Constantinos Koumenis - US grants

enzyme activity; apoptosis; physiologic stressor; hypoxia; DNA directed RNA polymerase; phosphorylation; protein kinase; tumor suppressor genes; cell line; microinjections;

Hypoxia is a well-characterized component of the solid tumor microenvironment. Hypoxic tumors are more resistant to radiation therapy and chemotherapy and have a poorer prognosis than better-oxygenated tumors. Classical biochemical studies have shown that exposure of tumor cells to hypoxia results in a pronounced decrease in the rate of protein synthesis, which is reversible upon reoxygenation. This process is hypothesized to constitute an efficient mechanism of cellular energy conservation that is critical for the survival of the tumor cell in this environment of reduced oxygen availability. While significant progress has been made in identifying individual gene products whose synthesis is altered by hypoxia, little is known about the mechanism by which hypoxia induces global downregulation of translation. Phosphorylation of the translation initiation factor eIF2alpha on ser51 plays a key role in the regulation of protein synthesis by other types of stress, such as heat-shock and brain ischemia/reperfusion injury. Preliminary data indicate that exposure of tumor cells to hypoxia increases the levels of eIF2alpha phosphorylation and decreases the rate of protein synthesis. The endoplasmic reticulum-resident kinase, PERK, becomes phosphorylated under hypoxia and may be responsible for hypoxia-induced eIF2alpha phosphorylation. We propose that phosphorylation of eIF2alpha plays a key role in the cellular adaptation to tumor hypoxia. Aim 1 will further characterize the kinetics and establish the oxygen dependency of elF2alpha phosphorylation in transformed and untransformed cells. Aim 2 will investigate whether inhibition of phosphorylation of eIF2alpha is required for inhibition of protein synthesis in cells exposed to hypoxia. In aim 3, genetic and biochemical means will be used to investigate whether the endoplasmic reticulum kinase PERK is responsible for hypoxia-induced phosphorylation of elF2alpha. Aim 4 will examine the consequences of deregulated eIF2alpha phosphorylation and translation on the most important endpoint of cellular adaptation, cell survival/cell death, by determining the short- and long-term viability of tumor cells exposed to moderate or extreme hypoxia. Accomplishing the aims of this application will increase our understanding of the process of cellular adaptation to hypoxic stress. Identification of the key players in this process and examination of the consequences of inhibition of their function on tumor cell survival, may lead to new therapeutic modalities that specifically target this adaptive in hypoxic areas of solid tumors.

DESCRIPTION (provided by applicant): Plant phenolic antioxidants related to the 3, 4-dihydroxycinammic acid exhibit potent antitumor activities, including anti-proliferative, anti-mitogenic and anti-angiogenic properties. Two structurally-related members of this family, Caffeic Acid Phenethyl Ester (CAPE) and curcumin, have been shown to preferentially induce apoptosis in transformed but not untransformed cells in vitro, and to inhibit polyp formation in experimental mouse tumor models with little or no overt toxicity. The anti-tumorigenic activity of CAPE and curcumin have been attributed to their antioxidant properties and their ability to inhibit the Nuclear Factor Kappa-B/COX-2 pathway. In preliminary studies, the PI has shown that CAPE and curcumin inhibit activation of the protooncogene kinase, Akt/PKB, that lies upstream of NF-kB activation. Akt is abnormally activated in a variety of tumors and promotes cell survival, cell proliferation and angiogenesis. The PI also has shown that CAPE exhibits potent radio- and chemosensitization properties against colorectal carcinoma cells. Since the Akt pathway is associated with resistance to ionizing radiation and increased angiogenesis, the PI proposes that CAPE and curcumin will be useful radiosensitizing, chemosensitizing, and anti-angiogenic agents for the treatment of solid tumors. In Aim 1, the preliminary studies in colorectal carcinoma cells will be extended to brain and prostate cell lines and tumor xenografts. Aim 2 will address whether the Akt signalling pathway or other anti-apoptotic pathways are the primary targets for CAPE and curcumin radiosensitization. In Aim 3, the role of increased oxidative stress induced preferentially in transformed cells by CAPE and curcumin will be evaluated as a potential radiosensitization mechanism. Aim 4 will extend the studies in Alms 1-3 to animal models of tumor radiosensitization and normal tissue injury to examine the ability of CAPE and curcumin to induce in vivo tumor radiosensitization and protect normal tissues fro radiation injury. Results from these studies will establish the molecular basis for the demonstrated radio/chemosensitizing properties of this class of naturally occurring compounds and will also set the stage for the design of clinical trials using CAPE and/or curcumin as chemotherapeutic agents and radiation dose modifiers.

