Kai Fu, M.D., Ph.D.
My research involves several aspects of hematopathology, with a strong emphasis on understanding the pathogenesis of aggressive B-cell lymphomas and developing target therapy based on their genetic alterations. Aggressive B-cell lymphomas are clinically and pathologically heterogeneous entities that mainly consist of diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL) and mantle cell lymphoma (MCL). Over the last few decades, R-CHOP based regimens have tremendously improved the outcome of these diseases. However, many patients still respond poorly to therapy, especially those with MCL or relapsed DLBCL. Moreover, toxicity and secondary cancer risk after chemotherapy are significant, particularly in patients treated with high-dose intensive regimens1,2. Therefore improvements in therapeutic regimens and strategies are urgently needed for the treatment of aggressive B-cell lymphomas.
One of the current studies in our lab is focusing on MCL. MCL is an aggressive non-Hodgkin's lymphoma which brings together the worst characteristics of both high-grade and low-grade lymphomas. The course is usually aggressive, and the disease is rarely curable. MCL is characterized by the chromosome translocation t(11;14)(q13;q32), which results in overexpression of cyclin D1. This translocation alone is not sufficient enough to result in lymphoma, and additional genetic alterations are necessary. Studies have shown that secondary genomic alterations are frequently detected in MCL, of which chromosome 13q31-q32 gain/amplification is one of the most frequent. Amplification at chromosome 13q31-q32 targets a microRNA cluster, miR-17~92, and overexpression of the cluster accelerates MYC-induced lymphomagenesis in mice. Based on gene expression profiling data, we found high-level expression of C13orf25, the primary transcript from which these microRNAs are processed, was associated with poor survival in MCL patients (p=0.0027). Enforced overexpression of miR-17~92 in retrovirally transduced MCL cell lines increased the phosphorylation of AKT and its downstream targets and reduced chemotherapy-induced apoptosis. PTEN was down-modulated in these cells, and our data suggest PTEN is a direct target of miR-17~92 in MCL. Furthermore, we discovered protein phosphatase PHLPP2, a negative regulator of the PI3K/AKT pathway, is also a direct target of miR-17~92. Its downregulation may act synergistically with PTEN downregulation to promote activation of the PI3K/AKT pathway in MCL. Also, we demonstrated that BIM is a direct target of the miR-17~92. In summary, overexpression of miR-17~92 down-modulates multiple proteins involved in PI3K/AKT signaling and apoptosis, and these effects collaboratively enhance resistance to chemotherapy in MCL cells.
More recently, our studies of miR-17-92 functions had extended to DLBCL, especially in the activated B-cell subtype of DLBCL (ABC-DLBCL). ABC-DLBCL is an aggressive lymphoma characterized by constitutive NF-κB activation, but whether oncogenic microRNAs contribute to this activation remains unclear. Herein, we reported the expression of MIR17HG (oncogenic miR-17~92 primary transcript) was positively correlated with NF-κB activation and expression of NF-κB downstream transcriptional target genes in ABC-DLBCL. Over-expression of miR-17~92 promoted ABC-DLBCL cell growth accelerated cell G1/G0 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, we found miR-17~92 promoted NF-κB activation through directly targeting multiple negative regulators of NF-κB pathway, including TNFAIP3, CYLD, and Rnf11, leading to increasing the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex. We further demonstrated miR-17~92 selectively activated IκB-α and NF-κB p65 phosphorylation, but not NF-κB p52/p100 phosphorylation. Moreover, higher miR-17~92 expression was associated with poorer clinical outcome in ABC-DLBCL patients. Our findings uncovered an innovative function of miR-17~92 and demonstrated the molecular mechanism in which miR-17~92 selectively activated the canonical NF-κB signaling in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy in ABC-DLBCL patients.
