (1) the reduced folate carrier (RFC, Kd ∼ 10-6 M), an anion transporter that delivers folates across the plasma membrane in a bidirectional fashion, and (2) the folate receptor (FR, Kd ∼ 10-10 M), which internalizes folate through active receptor-mediated endocytosis. The RFC, a membrane-spanning anion transporter, is present in virtually all tissues and is responsible for the majority of folate transport in and out of cells. In contrast, FR expression is largely restricted to malignant cells, activated macrophages, and the proximal tubule cells of the kidneys. Because a variety of important diseases are caused by the former two cell types, interest in exploiting FR for drug targeting applications has rapidly increased. And achievement of this targeting objective, primarily through conjugation of drugs to folic acid is believed to enable (1) enhanced net drug uptake by pathologic cells, and more importantly (2) reduction in drug deposition into non-pathologic cells, thereby mitigating collateral toxicity to normal tissues.

Glioblastomas: Hijacking Metabolism to Build a Flexible Shield for Therapy Resistance.

Significance: Glioblastomas (GBMs) are among the most lethal tumors despite the almost exclusive localization to the brain. This is largely due to therapeutic resistance. Radiation and chemotherapy significantly increase the survival for GBM patients, however, GBMs always recur, and the median overall survival is just over a year. Proposed reasons for such intractable resistance to therapy are numerous and include tumor metabolism, in particular, the ability of tumor cells to reconfigure metabolic fluxes on demand (metabolic plasticity). Understanding how the hard-wired, oncogene-driven metabolic tendencies of GBMs intersect with flexible, context-induced metabolic rewiring promises to reveal novel approaches for combating therapy resistance. Recent Advances: Personalized genome-scale metabolic flux models have recently provided evidence that metabolic flexibility promotes radiation resistance in cancer and identified tumor redox metabolism as a major predictor for resistance to radiation therapy (RT). It was demonstrated that radioresistant tumors, including GBM, reroute metabolic fluxes to boost the levels of reducing factors of the cell, thus enhancing clearance of reactive oxygen species that are generated during RT and promoting survival. Critical Issues: The current body of knowledge from published studies strongly supports the notion that robust metabolic plasticity can act as a (flexible) shield against the cytotoxic effects of standard GBM therapies, thus driving therapy resistance. The limited understanding of the critical drivers of such metabolic plasticity hampers the rational design of effective combination therapies. Future Directions: Identifying and targeting regulators of metabolic plasticity, rather than specific metabolic pathways, in combination with standard-of-care treatments have the potential to improve therapeutic outcomes in GBM. Antioxid. Redox Signal. 39, 957-979.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase Promotes Glioblastoma Radiation Resistance in a Phosphate and Tensin Homolog-Dependent Manner.

Aims: The goal of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-produced reactive oxygen species (ROS) enhance brain tumor growth of glioblastoma (GBM) under hypoxic conditions and during radiation treatment. Results: Exogenous ROS promoted brain tumor growth in gliomasphere cultures that expressed functional phosphate and tensin homolog (PTEN), but not in tumors that were PTEN deficient. Hypoxia induced the production of endogenous cytoplasmic ROS and tumor cell growth via activation of NOX. NOX activation resulted in oxidation of PTEN and downstream protein kinase B (Akt) activation. Radiation also promoted ROS production via NOX, which, in turn, resulted in cellular protection that could be abrogated by knockdown of the key NOX component, p22. Knockdown of p22 also inhibited tumor growth and enhanced the efficacy of radiation in PTEN-expressing GBM cells. Innovation: While other studies have implicated NOX function in GBM models, this study demonstrates NOX activation and function under physiological hypoxia and following radiation in GBM, two conditions that are seen in patients. NOX plays an important role in a PTEN-expressing GBM model system, but not in PTEN-nonfunctional systems, and provides a potential, patient-specific therapeutic opportunity. Conclusion: This study provides a strong basis for pursuing NOX inhibition in PTEN-expressing GBM cells as a possible adjunct to radiation therapy. Antioxid. Redox Signal. 39, 890-903.

M2 isoform of pyruvate kinase rewires glucose metabolism during radiation therapy to promote an antioxidant response and glioblastoma radioresistance.