[unreadable] DESCRIPTION (provided by applicant): Hypoxia is a well-characterized component of the solid tumor microenvironment that promotes resistance to radiation therapy and chemotherapy and is associated with a poorer overall prognosis. The hypoxic tumor cell elicits both HIF-dependent and HIF-independent mechanisms to adapt to and overcome the stress of low oxygen in tumors. Through experiments performed under the specific aims of the previously funded application and preliminary results in this proposal, we have shown that hypoxia/anoxia rapidly activates a translational control program mediated by activation of the endoplasmic reticulum (ER) kinase PERK and phosphorylation of the translation initiation factor elF2?. This pathway downregulates global protein synthesis but at the same time upregulates the expression of select stress-response proteins. These results together with those from other groups established that the Unfolded Protein Response, a cellular adaptation mechanism to ER stress, is activated by hypoxia and is required for hypoxia tolerance and tumor growth. In this application we propose to expand these findings to identify the signal for UPR activation by hypoxia, identify the role(s) of downstream effectors of PERK and elF2? phosphorylation, characterize pro- and anti- apoptotic pathways activated by ER stress and test whether the reliance of hypoxic tumor cells on UPR activation for survival can be therapeutically exploited. In Specific Aim 1 we will test whether lack of the downstream PERK effector ATF4 expression inhibits, and overexpression of ATF4 promotes, tumor growth and we will identify and characterize transcriptional targets of hypoxia-activated ATF4. Under Specific Aim 2 we will attempt to delineate the mechanism responsible for PERK and UPR activation under hypoxia by analyzing the status of-SH groups on the ER-resident folding enzymes Protein Disulfide Isomerase, and Erolp oxidase. In Specific Aim 3 we will investigate the role(s) of ER-targeted bcl-2 and ER-localized bax/bak in the induction of ER-dependent apoptosis by hypoxia/anoxia in UPR-proficient and -deficient cells. Under Specific Aim 4 we will test whether hypoxic tumor cells, which are resistant to genotoxic chemotherapeutic agents, are acutely sensitive to pharmacological ER stressors and whether the combination of ER stressors with ionizing radiation or chemotherapeutic agents results in better inhibition of tumor growth in animal tumor models. Solid tumor metastasis and resistance to most established chemotherapy regimens are key contributors to tumor morbidity and thus constitute a significant public health problem. Successful completion of the proposed studies will uncover a novel pathway that contributes to tumorigenesis and resistance to therapy and can potentially offer new targets and approaches to selectively attack these resistant cells and thus improve therapeutic outcome. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Hypoxia/anoxia is a well-characterized component of the microenvironment of most solid tumor. Considerable experimental and clinical evidence supports the notion that hypoxia fundamentally alters the physiology of the tumor towards a more aggressive phenotype. The Unfolded Protein Response (UPR) is a cellular stress adaptation pathway which promotes cell survival in the presence of Endoplasmic Reticulum (ER) stress, including physiological stress in the tumor microenvironment. The PERK/eIF2a/ATF4 pathway reduces the global rates of protein translation thereby alleviating metabolic stress under hypoxia while at the same time induces the translational upregulation of important chaperones and pro-survival genes. Another UPR component is the activation of the endonuclease/kinase IRE1 and its immediate effector XBP1, which activate a transcriptional program aimed at increasing the folding capacity of the ER. Recent in vitro and in vivo studies from the labs of the two co-PIs, have shown that transformed cells with ablated UPR responses exhibit reduced tolerance to hypoxia in vitro and form tumors that are slower growing compared to tumors grown from cells with an intact UPR, indicating that UPR activation contributes to tumorigenesis. In preliminary studies, we have developed cell-based and animal-based assays for UPR activation and its inhibition by small molecules. The overall goal of this proposal is to validate UPR activation as an important anti- tumor target and to use novel in vitro and in vivo assays to identify potent inhibitors of this response as novel chemotherapeutic agents. In Aim 1, we will employ in vitro reporter assays of PERK activity to identify specific inhibitors of the PERK/eIF21/ATF4 pathway and test the effect of combined administration of these inhibitors with inhibitors of the IRE-1 pathway (Irestatins) on tumor cell survival under normoxia and hypoxia. In Aim 2, we will investigate the potential synergy between inhibitors of the PERK and IRE1 pathways with the proteasome inhibitor Bortezomib in killing hypoxic tumor cells in vitro and in vivo. Studies under Aim 3, will evaluate the use of Zebrafish as a model system to analyze the ability of inhibitors of the PERK and IRE1 pathways to inhibit xenotransplanted human tumors and to inhibit angiogenesis without causing significant developmental abnormalities. Finally, in Aim 4 we will test the efficacy and potential toxicity of identified compounds in mouse tumor models. We expect that these efforts will culminate in the development of specific and potent inhibitors of the UPR which alone, or in combination with existing antitumor agents and modalities will be effective in reducing tumor burden in preclinical and clinical malignancies. PUBLIC HEALTH RELEVANCE: A hallmark of solid tumors is the requirement to adapt to, and eventually overcome the stressful environment of low oxygen, growth factors, glucose and pH in the growing tumor mass. The requirement for neoangiogenesis to support tumor growth is now well established and is the basis for several promising anti-tumor modalities. Based on published data from our labs and others, we propose that the Unfolded Protein Response also plays a crucial role in adaptation to hypoxic stress, and like angiogenesis, represents an encompassing and stable aspect of tumor development and thus provides a unique opportunity for therapeutic exploitation. This proposal aims to identify agents that target key components of this adaptive response has the potential to offer additional and novel approaches to target the very stresses that hinder existing anti-tumor treatments and thereby improve antitumor treatment efficacy.

