Leonidas C. Platanias - US grants

DESCRIPTION (Adapted from the applicant's abstract): Type I interferons (IFNs) are pleiotropic cytokines that exert multiple biological effects on normal and malignant cells, including antiproliferative, antiviral, and immunomodulatory activities. IFNalpha is used extensively in the treatment of neoplastic diseases and viral infections. Despite the widespread use of IFNalpha in clinical medicine, the specific mechanisms by which this cytokine exerts its effects have not been elucidated. Understanding of such mechanisms will provide valuable information that could be applied in the design of more effective means to use IFNs, and also in the development of novel antineoplastic and antiviral agents. Insulin receptor substrate (IRS)-proteins play a central role in insulin and insulin-like growth factor-1 (IGF-1) signaling by their src homology 2 (SH2)-docking function. These proteins are substrates for the insulin and IGF-1 receptors, and link these receptor tyrosine kinases to downstream signaling-proteins containing SH2 domains. IRS-proteins are also phosphorylated on tyrosine during IFNalpha treatment of cells. The best characterized member of this family, IRS-1, binds the SH2 domains of the p85 regulatory subunit of the phosphatidylinositol 3'-kinase in an IFNalpha-dependent manner, and such an association results in activation of this kinase. The goal of the studies proposed here is to precisely determine the function of IRS-proteins in Type I IFN signaling. Specific aim A is to determine whether the tyrosine residues of IRS-1 exhibit dual specificity, as tyrosine kinase substrates and as docking sites for IFN-regulated SH2-proteins. This will be achieved by establishing the interactions of IRS-1 with IFNalpha dependent kinases and downstream SH2-proteins, and by mapping the tyrosine sites of IRS-1 that are phosphorylated during IFNalpha stimulation. Specific aim B is to determine the biological consequences of activation of the IRS-system by IFNs. Studies will be performed to determine the responsiveness of cells that lack expression of IRS proteins to the antiproliferative and antiviral activities of Type 1 IFNs, and the effect of IRS-1 expression on such responses. Other studies will determine the effect of antisense IRS-1 RNA expression on the biological effects of Type I IFNs in IFNalpha-sensitive cells. The possibility that the antiproliferative effect of IFNalpha results in part by an antagonist inhibition of growth factor (insulin/IGF-1) signaling cascades will be also examined. The experiments proposed in specific aim C will determine the IFNalpha- dependent function of the related IRS-2 protein in cells that preferentially express this protein. They will determine whether IRS-2 also functions as a multisite SH2-docking protein, and whether the phosphatidylinositol 3'-kinase pathway is activated by engagement of this protein. Altogether, the proposed studies are aimed towards establishing a model in which signaling specificity for distinct ligands (IFNs, insulin, IGF-1) can be achieved at an early signaling point, through the differential phosphorylation of common docking proteins (IRS-proteins). Competition for the use of these proteins by tumor suppressor cytokines (IFNs) and neoplastic growth factors (insulin/IGF-1) may provide a mechanism by which the proliferative responses of neoplastic cells are regulated.

DESCRIPTION: (adapted from the investigator's abstract) Interferon (IFNa) is used extensively in the treatment of malignancies, but the mechanisms by which it exhibits its anti-tumor effects remain largely unknown. The applicants have identified a novel IFNa-signaling pathway involving engagement of the cbl proto-oncogene product and Crk-proteins (CrkL and CrkII). The working hypothesis is that this pathway plays a critical role in IFNa-signaling by providing a link between the functional Type I IFN receptor complex and downstream cascades that mediate the anti-tumor effects of IFNa. The current proposal is a systematic approach that will define the molecular functions of the Cbl-Crk pathway in IFNa-signaling, establish its biological relevance and determine its specific role in chronic myelogenous leukemia. Specific aim A is to identify the signaling functions of Cbl and Crk-proteins in IFNa sensitive cells. The hypothesis will be examined that Cbl, CrkL and CrkII play the role of adapter proteins, providing a link between the Type I IFN receptor and downstream pathways. This will be achieved by identifying the precise mechanisms by which IFNa-dependent tyrosine kinases regulate phosphorylation of these signaling molecules, and by identifying the elements activated downstream of them. Specific aim B is to determine the biological consequences of activation of this pathway. Two different methodologies will be used to achieve this goal. One approach will involve the generation of mutant, dominant-negative Cbl- and Crk-proteins and the expression of them in IFNa-responsive cell lines. The second approach will involve studies to determine the effect of antisense mRNA and antisense oligonucleotides against Cbl and Crk-proteins on the biological effects of Type I IFNs on normal and malignant cells. Specific aim C is to examine the potential role of Crk-proteins in mediating the growth inhibitory effects of IFNa in chronic myelogenous leukemia. The effect of IFNa on the interaction of BCR-ABL with CrkL and on the BCR-ABL-regulated activation of mitogenic signaing pathways. Further experiments determine the sensitivity of leukemic progenitors from CML-patients to the antiproliferative effect of IFNa, and will examine whether this sensitivity correlates with specific IFNa-induced signaling events. Altogether, these studies should provide valuable information the mechanisms by which signals generated at the Type I IFN receptor mediate inhibition of malignant cell proliferation.

DESCRIPTION: (provided by applicant): Interferon alpha (IFNa) has significant clinical activity in the treatment of chronic myelogenous leukemia (CML), but the mechanisms by which it exhibits its antileukemic effects remain unknown. We have identified a novel signaling cascade activated by the Type I IFN receptor, involving the small GTPase Rac1 and the p38 Map kinase. This pathway acts independently of the Stat-pathway, but in cooperation with it, to regulate transcriptional regulation of IFNa-sensitive genes. Our data demonstrate that this signaling cascade is activated in primary granulocytes from CML patients and that pharmacological blockade of its activation reverses the growth inhibitory effects of IFNa on primary leukemia bone marrow progenitors. This proposal is a systematic approach to identify the signaling mechanisms by which IFNa exhibits its antileukemic effects. Specific aim A is to determine the mechanisms of regulation of activation of the p38 pathway by the Type I IFN receptor in BCR-ABL expressing cells and to identify downstream effector mechanisms. Studies will be performed to determine the roles of Jak kinases and the vav proto-oncogene product on the activation of the Rac1/p38 pathway in BCR-ABL expressing cells and to define the role of p38-dependent nuclear histone serine phosphorylation in the induction of IFNa-responses in CML cells. Specific aim B is to determine the biological consequences of activation of p38 in CML. It will involve studies to determine whether Rac1 and p38 are essential for the generation of the growth inhibitory effects of IFNa on primary leukemic progenitors and whether defective activation of this pathway correlates with IFNa-resistance. It will also examine the hypothesis that IFNa downregulates BCR-ABL protein expression via a p38-dependent mechanism. Specific aim C includes studies to identify the mechanisms by which the BCR-ABL-tyrosine kinase antagonizes IFNa-dependent gene transcription and determine whether the BCR-ABL specific inhibitor, STI571, augments the growth inhibitory effects of IFNa via regulatory effects on the Rac1/p38 and Jak/Stat pathways. Altogether, these studies should provide important information on the mechanisms by which signals are transduced by the Type 1 IFN receptor in CML cells and advance our knowledge on the mechanisms of development of IFNa resistance. Identifying such mechanisms will facilitate the development of novel therapeutic approaches to overcome IFNa-resistance and the design of new pharmacologic agents for the treatment of CML.

DESCRIPTION: (provided by applicant) This is a competitive grant-renewal application, in which the overall goal is to understand the mechanisms of Type I IFN signaling in malignant cells. During the previous funding period, we identified several novel-IFN activated signaling cascades involving the CrkL-adapter and demonstrated that CrkL plays a critical role in Type I IFN signal transduction. We will now undertake studies to establish the functional roles of the different domains of CrkL in the context of its signaling capacity and define the contribution of distinct CrkL-cascades in the generation of the biological effects of IFNs. A major goal of this grant application is also to determine the mechanisms of regulation of Stat-serine phosphorylation. In addition to tyrosine phosphorylation, IFNalpha-dependent phosphorylation of Stat1 and Stat3 on serine 727 is required for transcriptional activation of target genes. Although it is well established that Jak-kinases regulate tyrosine phosphorylation of Stats, the IFNalpha-activated serine kinase that phosphorylates Stat-proteins is not known. The identification of such a serine kinase is an important outstanding issue, required to complete our understanding on the IFN-activated Jak-Stat pathway. Our data have established that a member of the protein kinase C-family of proteins, PKC-delta, is activated in a Type I IFN-dependent manner and regulates serine phosphorylation of Stat1. These findings, for the first time, provide the identity of the IFNalpha-activated Stat-serine kinase and form the basis for further studies. The objective of this section of this proposal is to define the mechanisms by which PKC-delta is activated by the Type I IFN receptor and interacts with Stat1, and possibly Stat3, to effect their phosphorylation on serine 727. Studies will be also performed to define the role of PKC-delta activation/Stat-serine phosphorylation in the induction of the antiproliferative, antiviral and immunomodulatory effects of Type I IFNs, as well as the induction of suppressive effects of IFNalpha on bone marrow leukemic progenitors from patients with chronic myelogenous leukemia. Altogether, this proposal will address important aspects of the mechanisms of Type I IFN-signal transduction and generation of antileukemic effects.

DESCRIPTION (provided by applicant): There is accumulating evidence that in addition to the classic IFN-activated Jak-Stat pathway, non-Stat pathways play important roles in the induction of the biological activities of interferons. We have provided the first evidence that Type I IFNs activate the p38 Map kinase pathway and that activation of this signaling cascade plays a critical role in Type I IFN-dependent transcriptional regulation. Our preliminary studies strongly suggest that the function of p38 MAPK is essential for the generation of IFN-responses, including the suppressive effects of IFNs on human hematopoiesis and the induction of an antiviral state. The overall goal of this proposal is to define the functional role of the p38 pathway in Type I IFN-signaling. The studies of specific aim A will identify upstream elements of the Type I IFN-dependent p38 MAPK pathway and will define the roles of such elements in the generation of IFN-responses. Specific aim B will determine whether the p38 MAPK cascade cooperates with the classic Jak-Stat pathway, or acts independently of it to regulate transcription of interferon-sensitive genes. The potential role that p38-mediated histone serine phosphorylation in the promoters of ISGs may exhibit in Type I IFN-dependent transcriptional activation, will be also examined. Specific aim C will define the role of p38 MAPK in the induction of Type I IFN-dependent antiviral activities against HBV and the generation of growth inhibitory responses on normal bone marrow progenitors. These studies will also identify the stage in the hematopoietic lineage cascade at which p38 is activated by Type I IFNs and will dissect the pathways downstream of p38 that mediate hematopoietic suppression. Specific aim D will determine whether the p38 pathway is activated by IFNalpha in tumor cells from patients suffering from malignant melanoma, and whether defective activation of this pathway accounts for the development of IFN-resistance in melanoma. This multifaceted approach should provide important insights on the mechanisms by which IFN-signals translate to specific biological responses, as well as on the mechanisms by which malignant cells develop resistance to the biological activities of interferons. Understanding such mechanisms may have important translational implications and facilitate the development of novel IFN-based therapeutic approaches in the future.

