Genetic Variants of Human Granzyme B Predict Transplant Outcomes after HLA Matched Unrelated Bone Marrow Transplantation for Myeloid Malignancies

Serine protease granzyme B plays important roles in infections, autoimmunity, transplant rejection, and antitumor immunity. A triple-mutated granzyme B variant that encodes three amino substitutions (Q48R, P88A, and Y245H) has been reported to have altered biological functions. In the polymorphism rs8192917 (2364A>G), the A and G alleles represent wild type QPY and RAH mutant variants, respectively. In this study, we analyzed the impact of granzyme B polymorphisms on transplant outcomes in recipients undergoing unrelated HLA-fully matched T-cell-replete bone marrow transplantation (BMT) through the Japan Donor Marrow Program. The granzyme B genotypes were retrospectively analyzed in a cohort of 613 pairs of recipients with hematological malignancies and their unrelated donors. In patients with myeloid malignancies consisting of acute myeloid leukemia and myelodysplastic syndrome, the donor G/G or A/G genotype was associated with improved overall survival (OS; adjusted hazard ratio [HR], 0.60; 95% confidence interval [CI], 0.41–0.89; P = 0.01) as well as transplant related mortality (TRM; adjusted HR, 0.48; 95% CI, 0.27–0.86, P = 0.01). The recipient G/G or A/G genotype was associated with a better OS (adjusted HR, 0.68; 95% CI, 0.47–0.99; P = 0.05) and a trend toward a reduced TRM (adjusted HR, 0.61; 95% CI, 0.35–1.06; P = 0.08). Granzyme B polymorphism did not have any effect on the transplant outcomes in patients with lymphoid malignancies consisting of acute lymphoid leukemia and malignant lymphoma. These data suggest that there is an association between the granzyme B genotype and better clinical outcomes in patients with myeloid malignancies after unrelated BMT

Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F

BACKGROUND: Despite significant progress in diagnostics and therapeutics, over fifty thousand patients die from colorectal cancer annually. Hence there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. METHODS: Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase(LDH) release and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F dependent genes, E2F1-Rb binding and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. RESULTS: Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically we demonstrate that at low concentrations, triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. CONCLUSION: Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models

Engineered Versions of Granzyme B and Angiogenin Overcome Intrinsic Resistance to Apoptosis Mediated by Human Cytolytic Fusion Proteins

The use of therapies based on antibody fusion proteins for the selective elimination of tumor cells has increased markedly over the last two decades because the severe side effects associated with conventional chemotherapy and radiotherapy are reduced or even eliminated. However, the initial development of immunotoxins suffered from a number of drawbacks such as nonspecific cytotoxicity and the induction of immune responses because the components were non-human in origin. The most recent iteration of this approach is a new class of targeted human cytolytic fusion proteins (hCFPs) comprising a tumor-specific targeting component such as a human antibody fragment fused to a human effector domain with pro-apoptotic activity. Certain tumors resist the activity of hCFPs by upregulating the intracellular expression of native inhibitors, which rapidly bind and inactivate the human effector domains. Higher doses of the hCFPs are, therefore, required to improve therapeutic efficacy. To circumvent these inhibitory processes, novel isoforms of the enzymes granzyme B and angiogenin have been designed to increase their intrinsic activity and reduce their interactions with native inhibitors resulting in more potent hCFPs that can be applied at lower doses. This chapter summarizes the basic scientific knowledge that can facilitate the rational development of human enzymes with novel and beneficial characteristics, including the ability to avoid neutralization by native inhibitors