DESCRIPTION (provided by applicant): The number of active research scientists with expertise in all aspects of radiation has been steadily decreasing for more than two decades, despite the increasing use of ionizing radiation in medical and non medical settings. To fill this need we propose the SUMMER UNDERGRADUATE PROGRAM TO EDUCATE RADIATION SCIENTISTS (SUPERS). We hypothesize that by (1) providing undergraduate students with a supportive environment that teaches them the underpinnings of cancer biology, radiation biology, radiation physics, and cancer imaging (2) introducing them to state-of-the-art laboratory techniques and equipment, and (3) exposing them to the clinical (translational) relevance of radiation research, we will encourage a significant number of these students to pursue cancer and radiation research career trajectories. Particular emphasis will be placed in the recruitment of minorities and women into the program, two populations who are substantially underrepresented in upper professional ranks of radiation research departments. There are 2 specific aims to our program: In Aim 1, we will provide a training program to expose talented undergraduate students to radiation biology, physics and cancer imaging. This program will combine both formal lecture-style education with hands-on research training in the laboratories of selected preceptors. The SUPERS program has the flexibility of allowing undergraduate students to participate for two consecutive summers. In Aim 2, we will establish strategies to engage students who take part in this program to pursue a career in cancer and radiation-related research. We will (a) expose students to scientists and physicians who have made significant contributions to cancer research, emphasizing the connection to clinical impact of these contributions. (b) encourage students to present their work at a research retreat to increase their confidence in their ability to communicate with other scientists, (c) establish personal relationships with the students to encourage them to stay in touch with preceptors and program leaders after completing the SUPERS program, and (d) establish a program to track outcome (career trajectories) of the SUPERS students and evaluate the programs success. By completing the goals of this program, we anticipate that we will increase both the number and quality of scientists engaged in cancer and radiation-related research, which should have a substantial impact in areas from healthcare to bioterrorism. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to identify, recruit and train talented undergraduate students, in the basic and translational aspects of Radiation Biology, Physics and Cancer Imaging. These three related fields have seen a steady decline in scholarly participation during the last two decades. Particular emphasis will be placed in the recruitment of racial minorities and women into the program, whom are substantially underrepresented in the radiation sciences. By completing the stated goals of this program, we anticipate that we will increase both the number and quality of future scientists engaged in Radiation-related research which should have a substantial impact in improving diverse life arenas such as health care and bioterrorism.