Second line of current studies in our lab is focusing on mTOR pathway signaling in DLBCL. Mechanistic target of rapamycin complex 1 (mTORC1) is a central integrator of nutrient and growth factor inputs that control cell growth in all eukaryotes. Rapamycin and its analogs (rapalogs) have been approved for the treatment of relapsed MCL. A large proportion of aggressive B-cell lymphoma patients, however, respond poorly to rapalogs. The second generation of mTOR inhibitors function as ATP-competitive inhibitors (TORi), directly targeting the mTOR catalytic site. TORis have been proven to be more effective than rapalogs in cancer treatment. However, the mechanism underlying the cytotoxic effect of TORis in aggressive B-cell lymphomas remains unclear. In our study, we demonstrated that TORi-induced apoptosis is predominantly dependent on loss of mTORC1-mediated 4EBP phosphorylation. Knocking out Rictor, a key component of mTORC2 or inhibiting p70S6K has little effect on TORi-induced apoptosis. In contrast, that increasing the EIF4E:4EBP ratio by either overexpressing EIF4E or knocking out 4EBP1/2 prevented lymphoma cells from TORi-induced cytotoxicity. Furthermore, down-regulation of MCL1 expression plays an important role in TORi-induced apoptosis whereas BCL2, in cells with high expression, confers resistance to TORi treatment. In addition, we found BH3 profiling to be effective in predicting the cytotoxicity of the TORi in lymphoma cells. Also, in combination with pro-apoptotic drugs, especially BCL2 inhibitors, the TORi exerted powerful anti-tumor effects both in vitro and in vivo. Taken together, this study provides rational support for TORi treatment in aggressive B-cell lymphoma and identified a mean to predict its effectiveness clinically.
Timothy Greiner, M.D.
My research involves the study of the etiology and progression of lymphoma by examining the mutational spectra of oncogenes and tumor suppresser genes. My laboratory has been involved in characterizing mutations in ATM, p53 and bcl-6. We are correlating mRNA expression patterns in lymphoma with patient survival, mutation status, and morphological subtypes. We are currently examining the methylation status of DNA in diffuse large cell and mantle cell lymphomas. The emphasis on molecular epidemiology involves the identification of EBV subtypes, the gene expression, and the molecular abnormalities in post-transplant lymphoproliferative disorders.
Other Specialized Cancer Research
Julia Bridge, M.D.
My research involves several aspects of genetics with special emphasis on cancer genetics of bone and soft tissue tumors. Over the last two decades, we have identified a number of tumor-specific chromosomal abnormalities for both benign and malignant bone and soft tissue tumors. These abnormalities are important diagnostically and prognostically, as well as for defining treatment strategies. These data have also contributed significantly to our understanding of the histopathogenesis of many of these neoplasms. Following identification of the anomalies cytogenetically, the key chromosomal breakpoint locations and underlying involved genes are further characterized with FISH positional cloning analysis and molecular techniques. Moreover, we custom design probe sets from these defined breakpoints for molecular cytogenetic assays and primer sets for RT-PCR analysis as additional diagnostic adjuncts.
Samuel Cohen, M.D., Ph.D.
My research involves several aspects of carcinogenesis, with an emphasis on urinary bladder as a model system in rodents and extrapolation between rodent models and human diseases. We have postulated that agents increase cancer risk by either directly interacting with DNA or increasing cell proliferation in appropriate target cells, allowing for more opportunity for spontaneous mutations to occur during DNA replication. Genotoxic chemicals, such as aromatic amines, nitrofurans, nitrosamines, and acrolein, require metabolic activation, DNA adduct formation, and mutagenesis. Numerous nongenotoxic chemicals have been identified, enhancing carcinogenesis by increasing urothelial cell proliferation, including arsenic, sodium salts, amino acids, calculus-forming chemicals, and phenolic chemicals. Increased proliferation occurs either by direct mitogenesis, such as by high doses of Propoxur, or by cellular toxicity and consequent regenerative hyperplasia, such as occurs with formation of calculi by chemicals like uracil, or by processes involving more subtle cytotoxicity, such as following high doses of sodium saccharin or arsenic. We successfully demonstrated the mode of action of sodium saccharin and related sodium salts. Based on our investigations, it is unlikely that these pose a carcinogenic hazard to humans. Studies with these salts involve various aspects of toxicology, basic chemistry, cell kinetics, electron microscopy, pathology, in vivo bioassays, renal physiology and molecular biology. These findings led to the delisting of saccharin from the National Toxicology Program’s List of Carcinogens.
PPARγ and dual PPAR agonists, developed as anti-diabetic and antilipidemic drugs, frequently produce bladder cancer in rats and hemangiosarcomas in mice. We are investigating the mechanisms involved for both of these targets, already showing that for the bladder the mechanism involves indirect formation of calcium-containing urinary solids that produce urothelial cytotoxicity and regeneration. The mechanism does not occur in humans. A variety of other nongenotoxic bladder carcinogens are being investigated in animal and in vitro systems. More recently, we have investigated the bladder carcinogenicity of dimethylarsinic acid (DMAV), an organic arsenical, and inorganic arsenic in rodent models and in cell culture. DMA and its metabolites are non-DNA reactive, but orally consumed DMA produces urothelial necrosis with consequent regeneration. Urothelial cytotoxicity is produced in vitro by trivalent arsenicals at concentrations less then 1μM. Urinary levels of DMAIII following carcinogenic doses of DMAV suggest that it may be critical to the urothelial toxicity induced in vivo. The relationship to growth factors, receptors, and cell cycle control mechanisms is being evaluated. A similar process is being evaluated for inorganic arsenic - a known human bladder carcinogen.
