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We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma. [ABSTRACT FROM AUTHOR]

CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Primarily expressed by activated CD4+ T cells, CD40L binds to CD40 on antigen presenting cells (APCs), thereby inducing APC activation. APCs, in turn, prime cytotoxic T lymphocytes (CTLs). Here, two tumor-associated antigen (TAA) animal models, p53-based and GP100-based, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8+ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another construct encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses as well as the potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8+ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the functional avidity of antigen-specific CD8+ T cells. Thus, our data show that vaccination with TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of better functioning antigen-specific CD8+ T cells. Our study serves as an important foundation for future clinical translation. [ABSTRACT FROM AUTHOR]

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8+ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8+ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8+ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8+ T cells. [ABSTRACT FROM AUTHOR]

CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Primarily expressed by activated CD4+ T cells, CD40L binds to CD40 on antigen presenting cells (APCs), thereby inducing APC activation. APCs, in turn, prime cytotoxic T lymphocytes (CTLs). Here, two tumor-associated antigen (TAA) animal models, p53-based and GP100-based, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8+ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another construct encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses as well as the potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8+ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the functional avidity of antigen-specific CD8+ T cells. Thus, our data show that vaccination with TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of better functioning antigen-specific CD8+ T cells. Our study serves as an important foundation for future clinical translation.

Hepatitis B virus (HBV) associated end-stage liver diseases are the leading causes of liver transplantation (LT) in Taiwan. Relapse of hepatitis B occurs after LT, raising the risk of graft failure and reducing patient survival. Although several oral antiviral agents have been approved for anti-HBV treatment, lamivudine (LAM) remained to be the most widely used preventive regimen in Taiwan. While several clinical predictors have been identified for hepatitis B relapse, the predictive roles of the histopathological characteristics in liver explants as well as the genotypic features of the viruses in pre-LT serum samples have not been assessed. Between September 2002 and August 2009, 150 consecutive hepatitis B surface antigen (HBsAg) positive patients undergoing LT were included for outcome analysis following assessment of the clinicopathological and virological factors prior to LT. Kaplan-Meier analyses discovered that pre-operative LAM treatment ≤3 months; membranous distribution and higher expression of tissue HBsAg in liver explants; preoperative viral load ≥10(6) copies/ml; and presence of large fragment (>100 base pairs) pre-S deletion (LFpreSDel) correlated significantly with hepatitis B relapse. Multivariate Cox regression analysis showed that the presence of LFpreSDel (P = 0.001) and viral load ≥10(6) copies/mL (P = 0.023) were independent predictors for hepatitis B relapse. In conclusion, besides high viral load, LFpreSDel mutation is an important independent predictor for hepatitis B relapse after LT. More aggressive preventive strategies should be applied for patients carrying these risk factors.