Lymphoma SPORE

NCI Specialized Program of Research Excellence (Lymphoma SPORE)

National Cancer Institute SPORE Information

SPORE Newsletter-Current Issue 

Holden Comprehensive Cancer Center at The University of Iowa and Mayo Comprehensive Cancer Center
Principal Investigator: George J. Weiner, MD, The University of Iowa
Co-Principal Investigator: Thomas E. Witzig, MD, Mayo Clinic

OVERALL ABSTRACT

The University of Iowa/Mayo Clinic (UI/MC) Lymphoma SPORE is a dynamic, productive, translational cancer research program based at two comprehensive cancer centers that was first funded in 2002 and competitively renewed in 2007. At the center of the ongoing success of the UI/MC SPORE is the collaborative interaction between investigators at Iowa and Mayo, as well as SPORE basic laboratory, clinical and population-based investigators.  The overall goal of the SPORE is to support innovative, interactive, translational research into lymphoma and chronic lymphocytic leukemia that leverages the expertise of laboratory, clinical and population-based research at both institutions.    Over the last funding period, the SPORE has been highly productive as demonstrated by identification of new tumor markers, scientific findings that led to additional translational lymphoma grants from the NCI, multiple publications including many authored by investigators from both institutions, and brisk accrual to translational clinical trials.  The UI/MC Molecular Epidemiology Resource is based in the SPORE, and serves as a vital resource for both SPORE research and research projects supported by other grants.  

The current proposal will build on past successes and support 4 major research projects that represent new concepts that emerged from research supported over the past funding period.  Support will also be provided to pursue novel translational concepts in lymphoma research and new investigators through the Developmental Research and Career Development programs.  

Projects are as follows:

  1. The Role of Monocytes in non-Hodgkin Lymphoma>
  2. In Situ Immunization using Nanoparticles
  3. Targeting JAK2 Kinase in Lymphoma
  4. Genetic Epidemiology and Function of Germline in Somatic Variants in DLBCL

Finally, the SPORE will enhance the infrastructure that supports translational lymphoma research at both institutions through shared core resources in Administration, Biostatistics and Bioinformatics, Biospecimens and Clinical Research. 

PROJECT 1: The Role of Monocytes in non-Hodgkin Lymphoma

Co-Principal Investigators: Stephen Ansell, MD, PhD (Mayo) and Clive Zent, MD Mayo)
Co-Investigators: Allan Dietz, PhD (Mayo) and Brian K. Link, MD (Iowa)

The tumor microenvironment plays an important role in non-Hodgkin lymphoma (NHL) and the role intratumoral immune cells play in the pathology of lymphoma has been significantly understated. Intratumoral monocytes and macrophages are particularly important and our data demonstrate that intratumoral monocytes in NHL are highly immunosuppressive and support malignant cell growth.

In preliminary work, we found that suppressive monocytic cells (SMCs) are abundant within the peripheral blood and tumor microenvironment in lymphoma patients and promote the survival of lymphoma cells. SMCs protect lymphoma cells from chemotherapy-induced cell death and promote lymphoma cell engraftment into severe combined immunodeficient (SCID) mice. Furthermore, we found that SMCs within lymph nodes express immunosuppressive ligands including B7-H1 (PD-L1, CD273), inhibit normal T-cell proliferation and promote the induction of FoxP3+ regulatory T cells. These preliminary studies suggest that SMCs have an effect on both malignant NHL cells and non-malignant intratumoral T-cells.

Based on our results, we hypothesize that the intersection between the immune system and the malignant cell in NHL is the SMCs. In this proposal, we plan to understand whether monocytes are specifically recruited to sites of lymphoma and which specific chemokines could be inhibited to prevent SMC migration; how lymphoma cells induce SMCs to support their malignant cell growth and to suppress the host’s anti-tumor immunity; and whether promoting monocyte/macrophage maturation or inhibiting their interaction with other cells, particularly in the presence of monoclonal antibodies, improves their anti-tumor function. Upon completion of this project, we expect to have a greater understanding of the role of monocytes and their progeny in NHL. Collectively our findings are likely to have a major impact by leading to an effective monocyte-directed therapeutic approach for patients with lymphoma.