Leah Cook, Ph.D.
My lab’s research is focused on investigation of cellular interactions within the tumor bone microenvironment that contribute to tumor progression, with a specific focus on immune cell-tumor interactions. Within bone, metastatic cancer cells highjack the normal couple process of bone remodeling, resulting in excess bone degradation and subsequent release of growth factors that promote tumor growth. Additionally, cancer cells progress and mediate bone turnover through molecular and cellular interactions with the surrounding bone stroma. My lab is currently focusing on identifying the importance of neutrophils and/or granulocytic MDSCs in prostate cancer progression and cancer-induced bone disease using a combination of transcriptome and proteomic profiling of patient samples and mouse in vivo models of bone metastasis. My goal is to identify novel immunotherapeutic targets for treating and curing bone metastatic cancers.
Rakesh Singh, Ph.D.
The overall goal of our research is to define the mechanism(s) that regulate the process of metastasis. We hypothesize that metastasis is a highly selective process that is regulated by interrelated mechanisms whose outcome is dependent upon both the intrinsic properties of tumor cells and the host response. Using human tumors xenografted in nude mice and murine tumor models, these studies have demonstrated the role of host-derived factors in regulating angiogenesis, resulting in site-specific expression of angiogenic factors, including basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and metastasis. Further characterization of the cellular and molecular mechanisms underlying these processes are currently ongoing in our laboratory. In addition, we are investigating the mechanism(s) of organ-specific metastasis. Recent reports suggest specific organ tissues carry unique marker molecules accessible to circulating cells. We have identified the molecule(s) expressed in organ tissues, which might be important to organ-specific metastasis using phage display libraries. Further characterization of organ-specific signature molecules will be useful in designing novel, highly targeted therapeutic approaches against organ-specific metastasis. In addition, our current research activities have also been focused on designing the strategies for inhibiting tumor-induced angiogenesis and activating anti-tumor immunity with the potential for synergizing the outcome of conventional therapeutic approaches, as well as understanding the role of tumor-stromal interaction in tumor progression and metastasis.
James Talmadge, Ph.D.
The Laboratory of Transplantation Immunology is focused on the process of metastasis, and interventional strategies that augment the host response against primary and systemic disease. We have demonstrated that the process of metastasis is selective, controlled by intrinsic properties of the tumor, and the host response against the tumor. Our research emphasizes molecular and cellular immunology, vaccines, transplantation, gene therapy, hematology and oncology. The primary focus is on host-immune interactions and the regulation of tumor progression, metastasis and therapeutic intervention with the goal of developing preclinical, clinical and surrogate-based hypotheses. Clinical and translational research includes studies focus on the tumor microenvironment including myeloid derived suppressor cells (MDSCs), dendritic cells (DCs) and T-cells; and gene therapeutic vaccines with a focus on breast cancer and melanoma. This includes clinical studies with Dr. Ken Cowan and Dr. Elizabeth Reed at UNMC, Dr. Dmitry Gabrilovich at Moffitt Cancer Center and collaborations with Intrexon, Inc. Early-stage studies have also focused on immune recovery following stem cell transplantation, at present, primarily in collaboration with Dr. Greg Bociek, Internal Medicine, as regards non-myeloablative, allogeneic, stem cell transplantation.
Basic/translational research studies are focused on host-tumor interactions during tumor progression, metastasis and cytoreductive therapy. These studies include a program in molecular therapeutics that is focused on cyclooxygenase (COX)-2 inhibitors, tyrosine kinase inhibitors, all-trans-retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) inhibition and their regulation of immune intervention. Our goal is to overcome tumor and iatrogenic suppression of DC function and T-cell responses to vaccines that is associated with MDSCs. As part of these studies, we have identified a critical regulatory role for a unique cellular phenotype strongly associated with tumor-associated immunosuppression that when removed slows tumor growth and prolongs survival. Studies into the mechanism of immunosuppression suggest a critical role for inducible nitric oxide synthase and arginase as a local/regional mediator of T-cell suppression. We are also very active (along with many others) in the development of a Biological Production Facility, which utilizes good manufacturing practices (GMPs) required for many of the above clinical studies. This includes the development of new vaccine manufacturing strategies and the validation of aspects of manufacture, such as release assays, freezing protocols, etc.