SPORE in Lymphoma Grant James P. Wilmot Cancer Center at the University of Rochester; Arizona Cancer Center at the University of Arizona; Massey Cancer Center at Virginia Commonwealth University Principal Investigator: Richard I. Fisher, M.D. Co-Principal Investigator: Thomas P. Miller, M.D. Overall Abstract: This Lymphoma SPORE application from the James P. Wilmot Cancer Center (JPWCC), University of Rochester, and the Arizona Cancer Center (ACC), University of Arizona provides a broad based, translational research program studying several major unresolved issues in the lymphoma biology. Specifically the projects will focus on several novel, targeted therapeutic agents either in development or approved for the treatment of three of the most common non-Hodgkin’s lymphomas, namely diffuse large B-cell, follicular and mantle cell lymphoma. We will initially seek to elucidate mechanisms of action and/or resistance both in vitro, and in carefully developed model systems; then pilot clinical trials with an emphasis on correlative studies which will be designed and conducted to validate these mechanisms. Investigators at the JPWCC and the ACC, long-time collaborators, lead each of the programs. The clinical and pathologic resources of the Lymphoma Committee of the Southwest Oncology Group are also integrated into this program. Projects include: Research Project 1 : Targeting Aurora Kinase in Aggressive B-cell non-Hodgkin’s Lymphoma, Daruka Mahadevan, M.D. Ph.D. (Laboratory); Daniel O. Persky, M.D. & Thomas P. Miller, M.D. (Clinical) Project 2 : Optimizing Redox Modulation as a Therapeutic Strategy For NHL. Co-Leaders: Steven Bernstein, M.D. & Margaret Briel, Ph.D. (Laboratory); Steven Bernstein, M.D. (Clinical); Co-Investigators: Shannon Hilchey, Ph.D., Paul Brookes, Ph.D. Project 3 : Potentiating Proteasome Inhibitor Activity in NHL. Co-Leaders: Steven Grant, M.D. (Laboratory); Jonathan Friedberg, M.D. (Clinical); Co-Investigators, Richard I. Fisher, M.D., Paul Dent, Ph.D., Yun Dai, Ph.D. Project 4 : Characterization of Lymphoma-Initiating Cells. Co-Leaders: Craig T. Jordan, Ph.D. (Laboratory); and Fay Young, M.D. (Clinical); Co-Investigator; Randall Rossi, M.S. Administrative Core, Director: Richard I. Fisher, M.D., JPWCC; Co-Director: Thomas P. Miller, M.D., ACC; Tissue Resource Core, Director: Lisa Rimsza, M.D., ACC; Co-Director: W. Richard Burack, M.D., Ph.D., JPWCC; Clinical Trials, Director: Jonathan Friedberg, M.D., JPWCC; Co-Director: Thomas P. Miller, M.D., ACC; Biostatistics, Director: Derick R. Peterson, PhD. (JPWCC) The Developmental Research Program is directed by Hartmut Land, Ph.D. (JPWCC) while the Career Development Program is Directed by Susan G. Fisher, Ph.D. (JPWCC) Project 1: Targeting Aurora Kinase in Aggressive B-cell non-Hodgkin’s Lymphoma Co-Leaders: Daruka Mahadevan, M.D. Ph.D; Thomas P. Miller, M.D.; Daniel O. Persky, M.D. Aurora kinases are a family of serine/threonine (S/T) kinases (Aurora A, B, C) that function predominantly in the mitotic-phase of the cell cycle. All 3 Auroras possess a highly conserved C-terminal kinase domain with a variable length N-terminal extension. These kinases regulate the completion of centrosome separation, bipolar spindle assembly, chromosome segregation and cytokenesis. Aberrant over-expression of Aurora A and B are oncogenic as they over-ride the mitotic and spindle check points leading to aneuploidy and genomic instability. Previously, we utilized small interference RNA (RNAi) techniques to validate Aurora A as an oncogenic target. We then evaluated the lymphoma, leukemia molecular profiling project (LLMPP) gene expression profile of ~100 mantle cell lymphoma (MCL) patients and demonstrated that over-expression of Aurora A and B, as ‘signature genes for proliferation’, predicts for an inferior overall survival and hence is a poor prognostic factor. Further, we showed that Aurora A is over-expressed in several of human MCL and DLBCL cell lines. Therefore, targeting Aurora with a specific small molecular inhibitor (SMI) might be of therapeutic value in aggressive B-cell non-Hodgkin’s lymphomas (NHL). Hence, we utilized a structure-based drug discovery approach to design and synthesize a specific ATP-competitive Aurora SMI (SGI-498). Treatment of MCL and DLBCL cell lines with SGI-498 led to reduced cell viability and apoptosis in the 5-10mM IC50 range. Large animal safety studies have been conducted and an IND application is in progress (in collaboration with Supergen). Collectively these data show that Aurora is an excellent oncogenic drug target in aggressive subsets of B-cell NHL. Despite encouraging data in cell lines, there has not been a comprehensive study evaluating the expression of Aurora kinases in fresh tumor specimens of aggressive B-cell NHL nor a clinical test of efficacy in lymphoma patients. Therefore, the hypotheses to be tested in this Project 1 are: (1). Aurora A and B are over-expressed in fresh human aggressive B-cell NHL (mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (TFL)) tissues, (2). Aurora inhibitor SGI-498 is effective in promoting apoptosis in cell culture and tumor regression in a mouse xenograft model(s) of NHL and (3). SGI-498 will be safe and effective in treating patients with relapsed aggressive B-cell NHL in a early Phase clinical trial. Project 2: Optimizing Redox Modulation as a Therapeutic Strategy For NHL Co-Leaders: Steven Bernstein, MD and Margaret Briehl, M.D. Co-Investigators: Paul Brookes, Ph.D. and Shannon Hilchey, Ph.D. The triterpenoids are novel electrophilic compounds that we have shown induce apoptotic cell death in mantle cell lymphoma (MCL) and diffuse large cell lymphoma (DLCL). Indeed our published and preliminary studies suggest that these compounds manifest their cytotoxic effects by both increasing the generation of reactive oxygen species (ROS), and decreasing the activity of such critical anti-oxidant defense mechanisms as thioredoxin (Trx) and the mitochondrial proteases, Lon and ClpXP. This suggests a novel therapeutic strategy for lymphoma based on modulating the lymphoma cell redox state. In addition, our proposed mechanism suggests that other redox active agents, such as proteosome inhibitors and histone deacetylase inhibitors, are rationale agents to combine with the triterpenoids. The Specific Aims of this proposal have been developed to provide the essential pre-clinical data needed to support the clinical evaluation of the optimal triterpenoid and other redox active agent combinations for patients with DLCL and MCL. Specifically, in Specific Aim 1, we will validate our proposed mechanism of activity of the triterpenoids so to optimize triterpenoid drug combinations in vitro. These combinations will be further optimized and the biological targets of ROS generation, Trx and mitochondrial proteases validated in vivo using DLCL and MCL/SCID xenograft models in Specific Aim 2. We anticipate that the data derived from these Specific Aims will suggest an optimal triterpenoid combination, and sequence of drug delivery to be studied in the context of a phase I/II trial as proposed in Specific Aim 3. A critical component of this trial will be the correlative laboratory studies whereby we propose to determine the in vivo effects of this drug combination on ROS, Trx and mitochondrial protease activity in patients with lymphoma. It is further anticipated that the lessons learned from these studies will support our long term goals which are to understand the clinical potential of the family of electrophilic compounds in NHL, which also includes the parthenolides, cucurmin, and the acivicins, and more importantly how best to exploit the redox state of lymphoma to generate new and effective approaches for the treatment of patients with NHL. Project 3: Potentiating Proteasome Inhibitor Activity in NHL Co-Leaders: Jonathan Friedberg, M.D. and Steven Grant, Ph.D. Co-Investigators: Richard I. Fisher, M.D., Yun Dai, Ph.D. and Paul Dent, Ph.D. Proteasome inhibitors (PIs) such as Bortezomib (Btzmb), induce apoptosis in neoplastic cells through multiple mechanisms, including NF-κB activation, oxidative injury induction, an ER stress promotion, among others. Btzmb displays marked activity in multiple myeloma, and several types of non-Hodkin’s lymphoma (NHL), prompting the development of second generation proteasome inhibitors (e.g., PR-171) which may have superior PK/PD characteristics compared to Btzmb. However, the need to improve PI activity in NHL persists. Recent evidence from our laboratory suggests that combining PIs with other clinically relevant targeted agents, including HDAC or small molecule Bcl-2 inhibitors results in synergistic induction of apoptosis in NHL cell lines. The goal of this project is to elucidate, using a diffuse large B-cell lymphoma (DLBCL) model, mechanisms of synergism between Btzmb or PR-171 and other targeted agents, extend these findings to an in vivo model system, identify laboratory-based response determinants, and use this information to initiate novel combination Phase I trials in patients with refractory NHL. In Specific Aim #1, we will determine whether synergism between PIs and HDAC inhibitors in NHL cells reflects NF-κB disruption, induction of oxidative injury, ER stress, or a combination of these factors. In Specific Aim #2, we will determine whether synergism between PIs and small molecule Bcl-2 inhibitors (e.g. ABT-737, GX15-070, HA14-1) stems from oxidative injury, activation of stress-related signaling pathways, and/or Bak and Bax activation. We will also establish a) whether these approaches are effective in Btzmb-resistant DLBCL cells; and b) which of these individual strategies might be most appropriate for specific DLBCL sub-types i.e., germinal center (GC) versus activated B-cell (ABC). In Specific Aim #3, we will extend these findings to a xenograft DLBCL model system, and determine whether in vitro determinants of synergism are operative in vivo . In Specific Aim #4, we will select the most promising of regimens emanating from Aims #1-3 to pursue as one or more Phase I trials combining PIs with HDAC or small molecule Bcl-2 inhibitors in patients with refractory NHL. We will also conduct correlative laboratory studies to validate mechanisms of in vivo synergism, and identify surrogate markers of disease responsiveness. It is anticipated that these studies will lay the foundation for novel and potentially more effective PI-based strategies in patients with refractory NHL. Project 4: Characterization of Lymphoma-Initiating Cells Co-Leaders: Craig T. Jordan, Ph.D. and Fay Young, M.D. Co-Investigator: Randall Rossi, M.S. Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential and the ability to reconstitute the tumor after transplantation. There is now compelling evidence that tumors from disparate malignancies harbor a minor population of tumor cells, termed ‘cancer stem cells’, that possess the unique stem cell property of self-renewal. We hypothesize that clinical non-Hodgkin’s lymphoma arises from such biologically distinct tumor-initiating cells – a lymphoma stem cell (LySC). The goal of this project is to identify, characterize, and target the tumor-initiating cells found within the context of primary human non-Hodgkin’s lymphoma (NHL). In malignancies of differentiated adult tissue such as breast, colon, prostate, and brain cancer, cell populations with the critical stem cell-like properties of self-renewal and differentiative capacity have been identified. Thus mutation/transformation of a B-lymphocyte could result in the acquisition of functionally defined stem cell properties. Preliminary data derived from NHL specimens present several lines of evidence in support of our hypothesis. In concordance with a central component of stem cell based diseases, we find clear phenotypic and functional heterogeneity in primary and cultured lymphoma specimens. These data strongly support a role for LySC in the pathogenesis of NHL. Further, because cancer stem cells are generally less sensitive to conventional cytotoxic therapy, we propose that the LySC population may represent a central reservoir for cells giving rise to clinical relapse and refractory disease. Therefore, the identification and subsequent targeting of LySC is a critical step toward optimizing therapeutic outcomes in NHL. Core A: Administrative Core Core Director: Richard I. Fisher, M.D. Core Co-Director: Thomas P. Miller, M.D. The major function of the Administrative Core is to provide an organizational structure through which the investigators can interact and communicate with each other to foster the fundamental goal of the SPORE, i.e., translation of basic biologic information to and from the clinic in order to improve the survival and quality of life for patients with lymphoma. This core will function via several standing committees of the SPORE including the Steering Committee, the Committee of Program and Core Leaders, the Developmental Research Program Review Committee, the Career Development Program Committee, the Institutional Advisory Committee, and External Advisory Board. These committees consist of the senior leadership of the SPORE and the Universities of Rochester and Arizona, as well as national lymphoma experts and lay advocates. The Administrative Core of the Lymphoma SPORE is the central administrative organization responsible for managing all aspects of the SPORE grant including communication with and reporting to the NCI, communication with other Lymphoma SPOREs, grant accounting and reconciliation, submission of grant related publications, etc. Dr. Richard Fisher will serve as Director of the Administrative Core, and Dr. Thomas Miller as Co-Director. Dr.'s Fisher and Miller will work collaboratively to provide expertise in translational and clinical lymphoma research. They have extensive administrative experience and are well qualified to provide leadership and direction for the Lymphoma SPORE. The Administrative Core schedules and coordinates all internal and external meetings (tabulated at the end of this section) including travel to the annual NCI SPORE meeting, creates and circulates minutes for those meetings, and coordinates the travel for all investigators and consultants. The administrative core will also establish a web site to facilitate the dissemination of information and to encourage collaborations with other investigators. The web site will be developed as part of the ongoing James P. Wilmot Cancer Center web site, and will be a key mechanism of communication between the University of Arizona and the University of Rochester. Other mechanisms of communication between these two centers, including regular conference calls, meetings, and videoconferences will be arranged by the administrative core. Core B: Tissue Resource Core Core Director: Lisa Rimsza, M.D. Core Co-Director: Richard Burack, M.D., Ph.D. The combined Tissue Resource