DESCRIPTION (provided by applicant): This application requests funds to purchase a precision X-Irradiator/ConeBeam CT for animal and cellular use at the University of Pennsylvania. This instrument (named Small Animal Radiation Research Platform-SARRP) represents a significant advance over any current instrument and was developed by Dr. John Wong at Johns Hopkins University, with support from the NCI through the Bioengineering Research Partnership mechanism. The SARRP addresses a critical need in Radiation Research for Image Guided Radiotherapy in rats and mice with resolution and accuracy comparable (on a scale- proportional basis) to therapy in humans. All former instruments for animal use have been derived from standard irradiators used for humans, large animals or industry. These represent, with minor improvements, 50-yr old technology. Until the development of this new machine (being commercially developed and sold by Gulmay Medical, Inc.) beam collimation for animal irradiators has been limited to shielding blocks and most irradiators had no freedom of movement for beam direction. The current instrument has the capability of 0.5 mm2 beam (a 400-fold improvement) and beam direction can be varied over 1200, with precise laser- based control of isocenter. The irradiation stage has 4-dimensional movement control (X-Y-Z and rotation) allowing complete flexibility of dose administration. Most importantly, the instrument is designed to be operated under low power as a cone- beam CT (CBCT) instrument and software for the instrument is modeled after human dose- planning, using the CBCT data. The instruments software control will also allow image fusion with other modalities, importantly PET and MRI. The instrument will be used in conjunction with other state-of-the art imaging equipment and will be initially installed in space occupied by the Radiation Research Division in the John Morgan Building and later moved to the new Perelman Center for Advanced Medicine at the University of Pennsylvania. Thus, in addition to providing advanced research capabilities for the Radiation Oncology Department, this instrument will open a world of opportunity for combined research with all other users of the Small Animal Imaging facilities at PENN. Importantly, numerous aspects of normal tissue research will become possible due to the precision of this device. Examples include realistic comparisons, using X-rays, of the scanned beam system designed for the Roberts Proton Radiotherapy center and selective irradiation of specific regions of mouse lung and brain. PUBLIC HEALTH RELEVANCE: This application requests support to purchase a unique animal irradiator, named the 'Small Animal Radiation Research Platform'(SARRP - Gulmay Medical). This instrument represents a unique, revolutionary, technical achievement that for the first time allows state-of-the art radiotherapy to be delivered to small animals (mice and rats) to closely recapitulate the parameters used for treatment of human cancer patients with radiotherapy. This will allow researchers in different fields (Radiation Oncology, Radiology, Pulmonary Medicine, etc), to study the effects of radiation on the tumor and surrounding normal tissues. This research will lead to better approaches to deliver radiation to the target, mitigate normal tissue toxicity and image changes in normal tissues before they manifest pathologically, thereby improving overall human health and quality of life.

DESCRIPTION (provided by applicant): The overall goal of this Program is to investigate the role of the Unfolded Protein Response (UPR) signaling pathway in tumor homeostasis and tumor progression. Rapidly proliferating cancer cells must thrive in a microenvironment wherein metabolic nutrients such as glucose, oxygen and growth factors become limiting as tumor volume expands beyond the established vascularity of the tissue. The UPR functions as a sensor of the availability of key cellular nutrients, such as glucose and oxygen that are criticall important for tumor growth and progression. The UPR and specifically the PERK kinase, has recently been shown to facilitate oncogene-mediated tumor progression, suggesting that the UPR may also respond to bioenergetic challenges triggered by aberrant oncogene-dependent signaling. The overall hypothesis being interrogated by this Program Project is that the UPR and more specifically, the PERK kinase, functions as a sensor of tumor ceil autonomous and non-autonomous bioenergetic stress; the ensuing activation of PERK catalytic function promotes tumor cell adaptation to this stress and thereby facilitates tumor progression. To test this hypothesis, three synergistic projects have been developed. Project 1 will evaluate mechanisms whereby a micro-RNA balances PERK-dependent pro-survival and pro-apoptotic functions. Key preliminary data suggest that miR-211 is a novel regulator of the pro-apoptotic factor, CHOP, and functions to temporally regulate CHOP expression. Project 2 will interrogate the function of PERK as a first response regulator of c- Myc-dependent bioenergetic and proteotoxic stress. Through its capacity to temper protein translation, PERK moderates cellular response to c-Myc thereby ensuring that bioenergetic capacity matches oncogenic demand resulting in tumor growth rather than apoptosis. Project 3 will test the hypothesis that tumor cells activate the UPR, and, perhaps, more broadly the Integrated Stress Response (ISR) due to oncogene activation or oxygen and/or nutritional deficit, and thereby acquire the ability to escape the anti proliferative and pro-apoptotic effects of Type 1 interferons, IFN?/ß. Through the synergistic functions of this Program, we will ascertain how PERK balances growth with apoptosis (Projects 1 and 2), how PERK responds to environmental challenge (Projects 1 and 3) and how tumor cells utilize PERK and the UPR to adapt to oncogene-triggered bioenergetic stress (Projects 1-2-3). All three projects will make extensive use of scientific Core B (Cell/Tissue Morphology Core) and have already established a working, highly collaborative relationship. It is our supposition that findings stemming from work proposed herein will provide a foundation for the design of novel anti-cancer treatment strategies targeting this pathway.