Three specific aims of this project are:

1. Define the mechanisms that result in the recruitment of monocytes to sites of lymphoma.
We will determine whether intratumoral monocytes/macrophages migrate in response to chemotactic ligands and establish a role for malignant cells in their recruitment. We will also determine whether monocytes undergo phenotypic and functional changes when recruited to lymphoma sites.
2. Determine the mechanisms by which monocytes promote malignant B-cell survival and suppress intratumoral immune effector cells.
We will identify the mechanisms responsible for the support of malignant cell growth and viability as well as the mechanisms responsible for suppression of effector T-cell function. We will then determine whether intratumoral monocytes remain immunosuppressive in the presence of monoclonal antibodies targeting lymphoma cells.
3. Block suppressive monocyte formation to improve immunity and impact clinical outcome.
We will determine whether maturation of intratumoral monocytes suppresses their ability to support lymphoma cell growth and survival, reverses their immunosuppressive properties and increases ADCC. We will confirm these results in patients treated with β-glucan, rituximab and alemtuzumab in a UI/MC SPORE clinical trial.

PROJECT 2: In Situ Immunization Using Nanoparticles

Co-Principal Investigators: George J. Weiner, MD (Iowa), Brian K. Link, MD (Iowa), and Aliasger Salem, PhD (Iowa)
Co-Investigators: Alicia Olivier, D.V.M., PhD (Iowa) and Suresh Veeramani, D.V.M., PhD (Iowa)

Immunotherapy of lymphoma through in situ manipulation of an involved lymph node can allow for development of an active anti-lymphoma immune response without the need for ex vivo handling or immunization with a specific antigen.   A comprehensive and successful approach to in situ immunization in lymphoma would require effective presentation of antigen by the lymphoma cells or professional antigen-presenting cells, activation of lymphoma-specific T cells and suppression of the regulatory arm of the immune response to enhance development of a sustained anti-lymphoma T cell response.  The current proposal evaluating a novel approach to in situ immunization is based on scientific advances made possible through the UI/MC SPORE over the prior funding period.  The overall hypothesis is that in situ immunization with nanoparticles will allow for induction and maintenance of a robust anti-lymphoma immune response with acceptable toxicity.  To test this hypothesis, this project will assess the effect of intratumoral injection of nanoparticles (NPs) containing doxorubicin (dox) on lymphoma cells, the immune microenvironment, and the anti-lymphoma immune response in animal models and in a Phase I clinical trial in subjects with lymphoma.  It will then assess the effect of intratumoral injection of NPs containing both dox and the toll-like receptor 9 (TLR9) agonist CpG ODN (CpG dox NPs) in mice and humans.  Finally, it will assess how agents capable of maintaining the T cell response impact on the success of in situ immunization with NP.  Successful development of such an approach to in situ immunization would be of great significance as a way to treat lymphoma, and could be applicable to other cancers as well.

To assess this hypothesis, we will explore the following specific aims.

Specific Aim 1 – Assess the effect of in situ injection of nanoparticles (NPs) containing doxorubicin (dox) on lymphoma cells, the immune microenvironment, and the anti-lymphoma immune response.  
Aim 1A – Refine the structure of dox NP to optimize the concentration and release of dox.
Aim 1B – Assess the toxicity, anti-tumor activity, and immune effects of dox NPs in mouse models of lymphoma.
Aim 1C – Conduct a Phase I clinical trial of dox NPs in subjects with lymphoma, and evaluate for toxicity, the local effects of the injected dox NP on the treated node and the development of an anti-lymphoma immune response.