Core at the Universities of Arizona and Rochester will have the responsibility of collecting, storing and cataloging pathological specimens from patients with lymphoproliferative disorders that are seen at the Arizona and James P Wilmot Cancer Centers at the Universities of Arizona and Rochester respectively. In addition, the Core will also collect and store normal lymphoid tissue and blood and bone marrow cells to be used as controls for biologic studies that use the corresponding malignant tissue. Specimens are stored as snap frozen blocks, paraffin blocks, viable cell suspensions, and frozen serum aliquots. Tissue procurement and data collection is done under the auspices of IRB approved protocols at each institution. All patients are required to sign an informed consent before abstracting clinical or epidemiologic information using procedures that maintain strict patient anonymity and that adhere to the guidelines established by the Health Insurance Portability and Accountability Act (HIPAA). In addition, at U of A, excess diagnostic tissue from non-consented patients is stored as a matter of routine practice. No clinical information is abstracted on these patients without direct IRB approval and waiver of informed consent. However, these tissues are more frequent and make valuable samples for test development and feasibility studies. A bank of hematolymphoid cell lines is also maintained with active collection of new cell lines of interest. These facilities are already integrated with each other regarding sample distribution and software development. The overall goal of the Tissue Resource Core is to provide necessary patient samples and cell lines to support the Lymphoma SPORE translational research projects. The specific aims are: To consent, collect, store, and annotate high quality samples in accordance with regulatory guidelines in order to maximize their potential usefulness; To characterize samples through pathologic evaluation, immunophenotyping, molecular studies, and clinical features; To conserve tissues through careful inventory control, creation of tissue microarrays, up-front extraction of DNA and RNA; To distribute samples in a timely and efficient manner to maximize their translational research potential. These combined resources at the University of Arizona and Rochester will be intimately involved with each of the projects proposed in this SPORE grant application and will facilitate the development of collaborative projects within and beyond the SPORE. Core B: Clinical Trials Core Core Director: Jonathan W. Friedberg, M.D. Core Co-Director: Thomas P. Miller, M.D. The primary objective of the clinical trials core is to provide assistance to the projects in all aspects of clinical trial design, implementation, regulation, data collection, and interpretation, pharmacokinetics, and other specimen collections for targeted therapy investigations and/or banking. Specifically, the clinical trials core facilitates protocol writing and preparation, submission, and regulatory review, interacting directly with the NCI, FDA, Industry sponsors and IRB as needed. The clinical trials core maintains the database and all aspects of data management for patients enrolled on clinical trials within the SPORE. Adverse event reporting and data and safety monitoring for all protocols on the SPORE application are coordinated through the clinical trials core. The clinical trials core seamlessly interacts with the specimen core, providing clinical, epidemiologic and demographic data on patients consenting to tissue banking, and with the biostatistics core, providing timely reports and data for analysis. Core C: Biostatistics Core Core Director: Derick R. Peterson, Ph.D. Co-Investigators: Changyong Feng, Ph.D., Ollivier Hyrien, Ph.D., Denise Roe, Dr. P.H. The objectives of the Biostatistics Core are to provide collaborative support for study design, data analysis, and dissemination of results for all SPORE projects, in addition to developing novel statistical methods to handle unique analysis problems, as needed. Statistical and methodological support is critical to ensure the quality of the design, conduct, analysis, and reporting of scientific trials and studies. With related experimental design and conceptual components, some of the quality control and analytic issues will be shared among the projects. By using the shared resource of the Biostatistics Core, all SPORE projects will benefit from the experience gained in each project. The Biostatistics Core investigators have extensive and complementary experience in quantitative methods for biomedical applications, including both clinical and basic science studies. They are committed to taking a direct interest in the substantive issues being investigated, to participating in regular project and program meetings, and to providing rigorous and innovative input on all quantitative matters arising in the projects. The 5 Specific Aims of the Biostatistics