Malignant Neoplasms; programs; Proteins; response;

The Unfolded Protein Response (UPR) is a cellular homeostatic program initiated by an excess of unfolded/misfolded client proteins in the Endoplasmic Reticulum (ER) lumen, with primarily a cytoprotective effect. We previously showed that tumor cell survival under hypoxic and nutrient deprivation stress is dependent on the ER resident protein and UPR effector PERK. In addition to the tumor microenvironment, oncogenes are also known to activate cellular stress responses, including metabolic stress, apoptosis, and senescence. MYC is the target of chromosomal translocation or gene amplification during the development of many human cancers. c-Myc expression has been associated with robust upregulation of both total cellular protein content and rates of protein synthesis. This finding raises the possibility that c-Myc-transformed cells experience a higher than normal level of ER stress. In preliminary studies, we have used multiple genetic models of regulated c-Myc activation, to demonstrate that Myc activates the PERK/elF2?/Atf4 arm of the UPR. Activation of the UPR leads to increased cell survival via the induction of cytoprotective autophagy and reduced release of Ca^* from the ER. PERK ablation significantly reduced Myc-induced autophagy, cell transformation and tumor formation in nude mice. Samples from E?-Myc mice and human lymphomas demonstrate higher levels of UPR activation, compared to corresponding normal tissues. We hypothesize that the increase in protein burden in cells overexpressing c-Myc results in ER stress and activation of the UPR which tempers ER stress and facilitates transformation and tumor growth. This hypothesis will be tested in four specific aims: In Aim 1, we will determine the requirement for elF2? phosphorylation, ATF4 and Chop induction, Nrf2 activation and miRNA 211 in c-Myc-induced transformation in vitro and in vivo. In specific Aim 2, we will elucidate the mechanism of cytoprotection afforded by UPR activation in the context of oncogenic transformation by Myc. Under specific Aim 3, we will investigate the impact of c-Myc overexpression on the activation of the other two major UPR pathways. In specific Aim 4, we will determine the role of PERK activation in c-Myc induced lymphomagenesis using a transgenic mouse model. Successful completion of these studies would establish, for the first time, Myc upregulation as a cell-autonomous activator of UPR and would unveil novel targets for inhibiting Myc-dependent tumorigenesis.

? DESCRIPTION (provided by applicant): Radiation therapy is commonly used to treat solid tumors including head and neck squamous cell cancer (HNSCC); however, many patients still fail locally. Therefore, we need to find new ways to increase its effectiveness. We have been investigating inhibitors of the PI3K/mTOR pathway. In preliminary studies we found that NVP-BEZ235, a dual PI3K/mTOR inhibitor, and NVP-BKM120, a PI3K inhibitor, radiosensitize cells in vitro and induce autophagy, which we hypothesize is a cytoprotective response rather than a mode of cell death. In Aim 1 we will test this hypothesis in vitro using BKM120 and also in vivo with flank and orthotopic tumors in nude mice. We will use both genetic approaches (knocking out key autophagy genes) and pharmacologic approaches (chemicals that inhibit autophagy, Spautin1 and chloroquine). We also have preliminary data that multiple drugs that inhibit PI3K/mTOR signaling including, the 2 above and GDC-0980, GDC-0068, and RAD001, decrease O2 consumption rate (OCR) in vitro. We have also shown that BEZ235 decreases tumor hypoxia in vivo; thereby, leading us to propose a new model by which oxygenation within tumors may be modulated to increase cell killing after radiation. In Aim 2 we will investigate the mechanism(s) by which these drugs decrease OCR. We have 2 hypotheses, the first of which is that they increase Ser293 phosphorylation of the E1? subunit of pyruvate dehydrogenase (PDH), which is a critical gatekeeper of mitochondrial respiration. Phosphorylation of PDH E1? inhibits its function, hence reduces entry of pyruvate into the citric acid cycle and consequently decreases OCR. Our second hypothesis is that drugs that inhibit mTOR downregulate the expression of mitochondrial proteins that are involved in cellular respiration. In Aim 3 we will investigate whether the decrease in O2 consumption by PI3K/mTOR inhibition leads to increased radiation sensitivity in vivo. One of the ways we will do this is by using the drug GDC-0980, which does not affect intrinsic (in vitro) radiosensitivity but does reduce OCR. Hence, if this drug leads to increased radiation response in vivo, it is likely through effects on oxygenation. In Aim 3 we will also continue our screen of a 426 chemical compound library of FDA- approved agents to search for other agents that decrease OCR. We will then test the top candidates (in terms of degree of reduction of OCR) for their effects on tumor hypoxia in vivo and determine whether they have an additive effect with PI3K/mTOR inhibitors on decreasing hypoxia. Successful completion of these aims will set the stage for PI3K/mTOR inhibitors currently being tested in the clinic to be used in combination with radiotherapy for HNSCC and generate new leads for translational drugs that impact upon tumor cell oxygen metabolism.