Specific Aim 2 – Assess the effect of in situ injection of NPs containing both dox and the TLR9 agonist CpG ODN (CpG dox NPs) on lymphoma cells, the immune microenvironment, and the anti-lymphoma immune response.
Aim 2A – Produce NPs containing dox and various concentrations of CpG ODN.
Aim 2B – Assess the toxicity, anti-tumor activity, and immune effects of CpG dox NPs in mouse models of lymphoma.
Aim 2C - Conduct a Phase I/II clinical trial of CpG dox NPs in subjects with lymphoma based on results of studies with the dox NPs.

Specific Aim 3 – Assess how agents capable of maintaining the T cell response impact on in situ immunization.
Aim 3A – Identify and incorporate the most effective agents that maintain T cell activation, block T cell checkpoints or block immunosuppressive cells into in situ immunization strategies. 
Aim 3B – Assess whether systemic or local blockade of T cell checkpoints or suppression of T regulatory cells enhances the efficacy of in situ immunization.

PROJECT 3: Targeting JAK2 Kinase in Lymphoma

Principal Investigators: Thomas E. Witzig, MD (Mayo) and Mamta Gupta, PhD (Mayo)

Recent advances in tumor biology have led to the identification of a variety of intracellular oncogenic pathways as potential targets for cancer therapy.  Specifically, many studies have found that activation of the JAK/STAT pathway promotes tumor cell proliferation and survival in various types of leukemia and lymphoma.  Our preliminary data demonstrate aberrantly activated JAK2 and STAT3 in more than 50% of diffuse large B-cell lymphoma patient samples.  In vitro inhibition of JAK2 with the novel JAK2 inhibitor TG101348 (TG) inhibited JAK2 and STAT3 phosphorylation and induced apoptosis in a variety of lymphoma cell lines and patient samples.  In this proposal the overall goal is to identify the molecular mechanisms underlying activation of the JAK/STAT pathway in lymphoma and to learn if inhibitors of this pathway can produce clinical benefit.  We have identified several novel missense mutations in JAK2 and STAT3 genes. In Aim 1 we will characterize the biological and therapeutic significance of these mutations with a site-directed mutagenesis approach. Suppressors of cytokine signaling (SOCS1) and protein tyrosine phosphatases (SHP1) are known key negative regulators of the JAK/STAT pathway.  Our preliminary data demonstrate silencing of SHP1 and SOCS1 genes in 33% and 86%, respectively, of DLBCL lymphoma samples.  In Aim 2, we will delineate the mechanisms of silencing and how this regulates JAK/STAT pathway activation. The JAK/STAT signaling pathway is utilized by a number of growth factors and cytokines.  We have identified increases in several JAK/STAT pathway-specific cytokines (IL-2, IL-6, IL-10 and EGF) in serum samples from patients with lymphoma compared to normal controls.  In vitro we found in lymphoma cells that JAK2 and STAT3 are rapidly activated in response to IL-10.   Aim 3 will investigate the role of signaling for these interleukins mediated through their receptors with a focus on IL-10.  This project is based on solid preliminary data demonstrating that the JAK/STAT pathway is a key mechanism for lymphoma growth and survival.  These data have guided the design of the phase II trial in Aim 4 that will test TG in patients with relapsed lymphoma.  Correlative research using patient samples pre- and post-therapy with JAK/STAT pathway inhibitors will increase our understanding of the mechanisms of how this pathway is regulated at the molecular and genetic level. These basic and clinical studies, working together, aim to offer a new therapeutic approach for patients with lymphoma.

The work is organized in 4 specific aims:

Aim 1:  To characterize the biological significance of novel mutations in the JAK/STAT pathway in lymphoma.
Aim 2:  To delineate the mechanism(s) of loss of negative regulation of the JAK/STAT pathway in lymphoma.
Aim 3:  Identify the role of JAK2 specific cytokine-mediated signaling in lymphoma.
Aim 4:  Determine the clinical activity of JAK inhibitors in a phase II trial.

This project is based on solid preliminary data demonstrating that the JAK/STAT pathway is a key mechanism for lymphoma growth and survival.  These data have guided the phase II trial design in Aim 4.  Aims 1-3 and the investigations using patient samples before and after therapy with JAK/STAT pathway inhibitors will increase our understanding of the mechanisms of how this pathway is regulated at the molecular and genetic level. These data will also guide the design of subsequent trials. These basic and clinical studies, working together, aim to offer a new therapeutic approach for patients with lymphoma.