[unreadable] DESCRIPTION (provided by applicant): Arsenic trioxide (AS2O3) is a heavy metal derivative that has potent antileukemic properties in vitro and in vivo. This agent is used in the treatment of patients with acute promyelocytic leukemia (APL), but the precise mechanisms by which it induces its antileukemic effects are not known. We have identified a novel signaling pathway activated by arsenic trioxide in APL cells, involving the p38 Map kinase. Our data suggest that activation of this signaling cascade exhibits a negative regulatory role on the induction of apoptosis and cell differentiation of APL cells. The overall goal of this grant application is to understand the mechanisms by which p38 negatively regulates the induction of AS2O3-responses in APL cells. Specific aim A is to determine the mechanisms of activation of the p38 Map kinase by AS2O3 in APL cells. Studies will be performed to examine the roles of the small GTPases Rac1 and Cdc42, and the Pak1 kinase, and to identify the Map kinase kinase (Mkk) that directly phosphorylates and activates p38. Specific aim B is to identify the downstream effector mechanisms by which the p38 Map kinase controls AS2O3-dependent apoptosis. Studies are proposed to examine the patterns of activation of different p38-isoforms in APL cells and to determine their roles in the generation of AS2O3-responses. Experiments will be also performed to dissect the contributions of different downstream effectors of the p38 pathway in the regulation of such responses. Specific aim C is to examine the activation of p38 in primary leukemic blasts from APL patients or patients with other subtypes of acute myeloid leukemia (AML), and determine whether such activation correlates with sensitivity or resistance to the effects of arsenic trioxide. Altogether, these studies should advance our overall understanding of the mechanisms by which AS2O3 generates its effects on malignant cells. They may also provide the basis for future clinical-translational efforts of combinations of AS2O3 and p38 inhibitors for the treatment of APL, and possibly other forms of AML. [unreadable] [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The anemia of chronic disease (ACD) is one of the most common types of anemia in the elderly and a frequent cause of morbidity in these patients. Overproduction of pro-inflammatory cytokines has been previously implicated in the pathogenesis of this syndrome, but the precise molecular mechanisms that lead to ineffective erythropoiesis are not known. We have shown that the p38 Map kinase pathway is a common mediator of the effects of different myelossuppressive cytokines on normal erythropoiesis and that its activation is essential for suppression of growth of human erythroid progenitors. We have also demonstrated that pharmacological inhibition of this pathway results in enhanced erythroid colony formation from the bone marrows of patients with ACD, suggesting a key role for this signaling cascade in the pathophysiology of this syndrome. The overall goal of this grant application is to determine the precise role of the p38 pathway in the pathogenesis of the anemias of chronic disease and to identify the mechanisms by which it mediates such effects. Specific aim A is to determine the role of p38 in the generation of the effects of the pro-inflammatory cytokine tumor necrosis factor a (TNFa) and the iron-regulatory hormone hepcidin on normal erythropoiesis. Specific aim B is to define the mechanisms of activation of the p38 pathway in bone marrows from patients with ACD, and to determine whether pharmacological or molecular inhibition of different p38-isotypes enhances the growth of erythroid progenitors from ACD-bone marrows in vitro. This will include studies using a novel inhibitor of p38, SCIO-469, that is currently under clinical development for other entities. Specific aim C is to dissect the roles of specific downstream effectors of p38 in the suppression of normal erythropoiesis in ACD bone marrows. The activation of known p38-regulated kinases, such as MapKapK2, MapKapKS, Msk1, and Mnk1, will be examined in ACD bone marrows, and their contributions to the suppression of erythropoiesis will be determined. Altogether, these studies should advance our overall understanding of the pathogenesis of chronic anemias in the elderly, and may provide the basis for the future development of novel therapeutic approaches for the treatment of ACD using as targets p38 and/or its effector kinases. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a competitive grant renewal application, whose overall goal is to understand the mechanisms of IFN-signaling. IFNs are pleiotropic cytokines that exhibit important antiviral and antineoplastic activities in vitro and in vivo. Although the mechanisms of IFN-dependent gene transcription via activation of the classic Jak/Stat-pathway are well understood, very little is known on the mechanisms of mRNA translation of IFN-sensitive genes and generation of protein products that mediate the biological effects of IFNs. We have identified a novel signaling pathway shared by both the Type I and II IFN receptors, involving activation of the PI 3'kinase and mTOR, and downstream engagement of the p70 S6 kinase. Our data indicate the function of this pathway is essential for mRNA translation of key IFN-sensitive genes and induction of the antiviral properties of IFNs. Interestingly, this pathway is also well-known to transduce mitogenic signals in response to growth factors and oncogenes. The surprising finding of engagement of this cascade by IFNs suggests that, under certain circumstances, mTOR-generated signals can mediate antiviral and, possibly, antitumor responses. The current proposal is a systematic approach to determine the role of this pathway in IFN-signaling. Specific aim A will examine the mechanisms of activation of mTOR by IFNs, and will define the role of Jak kinases in this process. Specific aim B will dissect the contributions of distinct IFN-regulated effectors downstream of mTOR in mRNA translation and induction of antiviral responses. Specific aim C will examine the functional relevance of 4E-BP1/2, Akt1/2 and p70S6k1/2 in the generation of the antiviral, immunoregulatory, and antitumor effects of IFNs in vivo, using different knockout mice. Finally, specific aim D will address the potential involvement of mTOR-pathways in the generation of the effects of IFNs on normal and leukemic hematopoiesis. Altogether, these studies should provide important information on the mechanisms by which signals are transduced by IFN receptors and advance our knowledge on the mechanisms of development of IFN-resistance. Identification of such mechanisms should prove valuable in designing better ways to use IFNs in clinical medicine and in the future development of novel, more potent and specific, pharmacological agents that target similar signaling pathways.

DESCRIPTION (provided by applicant): Extensive work over the last 3 decades has established the importance of interferons and other cytokines in health and disease. Several cytokines are implicated in development, regulation of innate immunity, control of normal hematopoiesis and other important cellular and biological functions, and there is rapid accumulation of knowledge in these areas. Greater understanding of mechanisms of disease and novel concepts for therapy will emerge from multi-disciplinary forums where advances can be presented and discussed by established and trainee scientists. The overall objective of this application is to obtain support for junior investigator attendance to the 2010 joint annual meeting of the two major international Societies addressing cytokine-mediated immune host modulation in health and disease: International Cytokine Society and the International Society of Interferon and Cytokine Research. The conference is entitled, "Cytokines in Infectious Diseases, Autoimmune Disorders and Cancer," to be held in Chicago, October 3-7, 2010. This event unites the biomedical expertise and energies of the two societies and has become the world's most important annual conference on basic and clinical research on cytokines. Recent annual meetings were held in Lisbon, Montreal and Vienna. The 2010 joint society meeting (Cytokines 2010) will bring together all members of both Societies to maximize interactions and foster collaborations. The goals of this meeting are: Specific Aim 1: To facilitate interactions of young investigators and trainees with well-established researchers in the cytokine and interferon research fields and to provide a forum for them to present their work. Specific Aim 2: To facilitate the development of a meeting that will attract scientists from academic and governmental institutions and from industry to discuss recent advances on the roles of cytokines and interferons in infectious diseases, autoimmune disorders and cancer. Oral and poster presentations will be made by both invited speakers as well as speakers selected from submitted abstracts. Specific Aim 3: To facilitate the development of a program which highlights the most important research developments and advances in the cytokine and/or interferon fields under the subject headings: inflammation, T cell biology/adaptive immunity, signal transduction, IL-1 biology, immunoregulation, infectious diseases, microbial pattern recognition, cytokines in immune cell development and differentiation, novel methods in cytokine detection, micro-RNAs and post-transcriptional regulation, interferons in the treatment of diseases, novel therapeutic targets in the treatment of malignancies, cytokines and cell death, viral mechanisms that block cytokine responses, cytokines and infectious diseases;interferons and viral infections, cytokines/chemokines and their receptors, structure/function and cytokine-based therapies. Specific Aim 4: To enable under-represented minorities and persons with disabilities to participate in a meeting that facilitates interactions with other scientists in their fields.

Abstract This proposal is a competitive renewal request submitted in response to PA-16-152 for continued funding of the Signal Transduction and Cancer Postdoctoral Training Program at the Robert H. Lurie Comprehensive Cancer Center (RHLCCC) of Northwestern University. The specific aims of the Program are: 1) to provide state of the art laboratory training to postdoctoral fellows in signal transduction and cancer, 2) to promote the recruitment of highly talented postdoctoral fellows with a special emphasis on attracting underserved minority trainees and 3) to support the career development of postdoctoral fellows in the program such that they can effectively contribute to translational cancer research. With strong Institutional support, the training program brings together a cadre of senior researchers, investigators who are new to the RHLCCC and three junior investigators, all of whom focus on aberrations in signaling pathways that contribute to the development of cancer. These preceptors are highly accomplished faculty at Northwestern University and members of the RHLCCC. A new feature of this program has been the development of a specialized curriculum in translational research that emanates from a newly established Translational Bridge Program at the RHLCCC. As described in the application, the curriculum consists of a core curriculum of coursework, seminars, symposia and journal clubs in addition to the mandatory course in Responsible Conduct of Research and training in scientific rigor and responsibility. The T32 has trained fifty postdoctoral fellows since inception of the Program in 1997; fourteen of our former trainees are in faculty positions, eighteen are senior scientists in industry and one is in a related admin position. The majority of our former trainees are engaged in cancer and signaling-related positions. We therefore request continued support for four postdoctoral fellows per year that will enable us to continue to offer focused training in signal transduction and cancer at the RHLCCC.

DESCRIPTION (provided by applicant): Interferons (IFNs) exhibit important antineoplastic properties in vitro and in vivo and are key elements in the immune surveillance against cancer. These cytokines have widespread applications in clinical medicine, but their utility in the treatment of many malignancies is frequently limited by the development of neoplastic cell resistance. We have identified a novel IFN-signaling cascade, involving the kinases Mnk1 and Mnk2. Our data have established that Mnk kinases play key roles in IFN-dependent mRNA translation and growth suppression, providing a direct link between MAPK pathways and mRNA translation of IFN-stimulated genes (ISGs). The engagement of this pathway ultimately regulates expression of protein products with key functional roles in the IFN-system, such as ISG15 which mediates ISGylation and other proteins with growth inhibitory properties. The current proposal is a systematic approach to establish the roles of Mnk pathways in the generation of IFN-mediated responses and to define the importance of defects in the activation of this cascade in the development of IFN-resistance in malignant cells. Specific aim 1 will identify upstream effector mechanisms and signaling events involved in the regulation of the Mnk pathway. Studies to define the roles of IFNR-associated Jak kinases in the activation of Mnk 1/2 will be performed, while the potential regulatory effects of Jak2 on the function of nuclear Mnk2b and mRNA nuclear export will be determined. The contribution of distinct MAPKKKs and MAPKKs in engagement of Mnks by the IFNR will be also assessed. Specific aim 2 will identify downstream elements of the pathway and will dissect their roles in the generation of IFN-dependent antineoplastic effects. The functional relevance of eIF4E phosphorylation and the involvement of hnRNPA1 and Sprouty proteins (Spry1 and 2) in IFN-signaling will be examined. Studies will be also performed to determine whether Mnks regulate mRNA translation of SLFN genes, a novel family of cell-cycle regulators that we have recently identified as mediators of IFN-antiproliferative responses. Specific aim 3 will determine the role of Mnk kinases in the generation of the suppressive effects of IFNa in Ph (-) myeloproliferative neoplasms with the JAK2V617F mutation. The requirement of Mnk activity in the induction of IFNa effects on primary clonal hematopoietic progenitors from patients with P. Vera and ET will be examined;and the ability of IFNa to induce responses in JAK2V617F mouse models with targeted disruption of the Mnk1 and/or Mnk2 genes will be determined. Altogether, these studies will advance our understanding of the mechanisms of generation of IFN-dependent antileukemic responses;provide important information on the events that lead to malignant cell resistance;and form the basis for the development of novel antineoplastic agents and approaches to overcome such resistance. PUBLIC HEALTH RELEVANCE: Interferons exhibit important antitumor effects in vitro and in vivo. These cytokines play key roles in the immune surveillance against cancer and have important activities in certain clinical settings, but the precise mechanisms by which they generate their effects remain to be defined. We have identified a novel IFN- activated signaling cascade involving Mnk kinases, which plays an important role in mRNA translation of IFN stimulated genes (ISGs), including genes with important biological properties, such Isg15 and Isg54. We have also identified a novel group of ISGs (SLFN genes) that regulate cell cycle progression and mediate IFN- dependent growth inhibitory responses. Our data suggest a model by which IFN-activated Mnk kinases complement the function of Jak-Stat pathways by regulating signals for mRNA translation of Stat-activated genes. The current proposal will precisely define the role of the Mnk pathway in the generation of the antileukemic properties of IFNs and will identify downstream effectors and targets of this cascade. Studies will be also performed to examine whether defects in the activation of Mnk kinases and/or their effectors result in leukemic cell resistance to the effects of IFNs in vitro and in vivo.

