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1.  BRAF Inhibition Increases Tumor Infiltration by T cells and Enhances the Anti-tumor Activity of Adoptive Immunotherapy in Mice 
Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors.
Experimental Design
BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T-cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of vascular endothelial growth factor (VEGF) was determined by enzyme-linked immunosorbent assay, protein array and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples.
We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the pre-clinical murine model.
These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell based immunotherapy for the treatment of melanoma patients.
PMCID: PMC4120472  PMID: 23204132
Melanoma; BRAF inhibitor; Adoptive cell transfer (ACT); immunotherapy; VEGF
2.  Broad Cross-Presentation of the Hematopoietically-Derived PR1 Antigen on Solid Tumors Leads to Susceptibility to PR1-Targeted Immunotherapy 
PR1 is a human leukocyte antigen (HLA)-A2 restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived antigen presenting cells, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific cytotoxic T lymphocytes (PR1-CTL) and the anti-PR1/HLA-A2 antibody 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an antigen become susceptible to therapies that target cross-presented antigens and suggest PR1 as a broadly expressed tumor antigen.
PMCID: PMC3504175  PMID: 23105141
5.  Adoptive T-cell Therapy Using Autologous Tumor-infiltrating Lymphocytes for Metastatic Melanoma: Current Status and Future Outlook 
Cancer Journal (Sudbury, Mass.)  2012;18(2):160-175.
Immunotherapy using autologous T-cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL), T-cells transduced with high-affinity T-cell receptors (TCR) against major melanosomal tumor antigens, and T cells transduced with chimeric antigen receptors (CAR) composed of hybrid immunoglobulin light chains with endo-domains of T-cell signaling molecules. Among these and other options for T-cell therapy, TIL together with high-dose IL-2 has had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined as well as un-defined tumors antigens against all possible MHC, rather than the single specificity and limited MHC coverage of the newer TCR and CAR transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trials are needed in an attempt to gain regulatory approval of TIL as standard-of-care. Second, improvements in how we expand TIL for therapy are needed, that minimize the time the T-cells are in culture and improve the memory and effector characteristics of the T cells for longer persistence and enhanced anti-tumor activity in vivo. Third, there is a critical need to identify surrogate and predictive biomarkers in order to better select suitable patients for TIL therapy in order to improve response rate and duration. Overall, the outlook for TIL therapy for melanoma is very bright. We predict that TIL will indeed emerge to become an approved treatment in the upcoming years through pivotal clinical trials. Moreover, new approaches combining TIL with targeted signaling pathway drugs, such as mutant B-RAF inhibitors, and synergistic immunomodulatory interventions enhancing T-cell costimulation and preventing negative regulation, should further increase therapeutic efficacy and durable complete response rates.
PMCID: PMC3315690  PMID: 22453018
6.  AP2β nucleolar localization predicts poor survival after stage I non–small cell lung cancer resection 
The Annals of Thoracic Surgery  2011;92(3):1044-1050.
Activating enhancer-binding protein-2β (AP2β) is a transcription factor involved in apoptosis. The purpose of the current study was to assess the cellular location and level of AP2β in Non-Small Cell Lung Cancer (NSCLC) and normal lung tissue and investigate whether the level and localization of AP2β expression is predictive of overall survival in patients with stage I NSCLC.
We performed immunohistochemical analysis of tissue microarrays (TMAs) prepared from stage I NSCLC specimens with adjacent normal lung tissue from two independent sets of patients who underwent lung resection with curative intent at our institution. AP2β intensity was assessed in TMAs, and AP2β staining patterns were classified as either diffuseor nucleolar in the TMAs. AP2β intensity and localization were analyzed for correlation with patients' survival.
Immunohistochemical analysis of TMAs showed that the intensity of AP2β immunohistochemical staining did not correlate with overall survival. When location of AP2β was analyzed in TMAs, all of the normal lung tissue had diffuse pattern of AP2β. In the first set of NSCLC, patients with nucleolar pattern had a significantly lower 5-year survival rate than patients with diffuse pattern (67% vs. 100%; P = 0.004); this finding was confirmed in the second set (64% vs. 91%; P = 0.02). Multivariate analysis revealed that nucleolar pattern was an independent predictor of poor overall survival in both sets.
The AP2β which is located in the nucleoplasm in normal lung tissue is found in either nucleoplasm or nucleoli in NSCLC. The patients with AP2β in the nucleoli had poor survival compared to patients with AP2β in the cytoplasm.
PMCID: PMC3272351  PMID: 21871297
Lung cancer biology; survival analysis
7.  Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells 
Nucleic Acids Research  2008;36(15):5021-5032.
Neural cells undergo glutamate-induced apoptosis in ischaemic brain tissue, in which prostate apoptosis response-4 gene (Par-4) is involved. Human-bone mesenchymal stem cells can be utilized as an effective therapy for ischemic brain injury. In this study, we found that glutamate could induce apoptosis in human-bone mesenchymal stem cells, accompanied by increased expression of Par-4 gene and Smac release from mitochondria. Repressing Par-4 expression attenuated the glutamate-induced apoptosis. Both Par-4 protein and E2F1 protein could bind to E2F1-binding BS3 site on Smac promoter and participated in the formation of a proteins-DNA complex. Moreover, in the complex, E2F1, not Par-4, was found to be directly bound to the Smac promoter, suggesting that Par-4 exerted indirectly its transcriptional control on the Smac gene though interacting with E2F1. Expression of full-length Par-4 in human-bone mesenchymal cells resulted in increased activity of the Smac promoter. In addition, the indirect transcripional regulation of Par-4 on Smac depended on its COOH terminus-mediated interaction between Par-4 and E2F1. We conclude that the formation of proteins–DNA complex, containing Par-4 protein, E2F1 protein and the Smac promoter, contributes to the pro-apoptotic effect on glutamate-treated human-bone mesenchymal stem cells.
PMCID: PMC2528162  PMID: 18660514

Results 1-7 (7)