PROJECT 4: Genetic Epidemiology and Function of Germline and Somatic Variants in DLBCL

Principal Investigators: James R. Cerhan, MD, PhD (Mayo) and Anne J. Novak, PhD (Mayo)
Co-investigator: Yan W. Asmann, PhD (Mayo)

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) subtype.  It is well established that there are driver somatic mutations in DLBCL etiology and prognosis, as well as a role for germline genetic susceptibility.  In fact, early results from candidate gene association studies have shown a promising role for common genetic variants in immune and apoptotic genes in NHL.  In the last funding cycle, we identified SNPs in BCL2L11 (BIM), CASP9, and APAF1 that were associated with increased risk in our SPORE case-control study.  Resequencing of these genes in the tumors of 40 DLBCL patients identified novel genomic alterations, and in Aim 1 we propose to characterize the etiologic and therapeutic significance of these novel mutations with laboratory-based studies.  In Aim 2 and 3, we propose a comprehensive and agnostic strategy that integrates both somatic and germline genetics in order to identify additional novel risk variants.  We will do this by leveraging our SPORE’s involvement in the large International Lymphoma Epidemiology (InterLymph) consortium genome-wide association study (GWAS) of DLBCL (>3000 cases and 10,000 controls) and our whole-exome next generation sequencing (NGS) study of paired tumor and germline DLBCL cases (N=77).  The GWAS is powered to identify common variants, and the NGS study will allow us to identify lower frequency variants, defined here as 0.5% to 5%.  Using a multistage design in Aim 2, we propose to identify novel germline low-frequency variants associated with risk of developing DLBCL, and in Aim 3, we identify and validate somatically acquired driver mutations in genes critical to DLBCL pathogenesis.  In exploratory analyses, we will evaluate pathways and the role of these variants in DLBCL prognosis.  This proposal utilizes the unique resources of our SPORE, the InterLymph GWAS, and our DLBCL whole-exome NGS project.  We have an outstanding team with a strong track record of interdisciplinary genetics work in lymphoma and have demonstrated the ability to integrate genetic epidemiology with lab-based functional work.  Our study builds on several novel findings from the prior study period, but also expands to fill an important need in the genetic epidemiology of DLBCL as the first comprehensive study of low frequency germline variants in risk.  Low frequency variants, either individually or cumulatively across a gene, and in combination with common variants, are likely to inform etiologic pathways and clinical risk assessment.  Furthermore, we will identify novel driver mutations in genes and pathways from DLBCL tumors that can inform tumor biology and identify novel treatment targets.  In summary, as the first comprehensive study of both germline and somatic genetic variants in DLBCL, we are likely to provide new and unexpected insights into lymphomagenesis, which can then be exploited clinically for risk assessment, prognostic stratification and identification of new treatment targets.

Our specific aims:

Aim 1.  To characterize the therapeutic significance of novel mutations in genes that regulate the intrinsic apoptosis pathway          
Hypothesis:  Mutations in genes identified through our resequencing work (CASP9, BCL2L11, and APAF1) will have functional impacts in the apoptosis pathway.
Approach:  We will assess the functional significance of 4 novel mutations in CASP9, BCL2L11, and APAF1 by determining if there is an inherent defect in the intrinsic apoptosis pathway and apoptosome formation in cells that express mutant forms of these proteins.  Similarly, we will follow-up in the laboratory the most promising variants identified in Aims 2 and 3.