BACKGROUND: Resistance to existing therapies is a significant challenge in improving outcomes for glioblastoma (GBM) patients. Metabolic plasticity has emerged as an important contributor to therapy resistance, including radiation therapy (RT). Here, we investigated how GBM cells reprogram their glucose metabolism in response to RT to promote radiation resistance. METHODS: Effects of radiation on glucose metabolism of human GBM specimens were examined in vitro and in vivo with the use of metabolic and enzymatic assays, targeted metabolomics, and FDG-PET. Radiosensitization potential of interfering with M2 isoform of pyruvate kinase (PKM2) activity was tested via gliomasphere formation assays and in vivo human GBM models. RESULTS: Here, we show that RT induces increased glucose utilization by GBM cells, and this is accompanied with translocation of GLUT3 transporters to the cell membrane. Irradiated GBM cells route glucose carbons through the pentose phosphate pathway (PPP) to harness the antioxidant power of the PPP and support survival after radiation. This response is regulated in part by the PKM2. Activators of PKM2 can antagonize the radiation-induced rewiring of glucose metabolism and radiosensitize GBM cells in vitro and in vivo. CONCLUSIONS: These findings open the possibility that interventions designed to target cancer-specific regulators of metabolic plasticity, such as PKM2, rather than specific metabolic pathways, have the potential to improve the radiotherapeutic outcomes in GBM patients.

NRF2 Mediates Cellular Resistance to Transformation, Radiation, and Inflammation in Mice.

Nuclear factor erythroid 2-related factor 2 (NRF2) is recognized as a master transcription factor that regulates expression of numerous detoxifying and antioxidant cytoprotective genes. In fact, models of NRF2 deficiency indicate roles not only in redox regulation, but also in metabolism, inflammatory/autoimmune disease, cancer, and radioresistancy. Since ionizing radiation (IR) generates reactive oxygen species (ROS), it is not surprising it activates NRF2 pathways. However, unexpectedly, activation is often delayed for many days after the initial ROS burst. Here, we demonstrate that, as assayed by γ-H2AX staining, rapid DNA double strand break (DSB) formation by IR in primary mouse Nrf2-/- MEFs was not affected by loss of NRF2, and neither was DSB repair to any great extent. In spite of this, basal and IR-induced transformation was greatly enhanced, suggesting that NRF2 protects against late IR-induced genomic instability, at least in murine MEFs. Another possible IR- and NRF2-related event that could be altered is inflammation and NRF2 deficiency increased IR-induced NF-κB pro-inflammatory responses mostly late after exposure. The proclivity of NRF2 to restrain inflammation is also reflected in the reprogramming of tumor antigen-specific lymphocyte responses in mice where Nrf2 k.o. switches Th2 responses to Th1 polarity. Delayed NRF2 responses to IR may be critical for the immune transition from prooxidant inflammation to antioxidant healing as well as in driving cellular radioresistance and survival. Targeting NRF2 to reprogram immunity could be of considerable therapeutic benefit in radiation and immunotherapy.

Radiosensitizing Pancreatic Cancer via Effective Autophagy Inhibition.

Despite aggressive treatments, pancreatic ductal adenocarcinoma (PDAC) remains an intractable disease, largely because it is refractory to therapeutic interventions. To overcome its nutrient-poor microenvironment, PDAC heavily relies on autophagy for metabolic needs to promote tumor growth and survival. Here, we explore autophagy inhibition as a method to enhance the effects of radiotherapy on PDAC tumors. Hydroxychloroquine is an autophagy inhibitor at the focus of many PDAC clinical trials, including in combination with radiotherapy. However, its acid-labile properties likely reduce its intratumoral efficacy. Here, we demonstrate that EAD1, a synthesized analogue of HCQ, is a more effective therapeutic for sensitizing PDAC tumors of various KRAS mutations to radiotherapy. Specifically, in vitro models show that EAD1 is an effective inhibitor of autophagic flux in PDAC cells, accompanied by a potent inhibition of proliferation. When combined with radiotherapy, EAD1 is consistently superior to HCQ not only as a single agent, but also in radiosensitizing PDAC cells, and perhaps most importantly, in decreasing the self-renewal capacity of PDAC cancer stem cells (PCSC). The more pronounced sensitizing effects of autophagy inhibitors on pancreatic stem over differentiated cells points to a new understanding that PCSCs may be more dependent on autophagy to counter the effects of radiation toxicity, a potential mechanism explaining the resistance of PCSCs to radiotherapy. Finally, in vivo subcutaneous tumor models demonstrate that combination of radiotherapy and EAD1 is the most successful at controlling tumor growth. The models also confirmed a similar toxicity profile between EAD1 and Hydroxychloroquine.