DESCRIPTION (provided by applicant): Type I interferons (IFNs) are pleiotropic cytokines with important antineoplastic and antiviral activities and constitute key elements in the immune surveillance against cancer and viral infections. Although several interferon stimulated genes (ISGs) that mediate the antiviral properties of IFNs have been identified, very little is known on the genes and protein products that mediate generation of the antiproliferative and antineoplastic effects of IFNs. We have identified a novel family of ISGs, the family of SLFN genes. Our data have provided the first evidence that engagement of the Type I IFN receptor (IFNR) results in expression of mouse and human SLFN genes, and have demonstrated that members of this family are involved in the suppression of anchorage-independent malignant cell growth and inhibition of collagen invasion by melanoma cells. The current proposal is a systematic approach to define the roles of distinct SLFN genes in suppression of oncogenesis and in the generation of the antineoplastic effects of IFNs;and to examine whether defects in the expression of specific SLFNs correlates with IFN-resistance in vitro and in vivo. Specific aim 1 will define the mechanisms of regulation of expression of SLFNs and will dissect the functional roles of distinct SLFNs in IFN-induced growth inhibitory effects. Studies to define effectors and mediators of responses downstream of SLFNs will be also performed. Specific aim 2 will determine whether induction of expression of SLFN proteins is essential for generation of the antineoplastic effects of IFN? in solid tumor mouse models in vivo. For that purpose malignant melanoma or renal cell carcinoma (RCC) mouse models will be established using mice with the elektra phenotype (Slfn2eka/eka) or Slfn1 or Slfn3 knockout mice. Finally, specific aim 3 will examine whether defective expression of human SLFNs correlates with resistance to the antineoplastic properties of IFN1 on primary malignant cells from patients with malignant melanoma or renal cell carcinoma (RCC), malignancies known to be responsive to immune modulation and IFN-treatment. Altogether, the proposed studies will address important issues on the role of SLFNs in the generation of antitumor responses and the means by which malignant cells develop resistance to IFNs. PUBLIC HEALTH RELEVANCE: Interferons (IFNs) play key roles in the immune surveillance against cancer and have important activities in certain clinical settings, but the precise mechanisms by which they generate their effects remain to be defined. We have identified a novel group of ISGs (SLFN genes) that regulate cell cycle progression and mediate IFN-dependent growth inhibitory responses. The current proposal will precisely define the role of these genes and their protein products in the generation of the antitumor properties of IFNs in vitro and in vivo. It will also determine whether defective expression of these genes correlates with resistance to the antitumor effects of IFNs against malignant melanoma and renal cell carcinoma.

DESCRIPTION (provided by applicant): This is a competing renewal application whose overall objective is to define the mechanisms of generation of the antileukemic effects of arsenic trioxide (As2O3) and to develop means and ways to enhance its antitumor properties. As2O3 is a heavy metal derivative with potent antileukemic activities in vitro and in vivo and it is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). Despite advances in the field and the high interest to expand the use of arsenic to other hematological malignancies, the precise mechanisms by which As2O3 induces antileukemic responses are not known. We have provided the first evidence that As2O3 -induced autophagy is essential for generation of its suppressive effects on primitive leukemic progenitors from patients with myeloid leukemias. The current proposal is a systematic approach to define the cellular events that lead to As2O3 -dependent autophagy and to identify the mechanisms of autophagy-mediated degradation of oncogenic proteins in primitive leukemic precursors and leukemia initiating stem cells (LICs). Specific aim 1 will examine the mechanisms of As2O3 -autophagy in leukemic progenitor cells. Studies will be performed to identify upstream kinases in the autophagic cascade in leukemia cells and to define the sequence of events leading to their activation in response to arsenic. Other studies will examine the relationship of autophagy with cellular negative feedback regulatory pathways (NFRPs) that are induced in response to As2O3. Specific aim 2 will identify effector mechanisms by which autophagy mediates arsenic- responses on primitive progenitors and leukemia initiating stem cells from patients with AML and CML. Experiments will be performed to define whether autophagy mediates arsenic-dependent degradation of different oncoproteins and to define the precise mechanisms by which this occurs. Other studies will determine whether pharmacological or molecular targeting of elements of the autophagic machinery can enhance arsenic-induced suppression of primitive leukemic precursors and LICs. Specific aim 3 will examine the role of autophagy in arsenic-dependent antileukemic responses in different leukemia mouse models in vivo. It involves experiments to determine whether autophagy is required for the antileukemic properties of As2O3 in vivo and to examine whether other inducers of autophagy exhibit synergistic antileukemic activities with As2O3. Altogether, this work will advance our understanding of the mechanisms by which As2O3 generates antileukemic responses. It should have important clinical-translational implications and possibly lead to the development of novel approaches to target early progenitors and LICs using combinations of As2O3 with other autophagy inducers. PUBLIC HEALTH RELEVANCE: Arsenic trioxide is a heavy metal derivative that exhibits important antileukemic effects in vitro and in vivo. This agent has major clinical activity in the treatment of acute promyelocytic leukemia (APL) and is approved by the FDA for the treatment of patients suffering from this subtype of AML. Despite the extensive clinical use of arsenic, the precise mechanisms by which it induces antileukemic responses are not known. Our studies have provided evidence that arsenic induces autophagic leukemic cell death and have suggested a potential mechanism by which it may target and eliminate leukemic stem cells (LIC), involving autophagic degradation of transforming oncoproteins. The work of this proposal will precisely determine the role of autophagy in arsenic- dependent LIC targeting using in vitro and in vivo models. It will also determine whether combinations of arsenic with other autophagy inducers provide an effective mode to eliminate primitive leukemia precursors and LICs.

DESCRIPTION (provided by applicant): This is a competing renewal application whose overall objective is to define the mechanisms of interferon (IFN)-signaling in malignant cells. IFNs exhibit important antineoplastic properties in vitro and in vivo and are key elements in the immune surveillance against cancer, but the mechanisms by which they generate such effects remain to be defined. We have provided the first evidence that the mTOR signaling cascade is engaged by IFN receptors (IFNRs) and regulates cap-dependent mRNA translation via control of the eukaryotic initiation factor 4E (eIF4E) and the eIF4F complex. Remarkably, our studies have provided evidence for signaling specificity and differential use of mTORC2 complexes by IFNs, as compared to oncogenic signals. Our studies suggest dual regulatory roles for mTORC2 complexes in IFN-signaling, controlling downstream pathways that regulate both transcription and mRNA translation of ISGs. The current proposal is a systematic approach to dissect the functions and roles of these complexes in IFN-signaling and to define their relevance in the generation of the antineoplastic effects of IFNs. Specific aim 1 will identify upstream IFNR-generated signals that lead to mTORC2 activation and will determine the mechanisms of specificity of mTORC2 engagement by the Type I IFNR. It includes studies to dissect the role of IFNR-associated kinases in the process; studies to define whether differential engagement of distinct Sin1 isoforms accounts for specificity in the IFN-system; and screening efforts to identif novel Rictor- and Sin1-interacting IFN-signaling elements. Specif ic aim 2 will define the mechanisms by which mTORC2 complexes control ISG expression and their roles in the generation of IFN-inhibitory responses in malignant cells. It includes experiments to define the roles of mTORC2 complexes in IFNR-activated signaling cascades that regulate transcriptional activation and mRNA translation of ISGs; experiments on the effects of IFN¿-activated mTORC2 complexes on AGC kinases; and studies to dissect the requirement of distinct downstream effector elements of mTOR pathways in the generation of IFN-antiproliferative responses. Specif ic aim 3 will examine the roles of mTOR-dependent signals in the antineoplastic effects of IFNs in Ph (-) myeloproliferative neoplasms (MPNs). JAK2V617F mouse models will be established in AKT -/-, S6K -/- and Pdcd4 -/- KO mice, and the ability of IFN¿ to induce antileukemic responses in vivo, in the presence or absence of distinct effectors of the pathway, will be assessed. The activation of IFN-dependent mTOR pathways in primary hematopoietic precursors from patients with MPNs in vitro and in vivo will be assessed and correlated with IFN-sensitivity. Altogether, these studies will advance our understanding of the signaling mechanisms controlling generation of IFN-antitumor responses and will provide important information on the events that lead to malignant cell resistance to IFNs. Ultimately, they may form the basis for new approaches to overcome IFN-resistance.

ABSTRACT ? OVERALL The Robert H. Lurie Comprehensive Cancer Center of Northwestern University is an NCI-designated, comprehensive matrix cancer center conducting a broad range of multidisciplinary clinical, basic, translational and population science research. The Lurie Cancer Center integrates the expertise and resources of the Feinberg School of Medicine (Chicago Campus) and its affiliated hospitals along with those of departments located on the University's Evanston Campus. The Lurie Cancer Center brings together 271 members from 31 Departments in 4 Schools. Established in 1974, the Lurie Cancer Center has been dedicated to the process of discovery, advancing medical knowledge and providing compassionate, patient-centered state-of-the-art cancer care, and conducting community outreach and survivorship programs. This is reflected in the following goals: 1. To conduct and support innovative basic, translational, clinical, and cancer prevention and control focused cancer research throughout the University. 2. To coordinate and integrate cancer-related activities, including community outreach initiatives. 3. To develop and conduct cancer education programs 4. To promote state-of-the-art cancer care at NU affiliated hospitals and drive precision cancer medicine initiatives. These goals are accomplished through the activities of 8 established programs and 14 shared resources: Over the current CCSG funding period there has been renewed institutional support and investment, new leadership, and significant growth in all divisions and programs of the LCC. Over the last funding period 88 new faculty have been recruited, including 30 clinical investigators and 58 laboratory or population scientists. There has been strengthened clinical and translational leadership and infrastructure and enhanced cancer focus. By leveraging the new Institutional commitment and support and significant philanthropic resources, the LCC will continue to develop innovative approaches to reduce the cancer burden in our catchment area and nationwide.

PROJECT SUMMARY (See instructions): The Senior Leadership is responsible for the overall planning and operations ofthe programs and activities ofthe Robert H. Lurie Comprehensive Cancer Center. The Senior Leadership is comprised ofthe Director, Deputy Director and nine Associate Directors, and has undergone a number of changes during the current project period. Steven Rosen, MD has continued as Director since 1989, and Leonidas Platanias, MD, PhD has continued as Deputy Director since his recruitment to Northwestern in 2002. Among the Associate Directors, also continuing in their positions are (1) Jonathan Licht, MD, for Clinical Sciences Research, (2) Mr. Timothy Volpe for Administration, (3) Al B. Benson, 111, MD for Clinical Investigations, and (4) Julian Schink, MD for Clinical Affairs. Changes among the Associate Directors include (1) Thomas O'Halloran, PhD, who assumed the role of Associate Director for Basic Sciences Research in September, 2007, (2) David Cella, PhD, who assumed the role of Associate Director for Cancer Prevention and Control Research in April, 2009, (3) John Crispino, PhD, who assumed the role of Associate Director for Education and Training in August, 2008. In addition, Jill Pelling, PhD, who served as Associate Director for Translational Research since 2004, retired on August 31, 2012, and a search is currently underway for her replacement. Finally, since the time of the last review, a new Senior Leadership position of Associate Director for Equity and Minority Health has been established to further strengthen the Lurie Cancer Center's long-standing commitment to outreach and service to minority and medically under-served populations. CCSG support is requested for all Senior Leadership positions except those of Associate Director for Clinical Investigations and Associate Director for Clinical Affairs.