Project Summary The Radiobiology and Imaging Program has been continuously approved by the NCI Cancer Center Support Grant since 1987. The Program seeks to improve patient outcomes through the advanced understanding of how ionizing and non-ionizing radiation interacts with cancer and normal tissues. The Programmatic goals are to: (1) Study molecular mechanisms of radiation response and identify targets to improve radiotherapy. (2) Elucidate mechanisms underlying use of Photodynamic Therapy (PDT) and translate to the clinic. (3) Develop methods for measuring and altering tumor oxygenation and metabolic status; understand the molecular events governing cell death by IR and physiological stresses. (4) Develop novel techniques to image the interaction between radiation, PDT and tissues. (5) Understand the biological effects of protons to inform their effective clinical use. The Program was rated as ?Exceptional? at the time of the 2010 CCSG renewal application and is led by Constantinos Koumenis, PhD, Professor and Director of the Research Division of Radiation Oncology and Amit Maity, MD, PhD, Professor of Radiation Oncology. Drs. Maity and Koumenis are NCI-funded researchers who bring their scientific vision to this Program, which is focused on basic and translational research and the development of investigator-initiated trials. Since the last renewal, the Co- Leaders have recruited new junior and senior scientists, enhanced collaborative peer-reviewed funding and increased the number of investigator-initiated clinical trials involving radiotherapy and imaging. Moreover, Drs. Koumenis and Maity have steered the Program towards new areas of emphasis including combined radiation and immunotherapy modalities and precision medicine and they have expanded the incorporation of imaging modalities into basic and translational efforts. Through this process, they increased interactions with the Immunobiology, Cancer Therapeutics, Breast Cancer and Cancer Control Programs. A major development has been the substantial expansion of both translational and clinical studies of proton therapy. Program members represent six departments from four schools at Penn. During the past five years, translational research has continued to be a major focus. The 33 Program members have $7.7M in research grant funding (annual direct costs), of which $7.5M is peer-reviewed and $3.7M is NCI-funded. There were a total of 405 cancer-related publications authored by Program members during the project period. Of these, 19% are intra- Programmatic, 28% are inter-Programmatic and 53% are multi-institutional.    

PROJECT SUMMARY The overall goal of the Small Animal Radiation Core (SARC) is to provide ?turn-key? animal housing and photon and proton radiation (including image-guided radiation) to tumors implanted into syngeneic mice as well as spontaneous, autocthonous tumors. Moreover, it will furnish accurate dosimetry and quality control necessary for successful completion of the studies proposed in Projects 2 and 3. A key feature of this Core is the centralization and standardization of services intended to minimize inter-animal variability in terms of immune system regulation and tumor response to radiation. We have devised a streamlined process for movement of animals from ordering (C57/Bl6) or procuring them from breeding (KPC pancreatic tumor model) to tumor radiation and back to housing until the animals are euthanized and tumors and tissues are harvested. At the center of this Core is a dedicated animal holding facility and a brand new renovated animal treatment room which will house and already available and established Small Animal Radiation Research Platform (SARRP) instrument from X-Strahl, Inc as well as dedicated proton Beam available in the Smilow Center for Translational Research (SCTR). The three main aims of this core are: Aim 1: To establish a dedicated housing suite with controlled environmental parameters. Aim 2: To deliver both Photon and Proton-based irradiation to mouse tumors in a conformal manner using the Small Animal Radiaiton Platform (SARRP) and available Proton Beam thereby avoiding radiation of normal tissues as much as possible. Aim 3: To develop precise dosimetry and quality-control analysis systems to ensure the prescribed dose was delivered to the target. The two co-Directors, Drs. Koumenis and Solberg, bring considerable expertise in small animal radiation as well as managing radiation facilities both at Penn and prior Institutions. Successful implementation of these services will provide the Project Leaders seamless and uniform treatment modalities and has the potential to serve as a model facility for other similar research efforts world-wide.