Core are to: Provide detailed consultation for the development of all study protocols, including clinical trials. This includes study design, defining outcome variables and important covariates, developing appropriate measures and methods to obtain the relevant data necessary to properly answer the study questions, identifying appropriate statistical methods for analysis, and performing power and sample size calculations. Provide biostatistical support during the conduct of the studies. This includes a synergistic relationship with the Clinical Trials Core to provide quality control and routine report generation as well as to assist in making any decisions related to protocol changes or revisions. Collaborate in the interim and final statistical analyses of the study data. This includes identifying appropriate statistical methodology, statistical programming, data analysis, assisting with the interpretation of the results of analyses, and producing final reports and graphical displays suitable for presentation and publication. Develop novel statistical methods to handle unique analysis problems, as needed. Collaborate in the preparation of manuscripts and presentations of the results of the studies. Development Research Program Director: Hartmut Land, Ph.D. The Developmental Research Program for the Lymphoma SPORE is designed to identify and selectively fund innovative and pilot studies of lymphoma translational research within and outside of the University of Rochester and the University of Arizona. Once annually, proposals will be requested. A senior faculty committee, which includes representation of lay advocates, under the direction of Hartmut Land, Ph.D., will review the detailed applications, gives them a priority score, and recommends projects for funding to the Lymphoma SPORE committee, who will make the final decision. The proposals will be evaluated for originality, scientific design, and particular attention to translational relevance. In addition, standard NIH review criteria for significance, approach, innovation, investigator, and environment will be applied. There will be no discrimination based on age, sex, race, religion, national origin, etc, and proposals from women and underrepresented minorities will be encouraged. Proposals with the highest scientific merit and directly related to lymphoma translational research will be considered for one or two years of funding. These projects may utilize the core facilities within the SPORE. The primary objectives of this developmental research program are as follows: Encourage laboratory and clinical investigators to apply their expertise toward lymphoma translational research. Foster collaboration between SPORE participants, and other lymphoma investigators. Provide seed money for research projects that will ultimately compete for peer-reviewed funding, or become main projects within the SPORE proposal. Career Development Program Director: Susan G. Fisher, Ph.D. Co-Director: Margaret Briehl, Ph.D. The Lymphoma SPORE Career Development program, based on the strong infrastructure for career development that has been a long tradition at both the University of Rochester and the University of Arizona, will provide comprehensive training and education to new investigators committed to careers in translational research in lymphoma. The goal of the SPORE Career Development Program will be to provide the necessary support, mentorship and research environment to enable young faculty to establish a translational research focus and independent funding in lymphoma. The specific aims of the Career Development Program are to: establish an environment that stimulates and facilitates multidisciplinary interactions among new lymphoma investigators, particularly minorities and women, and provides integrated research and education career development opportunities within a strong infrastructure of mentoring. The program’s educational component includes: lymphoma-related educational seminars (videocast to all SPORE participants), skill building workshops (webcast) through the CTSI; master’s degree in clinical investigation or translational research (visiting trainees); and intensive grant writing advisement/collaboration (individualized). A detailed, tailored mentoring plan will be implemented for each trainee since access to a strong mentor is critical to early career success. Funding will also be provided for costs related to the conduct of a translational study. A specific plan for program governance and implementation, trainee application, selection and evaluation, and evaluation of global program outcomes is outlined. The future expansion and success of translational research in lymphoma depends upon the support and enhancement of career development efforts that target individuals interested in hematologic malignancies and assure that they have continuous access to superb physician scientists. The proposed program will contribute to this important need.