Overview and Response to Previous Review The Robert H. Lurie Comprehensive Cancer Center of Northwestern University (Lurie Cancer Center) is dedicated to the conduct of innovative, translational clinical research. Novel investigator-initiated clinical trials (IITs) are a key element ofthe Lurie Cancer Center clinical research program and are of highest priority. In this competitive granting environment, funding for such studies is often either unavailable or only partially available. While industry sponsors are an important source of funds for such trials, the start-up process with our industry partners can be lengthy. The work of the Operational Efficiency Working Group (OEWG) has made it clear that rapid activation and enrollment to trials is vital. As such, the Lurie Cancer Center is committed to facilitating local, IITs through the CCSG mechanism of Protocol Specific Research Support (PSRS). PSRS funding is competitive in nature and supports protocol coordination and data management for early phase research trials. The goal of such support is to allow for the rapid development and completion of novel, translational hypothesis-driven trials that show promise for advancement to later phase clinical research deserving of independent funding. IITs are those institutional trials that are conceived of and written by Lurie Cancer Center investigators, and fall under the purview ofthe Lurie Cancer Center for ensuring appropriate conduct ofthe trial. Since the last competing application, the Lurie Cancer Center has provided support to 21 trials developed by Lurie Cancer Center members. The funding request for PSRS in this competing application consists of one Study Coordinator and one Data Manager.

The Robert H. Lurie Cancer Center (Lurie Cancer Center) has developed a comprehensive system of protocol review and oversight called the Clinical Protocol Scientific Review and Monitoring System (CPSRMS). This system is comprised of three distinct committees that work collaboratively to provide oversight of all aspects of clinical research conducted at the Lurie Cancer Center. These committees include: the Scientific Review Committee (SRC), the Data Monitoring Committee (DMC), and the Clinical Trial Audit Committee (CTAC). Each committee includes a Chair and/or Co-chair, but the CPSRMS overall is directed by the Associate Director for Clinical Investigations ofthe Lurie Cancer Center, Dr. Al B. Benson. This position is appointed by and reports to Dr. Steven Rosen, the Director ofthe Lurie Cancer Center. The three committees work in conjunction to form a robust system designed to ensure that research conducted at our center has scientific merit and meets the goals and priorities of the center, that clinical trial progress is maintained, and that patient safety and data integrity are maintained for our Northwestern University investigator-initiated trials (NU IITs). The CPSRMS committee responsible for Protocol Review and Monitoring (PRMS) is the SRC. The SRC is charged with the responsibility of evaluating all new and ongoing clinical research protocols for scientific merit, institutional priority and ongoing progress. The committee is a multidisciplinary committee that meets bimonthly to evaluate protocols. All cancer-relevant research studies conducted within the Lurie Cancer Center fall under the purview of this committee and require a level of review. Any faculty member within NU and all Lurie Cancer Center members are required to submit cancer-relevant clinical research protocols to the SRC for review. Protocols are classified into four types: institutional, external peer-reviewed, national/cooperative group, and industrial trials. The different types of protocols undergo different levels of review by the SRC. Any study that is reviewed and approved by an NCI approved peer-review committee (national trials and external peer-reviewed) is exempt from initial and ongoing SRC review for scientific merit. These studies are reviewed administratively for prioritization, and are reviewed annually for progress. All other studies (institutional and industrial) require initial and ongoing SRC review for scientific merit, and also are reviewed for prioritization and progress. Importantly, the NU Institutional Review Board (IRB) requires Lurie Cancer Center endorsement for all cancer-relevant protocols prior to IRB review, therefore ensuring the SRC review requirements have been appropriately met. The administrative support for SRC is coordinated through the Clinical Research Office (CRO).

The Robert H. Lurie Comprehensive Cancer Center of Northwestern University is an NCI-designated, university-based, matrix cancer center conducting a broad range of multidisciplinary clinical, laboratory and population science research. The Lurie Cancer Center integrates the expertise and resources of the Feinberg School of Medicine (Chicago Campus) and its affiliated hospitals along with those of departments located on the University's Evanston Campus. Established in 1974, the Lurie Cancer Center functions as a full organizational unit with the status of a department of the Feinberg School of Medicine. Since its inception, the Lurie Cancer Center has been dedicated to the process of discovery, advancing medical knowledge and providing compassionate, state-of-the-art cancer care, and conducting community outreach and survivorship programs as reflected in the following goals: 1. To conduct and support cancer research and to integrate cancer-related research throughout the University 2. To coordinate and integrate cancer-related activities of the University including community outreach initiatives 3. To develop and conduct cancer education programs 4. To promote and participate in state-of-the-art care of cancer patients at the affiliated hospitals, and 5. To develop and implement initiatives in cancer prevention and control research

PROJECT SUMMARY (See instructions): The Lurie Cancer Center has a well-developed system for planning and evaluation, the foundation of which is a formal strategic plan. The four major governance structures ofthe Lurie Cancer Center provide the primary means by which the Director and Senior Leadership conduct planning and evaluation on an ongoing basis, consistent with the strategic plan. The governance structures include the Executive Committee, the Center's primary policy and decision-making group, which coordinates overall planning efforts. Providing input to the Executive Committee are the Leadership Group, and the Internal and External Advisory Boards. The Leadership Group includes the Executive Committee and all Program and Shared Resource leaders/directors and meets quarterly to review and provide progress and input for research programs, shared resources and training/education. The Internal Advisory Board includes the Center Director, Deputy Director and Administrator; the Dean and other senior leaders of the Feinberg School of IVIedicine, Northwestern Memorial Hospital, Northwestern Medical Faculty Foundation, and Northwestern University; and key Department Chairs and Center/Institute Directors; and meets semi-annually to provide institution wide perspective and guidance to the Lurie Cancer Center Director. The External Advisory Board is comprised of recognized leaders in clinical, basic and population sciences, biostatistics and center administration as well as a patient advocate representative, and meets annually to review all aspects of Center operations. Additional planning input is provided by retreats, other Center advisory committees, surveys and ad hoc reviews. CCSG support is requested only for the activities of the External Advisory Board which plays an essential role in the evaluation ofthe Lurie Cancer Center's programs, activities, structure and organization.

PROJECT SUMMARY (See instructions): The Pathology Core Facility (PCF) is a centralized, comprehensive facility that provides professional expertise in pathology and human tissue-based studies and is specifically dedicated to the needs ofthe Cancer Center research community. The PCF has three main components: research histology, specimen procurement and protocol review. The research histology is composed of three units: routine histology, immunohistochemistry and molecular testing. This includes processing of fixed and frozen tissues, construction of hypothesis driven tissue microarrays, newly improved methods for TMA mapping and creation, sophisticated processes for the extraction of analytes such as RNA and DNA from samples, matched to end-point assay techniques. The research histology section also designs and validates the analytical assays, which include antibody selection and optimization for immunohistochemistry, a newly incorporated in situ hybridization for RNA and DNA with chromogenic and fluorescent detection. Adjoining to the research histology is microscopy, which provides pathologic verification of diagnoses, and quantitative interpretation of results, based on microscopic evaluation, including a new high-throughput digital slide scanner. An annotation and scribing software (Collibio¿) allows for distant review, retention of data with image, and conferencing. The biobanking component ofthe PCF has two main functions: human tissue procurement, and quality assurance and protection of research subjects. The tissue procurement section serves to obtain, process, store and distribute characterized neoplastic and benign human tissue. Specimens are not only procured for specific protocols and laboratory projects, but also there is an organized systematic effort for generalized procurement and storage for short and long-term cancer research. PCF maintains a universal IRB consent for procurement of all specimen types. As a member of ISBER and NCI Group Banking Committee, the PCF uses new information, ideas, technology, and regulations in further developing a non-renewable high quality biospecimens resource to meet all aspects of cancer research. Lastly, the PCF reviews all clinical protocols which involve human tissue specimens. The PCF is CAP-accredited, CLIA high-complexity certified, and has the capability to serve integral marker studies, which require biomarker-based treatment arm assignment. The PCF staff are members of institutional review committees, and in this manner provide input and oversight ofthe clinical trial protocol design, and the specifics of tissue release of retrospective specimens. These activities allow for incorporation of pathology-specific competencies into the design and conduct of translational studies, and integrate the PCF into multidisciplinary teams that serves the entire Cancer Center research community. In addition, the PCF also runs the NCI funded ECOG-ACRIN Pathology Coordinating Office - Reference Laboratory, which serves as the centralized center for processing and distribution of biospecimens obtained from patients enrolled in ECOG and intergroup clinical trials.

PROJECT SUMMARY (See instructions): The Structural Biology Facility provides: a) resources required for crystallographic structure determination, refinement and analysis, b) molecular graphics and computational support for structural biology, c) molecular graphics and computational support for structure-based drug discovery and d) highly specialized resources for macromolecular characterization related to structural biology. The Facility is essential for the research programs of investigators ofthe Cancer Center who are studying the relationship between macromolecular structure and function, or who are using macromolecular structure as the starting point for structure-based drug design. It is a unique resource at Northwestern University that capitalizes on the extensive expertise of a large group of users and the unique access to the synchrotron radiation X-ray source at Argonne National Laboratories. It also serves to nucleate the development of a local community with expertise in structural and computational biology. Such expertise will increasingly be called upon as the structures of more cancer related proteins become available. The Structural Biology Facility is located on both campuses of Northwestern University. It is based in the Department of Molecular Biosciences on the Evanston campus and in the Department of Molecular Pharmacology and Biological Chemistry in the Feinberg School of Medicine on the Chicago campus, and also at the Life Sciences Collaborative Access Team (LS-CAT) beam lines at Sector 21 of the Advanced Photon Source (APS). Dr. Alfonso Mondragon, a structural biologist at the Evanston Campus, directs the Facility. The Facility consists of three major components: 1) an outstation at the APS that is devoted to state of- the-art macromolecular crystallography, and, at both campuses, 2) automated facilities for setting up and visualizing crystallization experiments, and 3) computational facilities to support structural determination calculations, including NMR, crystallography and electron microscopy, computational drug-design, simulations and modeling efforts, including advanced graphical visualization and manipulation of models. The distributed nature ofthe facility reflects the means by which the data collection, computational, molecular visualization, and other scientific resources are networked, and thus integrated, for the structural biology research community at Northwestern. The Facility is continuously adapting to a changing environment During the last few years, it has modernized its entire computer infrastructure and increased significantly the number of crystallization robots and other modern tools for crystallography. The Facility plans to continue to grow and expand by adding more and better instruments, continue the upgrade/modernization ofthe existing equipment, and to incorporate new groups to its expanding user base.

PROJECT SUMMARY (See instructions): The Outcomes Measurement and Survey Core at the Robert H. Lurie Comprehensive Cancer Center is staffed with expertise on survey research design, outcomes measurement (psychometrics) and research operations support activities for cancer projects involving survey data. The Core Facility consists of four faculty social scientists and three research associates, with additional experienced and diverse faculty and staff available to support the activities of the Core. Core Facility faculty are members of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University and have appointments in academic departments in the Feinberg School of Medicine. The specific aims of the Core are: 1. to provide consultative and analytic expertise on self-reported outcome measurement, including special attention to literacy and cultural diversity upon data quality and interpretability; 2. to serve as a central resource for state-of-the-science instruments and measurement methods; and 3. to provide in-house research support services for the translation, collection and psychometric analysis of outcomes and survey data. During the current usage reporting period 08/01/11 - 07/31/12, the Core worked on 70 projects for 46 different Cancer Center members. The Core usage extends across seven different programs, and Core members interact with several other relevant Cores, particularly the Biostatistics Core Facility. In the past 5 years the Outcomes Measurement and Survey Core usage has grown by over 350% compared to the previous 5 years. Core members participated in numerous primary and subcontract federally-funded grant applications and co-authored 88 papers collectively. Over the next five years, the Core will continue to provide the Cancer Center with psychometric and survey research support in the areas of study design, data collection, and analysis and interpretation. State-of-the-science psychometric methods and novel multimedia tools will continue to be used. Collaboration with the Biostatistics, Bioinformatics and Clinical Research Office cores is expected to be strengthened due to shared collaborative interests and support of funded projects. The Outcomes Measurement and Survey Core Facility will continue to respond to user requests for participation in grant applications, expand faculty and staff effort as needed to respond to the changing needs of funded grants, strive to maintain a revenue profile where funded grants represent the majority revenue sources relative to CCSG and institutional funds, and strive to maintain the professional level of faculty and staff through their attendance at and participation in outcomes measurement, survey research, and statistical/psychometrics meetings and conferences.