Aim 2.  To identify novel germline low-frequency variants associated with DLBCL risk
Hypothesis:  Germline variants of low frequency will be associated with risk of developing DLBCL.
Approach:  In stage 1, we will use whole-exome sequencing data from 77 DLBCL cases and >500 controls from the 1000 Genomes Project to impute genotypes on 525 cases and 900 controls with GWAS data, and then test for association in the full case-control set.  We will next conduct a technical validation on the top 1536 significant imputed genotypes using the samples from stage 1.  In stage 2, we will conduct an in silico validation of all technically validated SNPs from stage 1 in 2,500 cases and 10,000 controls from the InterLymph GWAS.  In stage 3, we will resequence the top 20 gene regions from stage 2 in 500 cases and 500 controls and test for association.  In secondary analyses, we will test whether these validated variants impact prognosis (event-free and overall survival). 

Aim 3.  To identify somatically acquired driver mutations in genes critical to DLBCL pathogenesis
Hypothesis:  Somatically acquired mutations will be associated with risk of developing DLBCL.
Approach:  Using exome data from 77 paired tumor-normal DLBCL samples, we will identify novel and likely functional driver mutations by filtering against the paired germline DNA and bioinformatics analysis.  The top 20 genes will be sequenced to validate regions of interest in 500 DLBCL cases with paraffin tumor tissue in the SPORE.  In secondary analyses, we will also test whether these mutations impact prognosis. 

Core Areas

CORE A: Administration
Director: George J. Weiner, MD (Iowa)
Co-Director: Thomas Witzig, MD (Mayo)

The overall goal of the University of Iowa/Mayo Clinic Lymphoma SPORE (UI/MC SPORE) Administration Core is to stimulate research in lymphoma and to expedite the translation of discoveries into new and better methods of prevention, detection, and treatment of lymphoma. 

CORE B: Biospecimens
Director: Ahmet Dogan, MD, PhD (Mayo)
Co-Director: Sergei Syrbu, MD (Iowa)

The Iowa-Mayo SPORE Biospecimens Shared Resource provides a coordinated, centralized, and dedicated core for the procurement, processing and annotation of biospecimens from lymphoma patients and patients with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL).  The goal of the Biospecimens Core is to procure a variety of biologic specimens on all patients involved in UI/MC SPORE protocols and all newly diagnosed lymphoma patients seen at the University of Iowa and the Mayo Clinic Rochester, who are enrolled into the Molecular Epidemiology Resource (MER).

CORE C: Biostatistics and Bioinformatics
Director: Brian J. Smith, PhD (Iowa)
Co-Directors: Terry Braun, PhD (Iowa) and Susan Slager, PhD (Mayo)

The Biostatistics and Bioinformatics Core (Biostats Core) provides collaborative statistical and informatics support to the UI/MC SPORE projects, developmental projects, and other cores.  The comprehensive nature of the Core, which will have activities at both Iowa and Mayo, assures each SPORE investigator access to expertise that includes development of study designs and analysis plans, state of the art data analysis and interpretation, data management resources, and abstract and manuscript preparation. 

CORE D: Clinical Research
Director: Thomas Habermann, MD (Mayo)
Co-Director: Brian Link, MD (Iowa)

The UI/MC SPORE Clinical Research Core (CRC) has as its primary goal to be the direct translational link between research projects and clinical research emanating from these projects.  The specific aims of the CRC are to: 1) coordinate and perform SPORE clinical trials protocols; 2) manage SPORE observational epidemiology protocols and partner with the Molecular Epidemiology Resource (MER).  The CRC provides a critical link between clinical research and the specific projects, cores, and developmental research

DEVELOPMENTAL RESEARCH CORE
Director: Steven R. Lentz, MD, PhD (Iowa)
Co-Director: Stephen J. Russell, MD, PhD (Mayo)

The goal of the University of Iowa / Mayo Clinic Lymphoma SPORE (UI/MC SPORE) Developmental Research Program is to support innovative, scientifically sound projects that investigate a question pertaining to translational lymphoma research. 

CAREER DEVELOPMENT PROGRAM
Co-Director: Scott H. Kaufmann, MD, PhD (Mayo)

The goal of the University of Iowa/Mayo Clinic Lymphoma SPORE (UI/MC SPORE) Career Development Program (CDP) is to develop knowledgeable, well-trained scientists experienced in multidisciplinary research for future studies in translational lymphoma research.