Abstract The and functions, pathophysiology sheer enormity of the microbial biomass in the human intestinal tract, the co-evolution between humans the microbiota, and the established function of the gut microbes in regulating normal host physiologic are all consistent with the idea that alterations in gut microbial ecology play a role in the of several conditions including cancer.Radiotherapy (RT) is an established curative and palliative cancer treatment regimen, with approximately half of cancer patients with solid tumors receiving RT some time during their disease. Mounting evidence also suggests that high-dose hypofractionated radiation exerts potent immune modulatory effects, prompting immunological active tumor cell death inducing tumor associated antigen (TAA) cross priming with elicitation of anti-tumor CD8+ T cells, and abscopal effects. Although cancer whether be there have been groundbreaking responses to immunotherapy in certain malignancies such as lung and melanomas, so far, immunotherapy is effective only in a portion of patients. This raises the issue of there are additional important regulators of T cell function that are relevant to tumor control and could harnessed to enhance radiotherapy.In support of this hypothesis, we have generated preliminary results demonstrating that: a) Treatment with vancomycin caused a significant enhancement of the tumor inhibitory effect vancomycin enhanced the ability of RT to increase ovalbumin specific and IFN- ??producing CD8+ infiltrating T cells; c) gut gram+ depletion enhanced antigen presentation in the tumor draining lymph nodes and e) the observed synergy between vancomycin and RT in eliciting an anti-tumor immune response and inhibiting tumor growth was abrogated in IFN-? KO animals or by CD8+ cell depletion. Having established a direct link between the intestinal bacterial contents and radiotherapy, we propose the following three specific aims: Aim 1. To test whether the radiation enhancing effects of vancomycin are dependent on Gram+ bacteria, are transferable to another host and to identify the bacterial species responsible; Aim 2. To investigate the mechanism by which vancomycin treatment impacts both the priming and effector phases of RT-elicited antitumor immune responses; Aim 3. Perform a randomized pilot trial of antimicrobial therapy and stereotactic body radiation therapy in early-stage non-small cell lung cancer. Successful completion of these studies will firmly establish the microbiome as a target for therapeutic intervention in patients receiving radiotherapy and will identify markers of response in the clinical setting. of targeted radiation; b)

ABSTRACT/SUMMARY (Project 2) Tumor cell intrinsic stress including oncogene activation, as well as extrinsic stresses, such as low oxygen/nutrient availability, elicit perturbations in the endoplasmic reticulum (ER). Moreover, oncogenically transformed cells face increased burden placed by augmented biosynthetic pathways and rewired metabolism to meet the demands imposed by rapid proliferation. Adaptation to the ensuing stress and re-establishment of cellular homeostasis is achieved via activation of a coordinated signal transduction program termed the Integrated Stress Response (ISR). During the previous funded period, we demonstrated that increased rates of protein synthesis elicited by oncogenic MYC, activate the PERK/GCN2?eIF2? arm of the ISR, thereby supporting MYC-induced cell transformation. In preliminary unpublished studies, we have accumulated strong evidence supporting an essential role for the ISR effector and target of eIF2? ATF4, in transformation and tumorigenesis, particularly in tumors with activated MYC. However, how ATF4 elicits differential responses to various stresses in the context of MYC-dependent transformation is a critical question that remains unanswered. We will test the hypothesis that activation of ATF4 by the ISR plays a critical role in MYC- induced transformation and tumor progression by promoting metabolic and translational adaptation in coordination with MYC by focusing on three specific Aims. In Aim 1 we will identify critical nodes in cellular metabolism and translational regulation which are coordinately regulated by both ATF4 and c-MYC. Specifically, we will delineate the mechanism of Glut-1 and eIF4E transcriptional activation by ATF4 and determine functional requirements of GLUT1 and eIF4E in regulating glycolysis, translation and survival during MYC-dependent transformation in vitro and in vivo. Under Aim 2, we will delineate the mechanism of co- regulation of transcriptional targets between ATF4 and MYC by ChIP-seq analysis in lymphoma, colorectal (CRC) and prostate (PCa) cancer cells expressing inducible forms of MYC and analyze coordinately regulated genes. We will then determine the effects of knockout/knockdown of the identified co-regulated genes and newly identified targets in MYC-dependent proliferation, apoptosis and tumor growth. Finally, under Aim 3, we will determine the role of ATF4 in MYC-dependent transformation and tumorigenesis in PCa and CRC tumors using a conditional knockout ATF4 model crossed with PTENfl/fl:MycTg, and mouse CRC models (orthotopic, syngeneic and spontaneous) as well as 3D CRC organoids. We will also work with Project 1 to analyze the regulation of ATF4-dependent downregulation of BMAL1 and Clock genes and their role in translation and lymphomagenesis. Finally, with Project 3, we will analyze the effects of ATF4 ablation on type I interferon pathway and viability/effector functions of tumor-infiltrating cytotoxic T lymphocytes. Completion of these aims will provide a better understanding of the critical role of ATF4 in MYC-dependent pro-tumorigenic processes and may uncover new targets for therapeutic intervention in these malignancies.