PROJECT SUMMARY (See instructions) The Mouse Histology and Phenotyping Laboratory (MHPL) provides the Northwestern University research community with histopathology assessment of murine tissues by trained pathologists and comprehensive histology services performed by expert histotechnologists. In addition, training is provided for investigators to learn to detect gross anomalies in rodents, to harvest tissue from various organ systems and to perform immunohistochemical and special histological stains on tissue sections generated by the MHPL. The MHPL was developed to fulfill a need by the investigative community to assist with the analysis of new murine models, to leverage the expertise of pathologists in the histopathological assessment of tissue anomalies and neoplasms, and to increase the likelihood of extracting meaningful phenotypic information to guide future investigations. Murine tissue has histological characteristics distinct from human tissue and therefore accurate interpretation requires microscopic examination by.pathologists with an understanding of disease pathobiology, rodent histopathology and murine development. Moreover, tissue isolation, processing, and sectioning often require the involvement of highly skilled histotechnologists, especially when embryonic lethal phenotypes or complex developmental abnormalities are to be studied. The MHPL is directed by an anatomic pathologist and neuropathologist with more than twenty years of experience in diagnosing human tumors and developmental abnormalities. He has equally extensive training in rodent adult and developmental pathobiology and histopathology. Other members ofthe MHPL staff include a second fulltime associate pathologist, three full-time ASCP certified histotechnologists, two part-time histotechnologists and two part-time technical/administrative assistants. Together, this team provides a broad range of technical and diagnostic services to investigators throughout the Northwestern University research enterprise in a high-volume and fast-paced environment. These comprehensive histopathology support services enhance the ability of our Cancer Center investigators to characterize viable and embryonic lethal mouse models and to develop and analyze new in vivo model systems to study cancer biology.

PROJECT SUMMARY (See instructions): The Molecular and Translational Imaging Core (MTIC) fills a critical need for the Robert H. Lurie Comprehensive Cancer Center by providing a focus for all cancer imaging research and its clinical application in uniquely well-equipped and designed facilities that straddle the clinical and basic cancer research communities at Northwestern University. The core offers investigators access to a large scope of known imaging modalities, including 1.5, 3, 7 and 9 T MRIs, IVIS Spectrum for in vivo bioluminescence and fluorescence imaging, optical imaging, and X-ray digital subtraction angiography, as well as new imaging technologies developed within the core. The core features one ofthe most advanced 3-D visualization displays in the world along with the expertise required to manipulate and interpret image data. These unique capabilities are central to the advancement of efforts to understand and integrate cellular architecture, flow of information, regulation, and communication across length scales and their impact on tumorigenesis, metastasis, and response to treatment. The Imaging Core is closely intercalated with the Developmental Therapeutics Core and ChemCore, thereby providing cancer researchers with an efficient pipeline for developing, testing, and imaging new diagnostics and therapeutics. The Molecular and Translational Imaging Core will continue to catalyze the development of innovative new imaging technologies for the broad-scale interrogation of disease processes, guidance of therapeutic interventions, and the study human physiology and function. It will continue to provide unique educational opportunities thus fostering efficient, effective utilization of these valuable resources while training the next generation of clinicians, biomedical scientists and engineers. These services are integral to major NCI sponsored research efforts at Northwestern including the Center for Cancer Nanotechnology Excellence, a Cancer Nano Platform Program, and the Physical Sciences-Oncology Center

PROJECT SUMMARY (See instructions): The Northwestern University Transgenic and Targeted Mutagenesis Laboratory (TTML) is a shared resource designed to produce genetically engineered mice for research projects of investigators of the Robert H Lurie Comprehensive Cancer Center (RHLCCC), Feinberg School of Medicine (FSM), Evanston campus, and Lurie Children's Hospital of Chicago Research Center. The transgenic facility was initially founded at Northwestern University in 1989, as part of the Markey Program in Developmental Biology, to provide a resource for generating transgenic mice. Today, the facility has evolved into a well utilized laboratory that provides a broad range of services to NU investigators, including generation of transgenic mice, gene targeting of embryonic stem (ES) cells, generation of chimeric mice via ES cell microinjection into blastocysts, cryopreservation and recovery of mouse embryos/sperm, and rederivation of pathogen free mouse strains. In 2003, the facility was reorganized and expanded to include gene targeting services. Embryo cryopreservation and recovery, rederivation of mouse lines, and in vitro fertilization services were added in 2005. The embryo, and now sperm, cryopreservation and recovery services position NU and Cancer Center investigators to capitalize on emerging repositories of mutant mice generated around the world, most of which are stored as cryopreserved embryos. Innovative genome editing technologies have been introduced over the last 18 months. These technologies implore zinc finger nuclease (ZFN) and recombinase mediated cassette exchange (RMCE) mechanisms to create gene-specific modifications directly in the zygote via pronuclear microinjection. Cancer Center investigators save both monetary resources and time as these technologies eliminate the need for ES cell based gene targeting. The TTML provides the necessary infrastructure that allows most investigators access to transgenic technology that normally requires expensive microinjection equipment and highly skilled staff with expertise in microinjection, microsurgeries, embryo manipulation, animal husbandry, and ES cell culture. TTML staff provides consultation and guidance regarding transgenic and targeting vector design; appropriate screening strategies; DNA purification methods; breeding and analysis of transgenic founder and chimeric mice; and transgenic-related technologies. The oversight committee that governs the TTML meets at least twice a year and is comprised of a dynamic group of faculty with a wide range of transgenic-related expertise. They critically review facility data/progress and provide astute advice. Since the inclusion of the TTML as a resource within the Cancer Center in 1995, Cancer Center investigators have consistently been the primary group of NU investigators utilizing the TTML, emphasizing its pivotal role in the overall research mission of the RHLCCC.

The Robert H. Lurie Comprehensive Cancer Center Administration (RHLCCC) performs a vital role in assuring the Center achieves its goals and objectives through efficient and effective operations. The specific areas of responsibilities of the Administration are to: ? Provide administrative support to Director, Deputy Director Senior Leaders, Program Leaders, Shared/Resources Directors and Managers ? Manage all Center finances including grants, contracts, institutional and philanthropic resources ? Provide Human Resource administration for the Center ? Manage the Center's shared resources including oversight, usage patterns, and recharge rates ? Manage the Center's space, facilities and equipment ? Oversee information systems development and maintenance ? Manage public affairs, education, and communication programs ? Support the governance, planning and evaluation functions of the Center ? Support the membership application and review process ? Manage the Center's administrative support staff Overall, the Administration is organized to effectively respond to the needs of the leadership and the 267 full Members now participating in the research programs of the RHLCCC. With the rapid growth that has occurred, major resources have been committed both in staffing and information technology to assure that administrative operations are efficient and fully integrated with the central systems of Northwestern University. The Associate Director for Administration is supported in the conduct of these activities by a staff of 29.25 FTEs and has oversight of an annual operating budget of $42.0 million.

PROJECT SUMMARY (See instructions): The Biostatistics Core Facility is a defined group of faculty biostatisticians, staff statistical analysts and computer/administrative personnel whose mission is to provide state of the art biostatistical collaboration and support to Cancer Center members. The specific aims of the Core are to provide expertise in study design, data analysis and database management, interact with relevant Cancer Center shared resources, provide statistical input to investigator initiated protocols and statistical review of all new Cancer Center studies through the Protocol Review and Monitoring System, perform research in statistical methodology and implement innovative statistical techniques as they apply to Cancer Center members' projects, provide education in biostatistical methods to Cancer Center members and other persons performing cancer related research, and disseminate the teaching process to national and international entities beyond the Cancer Center. Over the past five years, the Core has achieved its primary goals by providing statistical collaboration and consultation to members of all Programs in the Cancer Center, with collaboration from other Cores. Two Core biostatisticians (one as co-chair) attend each of the bi-weekly Scientific Review Committee meetings. All protocols, chart reviews, letters of intent and amendments are reviewed by a biostatistician. The Core has been instrumental in recruiting two junior faculty statisticians with strength in statistical methodology development and application. The Core collaborated on the currently NCI funded Prostate SPORE, the Phase I and 11 Chemoprevention NCI Contract, the Physical Sciences Oncology Center and the Center of Cancer Nanotechnology Excellence. The Core participated in various Cancer Center education endeavors and has national and international presence in bioinformatics and clinical trials training. Core members co-authored 135 peer-reviewed publications over 5 years, worked on 182 projects for 106 unique users over the past year and co-directed (with other Center Cores) a seminar program of 49 seminars over 5 years. Key scientific contributions include: identification of two new physical protein interaction mechanisms in glioblastomas using network data analysis techniques, investigation of aberrant signaling in receptor tyrosine kinase (RTK) pathways by identifying genes with differential transcription dynamics in time course data using the Partition Decoupling Method, and use of concordance statistics to determine that MRI with enhanced reconstruction is superior to standard MRl as a measure of tumor necrotic fraction and viable tumor volume in animal studies of hepatocellular carcinoma. The Core aims to continue its collaboration with all Center programs and relevant shared resources, and broaden its development of statistical expertise through further education and recruitment.

The Cancer Informatics Core is comprised of informatics faculty and staff who are focused on providing informatics services and necessary computational infrastructure for the diverse informatics needs of Cancer Center members in the Robert H. Lurie Comprehensive Cancer Center. The Core works closely with RHLCCC governance committees to promulgate standards, provide advice and guidance, optimize systems and minimize redundancy through continued integration of data, databases, applications, software and computational infrastructure that is necessary to support cancer translational research. Since the last competitive renewal, the Core has established a scalable high performance cyber-infrastructure equipped with >200 TB of tiered storage and a virtualized data center to meet the data and computational needs of Cancer Center members. The Core also provides access and training for Cancer Center members on the 7000 core Northwestern Quest cluster for projects requiring high performance computing. During the past five years, the Core has met its primary goals of providing the necessary computational infrastructure for managing clinical trials with the Clinical Research Office, storage for microarray and next generation sequencing. The core has provided the necessary oversight, project management, and software development expertise to deliver data management and reporting applications for prostate cancer and breast cancer repositories. The core has also worked closely with the RHLCCC neuro-oncology investigators to deliver innovative patient-facing intake and assessment applications that are coupled to clinical data available through the Enterprise Data Warehouse with molecular data coming from biospecimens, including gene expression, copy number, and methylation data. We have also provided sophisticated gene expression analysis, pathway enrichment analysis, and methylation data analysis including visualization methods for more than 70 cancer center members and 160 projects during the past five years. In addition to providing these genomic analysis services to our cancer center members, we have released the tools developed for these projects as open source bioconductor packages [lumi, GeneAnswers, ChlPpeakAnno, MassSpecWavelet). The core has also developed, in conjunction with the Northwestern University Biomedical Informatics Center (part ofthe Northwestern CTSA) a number of web-based clinical research software modules that have been released as open source tools (Patient Study Calendar, Registar, eNOTIS, Surveyor). In addition, the core has developed and released tools for scientific network analysis (LatticeGrid) and competition management (NUCATS Assist). The Core will continue to support and extend these activities. We anticipate that during the next five year there will be additional member-driven demand in the area of next generation sequencing, high performance computing, and FISMA compliant computing.

PROJECT SUMMARY (See instructions): State-of-the-art imaging technologies are powerful tools that can be leveraged to study various cellular and molecular mechanisms in cancer progression and how tumor cells interact with the tumor microenvironment. The Cell Imaging Facility provides imaging modalities that cover the range from single molecule microscopy to whole animals, and from brightfield, to fluorescence and bioluminescence imaging. The Cell Imaging Facility offers bioluminescene/fluorescence in vivo imagers, microprobe lens-driven intravital microscope, multiphoton microscope, various fluorescence microscopes including macroview stereoscope, high content slide scanner and tissue cytometer, spinning disk confocal microscope, multispectral confocals with resonant scanner, integrated incubator microscope, structured illumination super-resolution microscope, laser microdissection system, and transmission electron microscope with 3D tomography capability. Collectively, this vast instrument portfolio offers core services in advanced microscopy on single molecules, macromolecular networks, live cells, FRET, FRAP, FLIP, photoconversion, TIRF, large tissue sections in bright field and fluorescence, specific tissue procurement, intravital imaging, and whole animal imaging. This creates a unique environment in which investigators can design workflow-based correlative experiments to study their cancer specimens all the way from whole animals to single molecules. This wide-ranging microscopy instrumentation and services is fully supported by the technical expertise of a team that has been consecutively recognized by the Northwestern University Office for Research as Outstanding Core for multiple years. The team is led by the facility director. Dr. Teng-Leong Chew. Dr. Chew is an expert in advanced live cell imaging, and has pioneered the development of several biosensors and imaging applications. Together the team provides active training and educational outreach in personalized instrument training, experimental consultation, seminars, journal clubs, hands-on workshops, as well as image analysis and programming boot camps. This 24-hr facility serves more than 180 labs throughout the university, and we maintain our leadership position in novel imaging technologies through a two-pronged approach: (i) aggressive and successful effort in securing shared instrument funds. We have added ten instruments over $300K since the last CCSG cycle; (ii) leveraging our expertise to attract leading manufacturers to establish beta test sites in the imaging center. These efforts have culminated in the facility being recognized by Nikon as one of only eight Nikon Imaging Centers in the world, allowing the cancer center to benefit immensely from the investment of Nikon's latest technologies. In 2012, we were selected by EuroBioimaging Consortium as the first American imaging center to join this trans-Atlantic multinational imaging network. These achievements place the Cancer Center uniquely at the forefront of cancer imaging technologies.

PROJECT SUMMARY (See instructions): The Clinical Research Office (CRO) o f t h e Robert H. Lurie Comprehensive Cancer Center (Lurie Cancer Center) of Northwestern University (NU) provides a centralized resource to facilitate the development, conduct and oversight of cancer-relevant clinical trials conducted at the Lurie Cancer Center. This includes assistance with protocol development, scientific review of protocols. Institutional Review Board submissions (initial and ongoing), clinical research coordination, data collection and management, quality assurance monitoring, coordinating procurement of biologic specimens for clinical/ laboratory correlates, and coordinating supervision of appropriate trials by statisticians and the Data Monitoring Committee. In the current reporting period (8/1/11-7/31/12) the CRO has provided primary clinical coordination, data management and/or regulatory support for 53 investigators, with 51 of these having external funding. The investigators came from 8 departments within Northwestern University's Feinberg School of Medicine; 45 of the investigators were Lurie Cancer Center members during the year. Since 2007, the number of studies open to accrual during each 1-year period ranged from 458 to 560 in the current reporting period; accrual ranged from 4424 to 6047 (5977 in the current reporting period). Of the 5977 subjects accrued in the current reporting period, 1848 were accrued to intervention studies, including 1456 subjects (79%) enrolled at the main institution and 392 (21%) at network institutions. Accrual was 149 subjects (8%) to national group trials, 1092 (59%) to externally peer reviewed trials, 439 (24%) to institutional trials, and 168 (9%) to industry trials. Considering non-intervention studies, 4129 subjects were accrued in total, with 98 subjects (2%) to national group trials, 1027 (25%) to externally peer reviewed trials, 3001 (73%) to institutional trials, and 3 to industry trials. During this grant cycle, six initiatives were undertaken: 1) continued development and enhancement of our clinical trial management system (Northwestern Oncology Trial Information System [NOTIS]), including NOTIS' electronic case report forms (eCRFs), designed for local investigator-initiated trials. Enhancements include expanded eCRFs and a Principal Investigator (PI) portal for PI review and sign-off of data; 2) roll-out of NOTIS to Lurie Cancer Center members sites and ECOG-ACRIN affiliate sites; 3) development of standard operating procedures for all CRO services; 4) restructuring of the CRO to include dedicated data management staff. The resulting coordinator/data management teams have been restructured to be disease focused; 5) enhanced focus on protocol development, with the creation of a Protocol Development Coordinator who assists investigators in protocol writing, and also in activation and implementation of new studies; and 6) most recently, the additional o f a Clinical Research Recruitment Coordinator, whose focus is recruitment in general, but with a top priority being the recruitment of under-represented populations.

PROJECT SUMMARY (See instructions): The Medicinal and Synthetic Chemistry Core (ChemCore) is a shared resource facility dedicated to providing chemistry services for advancing drug discovery and chemical biology research. The facility serves investigators ofthe Robert H. Lurie Comprehensive Cancer Center (RHLCCC), Northwestern University, and external organizations by providing a range of customized services in three primary areas: Cheminformatics, Medicinal and synthetic chemistry, and Compound purification. The cheminformatics service provides computational chemistry, molecular modeling, and computer-aided drug design. These services provide investigators with unique insights into fundamental processes of cancer biology and allow efficient access to powerful platforms to advance early stage drug discovery efforts prior to committing larger resources. The medicinal chemistry service provides researchers with access to high-level resources for the custom synthesis of molecular probe and tools compounds, hit-to-lead chemistry, lead optimization medicinal chemistry, and consulting on drug discovery projects. This service is staffed by professional medicinal chemists who help enable translational research based on the basic research of RHLCCC members. Finally, ChemCore also provides comprehensive instrumentation and expertise to purify and reformat small molecules. Purification services include a full-service custom dual-purpose analytical-to preparative mass-directed HPLC (A2Prep LCMS) and self-service analytical and preparative HPLCs, each equipped with UV and ELS detection. This equipment helps RHLCCC members carry out small molecule chemical biology and drug discovery research much more rapidly. ChemCore works closely with the affiliated RHLCCC High Throughput Analysis Laboratory (HTAL) and Developmental Therapeutics Core (DTC) facilities, which carry out high-throughput screening, in vivo testing, and pre-IND development. Together with these cores, ChemCore provides RHLCCC members and their collaborators with a pipeline to translate basic discoveries into the clinic.

The Robert H. Lurie Comprehensive Cancer Center (Lurie Cancer Center) has developed a robust system for data and safety monitoring, accomplished through the collaborative efforts of the three committees of the Clinical Protocol Scientific Review and Monitoring System (CPSRMS). These committees are: the Scientific Review Committee (SRC), the Data Monitoring Committee (DMC), and the Clinical Trial Audit Committee (CTAC). These committees are responsible for administering the Lurie Cancer Center's Data and Safety Monitoring Plan (DSMP), which was originally approved by the NCI in August of 2001, and has since undergone 10 revisions. The most recent revision to the plan was submitted to the NCI in May of 2012 and was accepted without comment at that time. In the previous competing CCSG application the assessment of Data and Safety Monitoring resulted in an Acceptable [Approved] status with no deficiencies noted. However, in section 9.2 PRMS, the previous CCSG competing submission included comments in the critique that relate directly to data and safety monitoring. First, it was suggested that the CTAC expand the auditing program to be more comprehensive. This has been done, and the audit strategy is described below. The second comment relates to DMC review for phase I studies and offered that a medical monitor may provide greater oversight for these trials if our phase I studies increase in number. A significant increase has not been seen, so this change has not been made, however both the SRC and DMC are mindful of and appreciate this recommendation. If our phase I program increases substantially, this change will be considered.

The Cancer Control and Survivorship Program ofthe Robert H. Lurie Comprehensive Cancer Center is a multi-disciplinary program focusing on three thematic areas: (a) Measure, Analyze and Interpret Quality of Life (QOL), (b) Understand and Improve Cancer Survivorship, and (c) Symptom Management and Supportive Care. The Program Leader is Frank J. Penedo, PhD, an accomplished clinical health psychologist whose research focuses on psychosocial and biobehavioral determinants of adjustment to cancer and the efficacy of manualized interventions on QOL and health outcomes with emphasis on ethnic minorities, and the Program Co-Leader is Jamie Von Roenn, MD, a distinguished medical oncologist with expertise in palliative oncology education and research. The objective ofthe Cancer Control and Survivorship Program, which is comprised of 38 faculty members from 13 departments and 2 schools is to facilitate and focus research on secondary cancer control and survivorship within the Cancer Center. Between August 2007 and July 2012, there were 545 cancer-relevant publications from the program members, with 26.6% representing intra-programmatic and 31.4% representing inter-programmatic collaborations. A total of 7,138 participants were enrolled in program studies with 2,802 enrolled on intervention studies and 4,336 enrolled on observational or correlative studies. Total cancer relevant funding for the Program is $14,818,344, with 70% coming from peer reviewed funding. Peer reviewed funding is $10,367,715 (total) and $7,612,441 (direct), with $1,819,740 (23.9%) from NCI and $5,792,702 from other peer reviewed sources. Areas of research include: measurement science, determinants of optimal survivorship including basic mechanisms, psychosocial interventions and educational programs, and symptom palliation. The contribution of program efforts in improving quality of life, state ofthe art assessment of patient reported outcomes and palliation of cancer-related physical and psychosocial symptom burden via psychosocial interventions, coupled with community based activities, support the significance of this work. The Program is highly innovative as it involves multiple projects implementing cutting edge technology. The Program actively seeks to improve cancer control and survivorship by developing and implementing methodologically sound, clinically relevant and highly innovative research initiatives that are clinically effective and translational, aimed to reduce the burden of cancer across multiple communities. Members' interests and capabilities span many disciplines. Their integrated focus upon cancer control and survivorship promotes an environment of intra- and inter-program collaboration and productivity.

PROJECT SUMMARY (See instructions): The Developmental Therapeutics Core (DTC) works with RHLCCC investigators to facilitate translation of new oncology therapeutic agents to the clinic. DTC provides a range of services that include in vitro tumor cell line assays such as the NCI60 panel, proliferation, apoptosis, cytotoxicity, migration and invasion assays. Animal studies run by DTC include a variety of subcutaneous and orthotopic models of tumor growth and metastasis that can be used to study mechanism and evaluate new drug candidates, exploratory pharmacokinetics (in collaboration with the RHLCCC funded Clinical Pharmacology Core), and exploratory toxicology including histopathological evaluation. In addition, DTC assist investigators with drug formulation and initial assessments of drug stability. DTC staff are proficient using all routes of administration including PO by gavage, IV, IP, and SC and in small animal surgery, which allows for implantation of osmotic minipumps for continuous infusion. To date, DTC has collaborated with a broad spectrum of different investigators including clinicians looking to evaluate new drugs or combinations before moving these into the clinic and basic science researchers such as chemists or individuals who lack tumor biology or animal model expertise. DTC has also collaborated with the Center for Molecular Innovation and Drug Discovery (CMIDD) to carry out initial in vitro evaluation for a number of early discovery projects. DTC also advises on study design and provides consulting and training to faculty and students. DTC has been able to accelerate implementation of studies by working with the lACUC to set up and manage a number of blanket proposals under which it can run most of these studies that it engages in simply by filing a one page study-specific addendum describing the cell line used, if applicable, and the drug to be studied. DTG routinely adds fellows and students to these protocols in order to facilitate hands-on training. DTC currently has one full time and one part time FTE in addition to the director but is in the process of hiring two additional research associates to meet the growing demand for its services. DTC currently occupies bench and office space in Silverman Hall West it carries out all of its animal work in the vivarium that is located in Pancoe Hall, which adjoins Silverman Hall. In addition, DTC carries out a number of studies in collaboration with the Molecular and Translational Imaging Core (MTIC). DTC routinely labels most of its cell lines using Luc2 and dTomato (bioluminescence and fluorescence, respectively), which allows real time monitoring of orthotopic tumor growth and metastasis using the IMTIC facilities for this purpose. Finally, in addition to increasing staff to meet the demand for existing models, DTC is collaborating with the pathology core facility (PCF) to develop a novel repository of patient-derived xenografts across a variety of histologies that will only be passaged in vivo.