ABSTRACT/SUMMARY (Overall) The overall goal of this Program is to investigate the role of the Integrated Stress Response (ISR) signaling pathway in tumor cell fate and tumor progression. Rapidly proliferating cancer cells must thrive in a microenvironment wherein metabolic nutrients such as glucose, oxygen and growth factors become limiting as tumor volume expands beyond the established vascularity of the tissue. The ISR integrates signals from sensors (such as the endoplasmic reticulum kinase PERK and cytoplasmic kinase GCN2) of cellular nutrients to homeostatic processes including translational control, carbon and oxygen metabolism and receptor signaling. The ISR has also been shown to facilitate oncogene-mediated tumor progression, suggesting that it may also respond to bioenergetic challenges triggered by aberrant oncogene-dependent signaling. The overall hypothesis to be tested in the proposed studies is that the Integrated Stress Response plays a pivotal role in mediating MYC-dependent and hypoxia-dependent tumor progression through its capacity to engage and regulate key pathways involved in circadian, translational, metabolic and immune functions thereby facilitating tumor cell survival and growth. The above hypothesis will be tested by three highly integrated projects: Project 1 will define miRNAs subject to ISR control whose function is to fine-tune protein synthesis during an ISR/UPR response. Two key, microRNAs, miR-211 and miR-217, are the focus; collectively, they function as regulators of Bmal1 during ER stress and their contribution to Bmal1 repression to lymphoma progression is critical for tumorigenesis. Project 2 will identify critical nodes in metabolism and translation control which are coordinately regulated by both ATF4 and c-MYC and delineate the mechanism of co-regulation of common transcriptional targets. It will also functionally test the role of ATF4 in MYC-dependent transformation and tumorigenesis. Project 3 will delineate the mechanisms underlying ISR-induced IFNAR1-dependent and independent inactivation of the IFN1 pathway, its role in the loss of viability of intratumoral cytotoxic lymphocytes and the generation of the immune privileged niches. It will also determine whether targeting these mechanisms can augment anti-cancer immunity. All three projects will make extensive use of Core A (Administrative) and scientific Cores B (Metabolomics/Genomics) and C (Biostatistics) and have already established a working, highly collaborative relationship. Collectively, our three integrated and synergistic Projects will provide a molecular framework that addresses the potential efficacy of targeting the ISR to antagonize malignancy in three highly prevalent and lethal types of tumors.

ABSTRACT (Core A) The Administrative Core (Core A) will provide support and effort coordination for each of the projects in this P01, as well as the two scientific cores. The aims of the Administrative Core are to ensure that the resources within the P01 are distributed equitably to the investigators and that all rules and regulations of the National Cancer Institute and the National Institutes of Health are followed. The Administrative Core will coordinate meetings of the investigators and their laboratories and facilitate communication between our investigators with the External Advisory Board. The finances of the P01 will be organized by Ms. Jenine Iannaccone, Grants Administrator in the Department of Radiation Oncology, who will meet monthly with the P01 Principle Investigator and quarterly with each Project and Core Leader. Ms. Andrea Rycroft, Administrative Coordinator, will coordinate travel arrangements for the members of the P01, as well as the advisors who will visit the University of Pennsylvania. She will also provide administrative support for each of the P01 investigators for efforts related to the Program. It is anticipated that through the combined efforts of the Core Director Ms. Rycroft and Ms. Iannaccone, the research operations and communications of the P01 will run with optimum efficiency