PROJECT SUMMARY (See instructions): The Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core Facility provides comprehensive flow cytometry and cell sorting services. The Flow Cytometry Core Facility serves investigators of the Cancer Center, Northwestern University Medical School, Northwestern University and other affiliated institutions by providing access to routine flow cytometry assays such as immunophenotyping and DNA analysis. However, the major mission of the facility is to provide the guidance, technical assistance, and equipment for investigators to utilize more complex multi-parametric, multi-laser measurements as well as cell sorting in their research regardless of their level of cytometry expertise. The Core Facility has an international reputation and extensive experience in developing and implementing complex multi-parametric assays with particular expertise in intracellular liable antigen detection such as phosphorylated protein epitopes. This level of expertise along with the equipment housed within the Core Facility permits implementation of most flow cytometry assays that have been published. Services provided by the facility extend from consultation on experimental design, sample preparation and data analysis to instrument operation and set-up for cell sorting and multi-laser operation. In addition, the Core Facility has an active program of assay development guided by the interests/needs of the investigators. Reflective of the facility's commitment to flow cytometry education/ training of the next generation of researchers, user training for operation of not only less complex analysis instruments but also the more complex analyzer and sorting instruments is offered. Thus, the Flow Cytometry Core Facility serves as a focus for individuals interested in cellular based measurements and cellular heterogeneity in disease providing critical support for cancer related research within the institution. The Chicago campus facility includes three cell sorters; a 5 laser/15 parameter MoFlo high speed cell sorter (Beckman-Coulter. BC) along with two new Becton-Dickinson (BD) FACSAria 111 cell sorters, a 4 laser/13 parameter instrument and a 5 laser/17 parameter instrument housed in a Bio-containment hood and negative pressure room; and a 3 laser, 10 parameter FACSAria II (BD) on the Evanston campus. The Chicago campus facility also has three benchtop flow cytometry analyzers; a 6 laser/18 parameter FORTESSA (BD), a 3 laser/11 parameter Cyan (BC) and a single laser/six parameter XL (BC) with the Evanston facility having a 4 laser/13 parameter LSRII (BD). In addition, the Chicago facility has a ViCell cell counter/viability instrument (BC). The facility has multiple offline analysis workstations providing the components for post acquisition data analysis and graphics generation. The Core Facility is now heavily utilized by a wide number of investigators (148), approximately 62% of which are Cancer Center members who account for approximately 74% of core utilization.

The High Throughput Analysis (HTA) Laboratory was a developmental facility in the past CCSG renewal, in which it was referred to as the Diagnostics and Therapeutics Screening Core. It is located on the Evanston Campus. The HTA Laboratory's mission is to provide Lurie Cancer Center researchers with advanced technology and expertise for large scale experiments that elucidate the basic biology of cancer and discover novel therapeutic agents. Such experiments can involve parallel manipulation of thousands of samples and the gathering and analysis of data at a similar scale. They include cell-based screening of large compound libraries, massively parallel investigation of gene function using RNAi and gene disruption collections, and high throughput biochemical assays. The facility offers advanced technologies for automated liquid handling, strain collection manipulation, and data acquisition. It provides access to 17 major instruments. These include four advanced platforms for robotic liquid handling, two of which specialize in highly accurate nanoliter liquid dispensing. The facility's analytical equipment includes four photometric plate readers that provide facility users all major detection modes, including simultaneous whole-plate fluorescence measurement for parallel analysis of ion currents and other kinetic phenomena. Additionally, the facility offers access to a Cellomics Arrayscan high-content screening system. The HTA Laboratory integrates these platforms with laboratory infrastructure such as tissue culture, wet bench, and microbial growth chambers, allowing complex in-house work flows. Together these make a broad spectrum of molecular and cell-based assays possible. Importantly, In addition to training and access to instruments, the core offers extensive service for design, validation, and execution of diverse large-scale experiments, including compound library screening and genome-scale functional analysis. The facility also houses biological and chemical libraries for distribution or screening, including a collection of bacterial stocks of snRNAmir lentiviral vector constructs that target nearly all genes in the human and mouse genomes. This capability includes a recently installed tube-based chemical library storage system The HTA Laboratory currently has 251 active users from 76 research groups. Over the last funding period, 79 to 95% of instrument was by Cancer Center member groups. Thus, in its developmental period the facility has become an important established resource for the cancer research community at Northwestern University.

PROJECT SUMMARY (See instructions): The Keck Biophysics Facility is a center for molecular biophysical research which provides Northwestern groups with advanced equipment outstanding services, specialized training, and technical expertise. Created in 1998 by Northwestern University's Center for Structural Biology with a grant from the W.M. Keck Foundation and additional support from the NIH, the Rice Foundation, and the Robert H. Lurie Comprehensive Cancer Center (RHLCCC), Keck has been one of Northwestern's Shared Resources since April 1,1999. The Keck Facility has a set of 20 advanced instruments that together allow for integrated biophysical analyses of macromolecular structure, interactions, and function. Each particular biophysical instrument, such as a circular dichroism spectrometer, a fluorimeter, or a calorimeter, provides just one or a few individual facets of a complex overall picture. Data from many individual biophysical studies - using a diverse set of particular instruments - must be combined to yield the needed comprehensive picture. In the past 5 years, the facility has been thoroughly modernized with state of the art equipment that replaced older instruments, addition of new technologies and capabilities, a modern internet-based reservation/administration system and many upgrades. In parallel, the Facility has invested in personnel training in order to provide users with outstanding technical expertise and assistance. To better accommodate the needs of our diverse user base, we are offering a flexible array of services. Besides counseling, training and assistance, full service options are also available on all the Facility's instruments. The Keck Facility currently has 506 active users from 90 research groups (students, postdoctoral fellows, and faculty who are authorized to use Keck Facility equipment). Researchers affiliated with the RHLCCC account for over 80% of the total utilization ofthe Facility. As such, the Keck Biophysics Facility is a critical resource for cancer research.

? DESCRIPTION (provided by applicant): The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in diverse physiological and pathological processes such as HIV infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. Although the CXCR4 receptor and its unique ligand SDF-1 have been widely studied, all small molecule modulators of the SDF-1/CXCR4 axis have been antagonists. The lack of available small molecule agonists constitutes a substantial gap in the ability to probe the biology of CXCR4. Using new in silico screening strategies, we have recently discovered the first series of small molecule CXCR4 agonists and have demonstrated their unique behavior in a variety of biological settings. Notably, our small molecules cause internalization of the CXCR4 receptor, a strong chemotactic response, and chemosensitization of tumor cell lines. Development of these small molecule agonists and structurally related antagonists will provide a unique and powerful means to study the function of the CXCR4 receptor and how this relates to disease processes. Acute myeloid leukemia (AML) is a group of myeloid leukemias with a very aggressive and fatal course if left untreated. The bone marrow (BM) microenvironment provides an important protective effect against chemotherapy and disruption of this interaction renders AML cells sensitive to chemotherapy in vitro and in vivo. The SDF-1/CXCR4 axis plays a key role in regulating stem cell mobilization and trafficking and its expression has been shown to negatively correlate to the prognosis of many cancers. Our lead agonist significantly enhances chemosensitivity of multiple leukemic cell lines to several chemotherapies, suggesting that CXCR4 agonists may provide a novel therapeutic approach for the treatment of AML. The overall goal of this project is to optimize small molecule CXCR4 agonist probes and characterize their activity against the CXCR4 receptor and AML in vitro and in vivo. Our unique small molecule CXCR4 agonists and antagonists give us a set of unique molecular tools to understand how CXCR4 receptor pharmacology impacts AML. In Aim 1 we will use rational medicinal chemistry to optimize our lead series for potency and drug-like properties, incorporating in silico design and robust biological testing into an iterative process. We will characterize new CXCR4 modulators in Aim 2 by evaluating their behavior against a number of in vitro systems including calcium mobilization, receptor binding, receptor internalization, chemotaxis, and signaling through various pathways. Aim 3 will evaluate the effects of CXCR4 modulation on AML using leukemia cell lines and primary human tumor cells as well as in vivo using patient-derived xenografts models of AML. The proposed studies will generate new molecular probes to investigate the pharmacology of CXCR4 and understand how this important receptor is involved in AML. These results will provide new insights into AML and potentially open up new avenues for therapeutic development.

? DESCRIPTION (provided by applicant): Northwestern University Center of Cancer Nanotechnology Excellence (NU-CCNE) for Nucleic Acid-Based Nanoconstructs for the Treatment of Cancer Northwestern University PIs: Chad A. Mirkin and Leonidas Platanias Much still remains to be learned about the genetic basis of cancer and how it can be analyzed and treated. Current treatment methodologies fall short of providing efficacious, targeted, precision therapies geared towards the individual patient. Due to their novel chemical, biological, and physical properties, nucleic-acid based nanoconstructs can be used to gain access to privileged intracellular environments, discover new aspects of cancer biology, and exploit nanostructure-biomolecular interactions to create effective treatment options. The Center of Cancer Nanotechnology Excellence for Nucleic Acid-Based Nanoconstructs for the Treatment of Cancer, headquartered at Northwestern University (NU), proposes to explore these vast possibilities by applying a novel class of nanostructure genetic constructs - the spherical nucleic acid and variants of it - for the study and treatment of brain and prostate cancer. The proposed Center will feature three projects (one discovery-based and two translational), one core facility, and two for-profit partners united to provide novel nanotechnology-based solutions to daunting and complex issues in cancer research and treatment. Under the direction of Principal Investigators, Professor Chad A. Mirkin and Dr. Leonidas Platanias the CCNE represents an integrated partnership between the NU Robert H. Lurie Comprehensive Cancer Center (RHLCCC) and the NU International Institute for Nanotechnology (IIN). The RHLCCC is an NCI-designated, comprehensive, University-based, matrix cancer center conducting a broad range of multidisciplinary basic, clinical, and population science research with over $167 million dollar in annual extramural funding. The NU International Institute for Nanotechnology (IIN) is an umbrella organization which unites all of the nanotechnology research and educational programs at NU, and currently represents more than $600 million in funding for nanotechnology research and infrastructure. Building upon the significant advances in cancer research and in nanotechnology obtained at NU over the past nine years, and operating within the framework of a single university, the NU-CCNE will optimize the intensive level of integration and collaboration required to create an accelerated pathway-from conception to clinical trial-for development of nanomaterials and nanodevices to overcome cancer.

PROJECT SUMMARY/ABSTRACT This is a competing renewal application whose overall objective is to define the mechanisms of interferon (IFN)- signaling in malignant cells. We have provided the first evidence for the existence of a novel pathway engaged by the Type I IFN receptor (IFNR), involving the ULK1 kinase. This ULK1 pathway operates in an autophagy- independent manner to regulate expression of IFN stimulated genes (ISGs) and is essential for the generation of the suppressive effects of IFN??on primary malignant hematopoietic precursors from patients with myeloproliferative neoplasms (MPNs). Remarkably, ULK1 forms complexes with unique partners that have distinct structures and functional capabilities, suggesting that coordinated operation of such complexes is required for optimal control of IFN-induced antineoplastic effects. Aim 1 will identify upstream IFNR-generated signals and mechanisms that control ULK1 activation. It includes studies to map the IFN-phosphorylation sites on ULK1 and identify direct upstream kinases that modify its function. Aim 2 will define ULK1 effector pathways that mediate IFN-responses. It includes experiments to determine the roles of novel effectors that we have identified, including ROCK1/2, PCM1 and CARD9. A combination of approaches will be used, including generation of selective knockouts by the CRISPR-Cas9 methodology and mutation of putative ULK1 phosphorylation sites in the structures of target proteins, followed by assessment of its effects on IFN signaling and antineoplastic responses. Aim 3 will define the role of ULK1 pathways in the generation of the antineoplastic effects of IFN? in MPNs. Jak2V617F-driven MPN mouse models will be established using mice with Ulk1-/-, Pcm1-/-, or Card9-/- backgrounds, and the ability of IFN? to induce antineoplastic responses in such mice will be examined. The therapeutic effects of a ROCK1/2 pharmacological inhibitor alone and in combination with IFN??in mediating anti-MPN responses in vivo will be also determined. In other studies, the activation of IFN- dependent ULK1 effector signals will be determined in primary malignant precursors from patients with different MPNs participating in IFN-clinical trials and correlated with molecular characteristics and clinical responses to IFN?-treatment. Altogether, this proposal introduces highly novel concepts in the IFN-field and the mechanisms of IFN-generated antitumor effects and may lead to the development of novel therapeutic strategies and approaches for the treatment of MPNs.

ABSTRACT ? DEVELOPMENTAL FUNDS Developmental funds provide an essential source of support for the strategic initiatives of the Lurie Cancer Center (LCC), consistent with its overall planning processes and priorities. In the current project period, we used CCSG developmental funds in two major areas: recruitment of new investigators and development of one new shared resource. CCSG funds were leveraged by additional institutional resources and philanthropy to support faculty recruitment and retention, pilot projects, and equipment and technology upgrades of existing shared resource facilities. The LCC Executive Committee determines the priorities for the allocation of Developmental Funds and supplemental institutional support, with input from the Program Leaders and the External Advisory Board, to ensure alignment with CCSG guidelines and the LCC strategic plan. Eight faculty received CCSG recruitment support during the current cycle. In addition, during the current cycle, we used CCSG Developmental Funds to support one new shared resource, the Proteomics Core Facility, which is presented as an established facility in this application. Although CCSG funding was not used for pilot projects, the LCC invested substantially in intramural awards. This value-added investment has stimulated and promoted innovative research efforts in basic, clinical, translational and population sciences research, leading to new extramural grant funding, new transdisciplinary collaborations and important cancer relevant publications. Developmental Funds have leveraged additional institutional support and provided a superb return on investment, with approximately $1 million CCSG funds allocated to new faculty recruits over this grant period, generating a total of $33.3 million in grants and clinical trials; a 33-fold return on the investment (ROI). The $33.3 million includes $15.7 million in NIH funding, greater than 15 times the ROI of the CCSG funds invested. Given the continued substantial growth of the LCC?s overall research enterprise, and the successful use of Developmental Funds in the current project period, we request $400,000 per year to be used in two categories: 1. Recruitment of investigators in areas of strategic priority for the LCC, including cancer immunology/immunotherapy, cancer epigenetics, cancer epidemiology and drug discovery and development. 2. Development of a Metabolomics Core that will be of high value to LCC members.

ABSTRACT ? PLANNING AND EVALUATION The Lurie Cancer Center (LCC) has a well-developed system for planning and evaluation. As new LCC Director, Dr. Platanias established a new Senior Leadership team, with well-defined roles and responsibilities: (1) Maha Hussain, M.D. was appointed as Deputy Director, (2) Kathleen Green, Ph.D. as Associate Director for Basic Sciences Research, (3) Massimo Cristofanilli, M.D., as Associate Director for Translational Research, (4) Milan Mrksich, Ph.D. as Associate Director for Shared Resources, (5) Aleksandar Zafirovski, M.B.A., as Associate Director for Administration and (6) Jeffrey Wayne, M.D. as Associate Director for Clinical Operations. Among the Associate Directors continuing in their positions are (1) David Cella, Ph.D., for Cancer Prevention and Control Research, (2) John Crispino, Ph.D., for Education and Training and (3) Alexis Thompson, M.D., for Equity and Minority Health. This team works collectively as the Executive Committee (EC), which meets every two weeks and is the primary decision making body that sets the overall strategic direction of the Center. The EC provides direction in matters relevant to LCC operations, management, and policy and oversees all research activities. It also sets budgetary and fiscal priorities and reviews LCC membership. CCSG support is requested for all Senior Leadership positions except the position of Associate Director for Clinical Operations. In addition to the EC, there are several other governance structures that provide the primary means by which the LCC conducts planning and evaluation on an ongoing basis. These are the Scientific Research Council (SCRC), the Clinical Cancer Center Executive Council (CCCEC), the Program Leaders Committee (PLC) and the Internal and External Advisory Boards. The SCRC includes the Associate Directors and all Program and Shared Resource leaders/directors, and meets quarterly to review initiatives and provide input for research programs, shared resources and training/education. The PLC, a new committee established by Dr. Platanias, meets monthly to discuss joint initiatives within and between programs and provides important input to the EC. The External Advisory Board (EAB) is comprised of recognized leaders in clinical, basic and population sciences, biostatistics and center administration, and meets at least annually to review all aspects of LCC operations and initiatives. After his appointment as Director, Dr. Platanias increased the number of EAB members and made several new EAB appointments with input from other senior leaders. Additional planning input is provided by retreats, other Center advisory committees, surveys and ad hoc reviews. With careful planning and critical input from the different advisory groups, the Director initiated a new five-year strategic planning process, reorganized center research program membership, and created working groups to continue improving oversight and governance. These coordinated teams provide critical advice and guidance, ensuring that the LCC most effectively sets priorities and pursues objectives that promote its mission. CCSG support is requested for the activities of the EAB, which plays an essential role in the evaluation of the LCC?s programs and organization.

Glioblastoma (GBM) is most common central nervous system neoplasm in adults and one of the most aggressive and fatal malignancies in humans. The limited number of available therapies almost always fails, due to resistance of GBM stem cells (GSCs). We have found that expression of MNK kinases correlates with GBM grade and overall survival. Our studies demonstrate that these kinases play key and essential roles for survival of GSCs, raising the possibility that MNK targeting may provide a unique approach for the treatment of GBM. The current proposal aims to identify effector mechanisms by which MNK pathways promote GSC survival and to develop novel, specific, and effective MNK inhibitors that could be ultimately developed clinically for the treatment of GBM. Different GBM models and primary samples from GBM patients will be used for that purpose. Aim 1 will define MNK effector pathways in GSCs and will dissect their contributions in GBM pathophysiology. Experiments will be performed to define the roles of MNK-regulated effectors in controlling oncogenic mRNA translation, cell proliferation, and survival of GSCs. In addition, the differential requirement of MNK1 versus MNK2 in GSC growth and survival and their regulatory effects on downstream pathways will be dissected. Aim 2 will develop potent and selective MNK inhibitors through rational medicinal chemistry optimization. For this purpose, optimization of the MNK inhibitors that we have already developed will be pursued to improve potency, selectivity, and pharmaceutical properties. In addition, crystallization studies will be performed to understand the binding mode and support structure-based drug design. Aim 3 will evaluate the effects of MNK inhibition in orthotopic GBM mouse models. Compounds selected for adequate toxicity profiles and pharmacokinetics will be tested for efficacy against GBM using orthotopic xenograft mouse models for GBM. Altogether, the results of this work will provide important information on the mechanisms by which MNK kinases promote survival of GBM stem cells and will drive the development of novel pharmacological agents targeting the MNK kinase pathway for the treatment of GBM.

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is a highly aggressive malignancy with very high morbidity and mortality, due to lack of effective therapies. The overall goal of this proposal is to identify novel cellular targets in GBM cells that could lead to new therapeutic approaches. We have found that a member of the Schlafen (SLFN) gene family, SLFN5, is significantly overexpressed in GBM as compared to normal brain, and that high levels of SLFN5 expression correlate with poor survival among GBM patients. Our data indicates that SLFN5 promotes GBM growth by repressing IFN signaling and IFN stimulated gene (ISG) expression via an interaction with the transcriptional activator STAT1. This highly novel finding forms the basis of the current proposal. Aim 1 will identify elements of SLFN5-STAT1 complexes, define upstream regulatory signals required for the formation of such complexes, and determine relationships between elements of these complexes and SLFN5-associated transcriptional repression. The functions of different SLFN5 structural motifs and their importance to the suppression of IFN-responses will be examined. Studies using primary samples from GBM patients will be also employed for such studies. Aim 2 will define the effects of SLFN5 expression in vivo using three distinct orthotopic engraftment models and GBM cell pairs with and without SLFN5 expression: i) conventional xenograft models using athymic mice for examining the effects of SLFN5 on tumor establishment and growth; ii) humanized orthotopic PDX models to examine SLFN5 effects for human-on-human immune response against tumor, as well as to examine tumor response to immune checkpoint therapy; and iii) same a ii but using mouse GBM syngeneic models in which the host animals have a fully functional immune system. Altogether, the results of this work will provide important information on the mechanisms by which SLFN5 expression suppresses IFN-responses and promotes GBM growth. The successful performance of this work should facilitate development of highly novel approaches for the treatment of GBM using SLFN5 as a target.

PROJECT SUMMARY/ABSTRACT Despite recent advances in the treatment of acute myeloid leukemia (AML), the morbidity and mortality of AML remains very high and novel therapeutic approaches are urgently needed. This is a competing renewal application whose overall objective is to define the functional significance of novel protein complexes in AML cells and to exploit their potential therapeutic targeting. We have identified novel regulatory protein complexes involving the cyclin dependent kinase 9 (CDK9). We found that CDK9 is a novel binding partner of the mTOR complex scaffold protein, mLST8 and is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to Raptor and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to Rictor, SIN1 and mLST8, forming CTORC2 complexes that control mRNA translation through phosphorylation of LARP1 and rpS6. Targeting CTOR complexes results in suppression of growth of primary human AML progenitors in vitro and generation strong antileukemic responses in AML xenografts in vivo, suggesting an essential role for CTOR complexes in the survival of AML leukemic precursors. Specific aim 1 will define the functions of nuclear CTORC1 complexes and mechanisms by which they control expression of genes that promote leukemogenesis. Studies will be performed to map the interactions between CDK9 and other CTORC1 elements and define mechanisms by which CTORC1 complexes control transcriptional activation of mitogenic genes and mRNA splicing. Specific Aim 2 will determine the functions of CTORC2 complexes and their roles in mRNA translation of target genes and survival of primitive leukemic precursors. Experiments will be performed to define components and effectors of CTORC2 complexes and their roles in mRNA translation, protein expression, and leukemic cell survival. Specific aim 3 will examine the antileukemic properties of CTORC1 and CTORC2 targeting on primary leukemic precursors in vitro and in vivo. It will involve in vitro studies using primary leukemic progenitors from a large group of AML patients and in vivo experiments using AML mouse models, all aimed to determine the impact of the different CTOR complexes on leukemogenesis and the potential synergistic effects of CTORC targeting with other antileukemic agents. Altogether, the studies of this competing renewal application will advance our understanding of the mechanisms of leukemogenesis and will provide the basis for important future clinical-translational efforts, involving targeting of CTOR complexes for the treatment of AML.