Richard Dondero

The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice

In both type 1 and type 2 diabetes, pancreatic islet dysfunction results in part from cytokine-mediated inflammation. The ubiquitous eukaryotic translation initiation factor 5A (eIF5A), which is the only protein to contain the amino acid hypusine, contributes to the production of proinflammatory cytokines. We therefore investigated whether eIF5A participates in the inflammatory cascade leading to islet dysfunction during the development of diabetes. As described herein, we found that eIF5A regulates iNOS levels and that eIF5A depletion as well as the inhibition of hypusination protects against glucose intolerance in inflammatory mouse models of diabetes. We observed that following knockdown of eIF5A expression, mice were resistant to beta cell loss and the development of hyperglycemia in the low-dose streptozotocin model of diabetes. The depletion of eIF5A led to impaired translation of iNOS-encoding mRNA within the islet. A role for the hypusine residue of eIF5A in islet inflammatory responses was suggested by the observation that inhibition of hypusine synthesis reduced translation of iNOS-encoding mRNA in rodent beta cells and human islets and protected mice against the development of glucose intolerance the low-dose streptozotocin model of diabetes. Further analysis revealed that hypusine is required in part for nuclear export of iNOS-encoding mRNA, a process that involved the export protein exportin1. These observations identify the hypusine modification of eIF5A as a potential therapeutic target for preserving islet function under inflammatory conditions.

Eukaryotic Translation Initiation Factor 5A Small Interference RNA-Liposome Complexes Reduce Inflammation and Increase Survival in Murine Models of Severe Sepsis and Acute Lung Injury

BACKGROUND Many novel therapeutics have failed to reduce all-cause mortality associated with severe sepsis. Eukaryotic translation initiation factor 5A (eIF5A) is a regulator of apoptosis as well as inflammatory cell activation, making it a potential target for sepsis therapy. METHODS In a murine model of severe sepsis, mice were intraperitoneally challenged with lipopolysaccharide (LPS). Mice were treated both before and after LPS challenge with liposome complexes containing either an eIF5A-specific or control small interference RNA (siRNA), and both survival and serum concentrations of inflammatory cytokines were monitored. The ability of eIF5A siRNA to reduce inflammatory cytokines was also tested in a model of acute lung injury established by intranasal administration of LPS to mice. RESULTS There was a statistically significant increase in the rate of survival for mice intraperitoneally challenged with LPS that received eIF5A siRNA, compared with that noted for mice that received control siRNA (71% vs. 5%; P< .001), as well as a reduction in cytokine expression in serum. Concentrations of proinflammatory cytokines were also reduced in the lung homogenates and serum of mice that were intranasally challenged with LPS and received eIF5A siRNA (P< or = .05). CONCLUSIONS eIF5A siRNA-liposome complexes reduced inflammation and contributed to increased survival in a model of severe sepsis, decreased inflammation in a model of acute lung injury, and should be considered for clinical use.

SNS01-T Modulation of eIF5A Inhibits B-cell Cancer Progression and Synergizes With Bortezomib and Lenalidomide

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA–mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.

Modulation of eIF5A Expression Using SNS01 Nanoparticles Inhibits NF-κB Activity and Tumor Growth in Murine Models of Multiple Myeloma

Despite recent advances in the first-line treatment of multiple myeloma, almost all patients eventually experience relapse with drug-resistant disease. New therapeutic modalities are needed, and to this end, SNS01, a therapeutic nanoparticle, is being investigated for treatment of multiple myeloma. The antitumoral activity of SNS01 is based upon modulation of eukaryotic translation initiation factor 5A (eIF5A), a highly conserved protein that is involved in many cellular processes including proliferation, apoptosis, differentiation and inflammation. eIF5A is regulated by post-translational hypusine modification, and overexpression of hypusination-resistant mutants of eIF5A induces apoptosis in many types of cancer cells. SNS01 is a polyethylenimine (PEI)-based nanoparticle that contains both a B-cell-specific expression plasmid expressing a non-hypusinable mutant of eIF5A and a small interfering RNA (siRNA) which depletes endogenous hypusinated eIF5A. Reducing hypusine-modified eIF5A levels was found to inhibit phosphorylation and activity of ERK MAPK and nuclear factor-κB (NF-κB), and thus sensitize myeloma cells to apoptosis resulting from transfection of a plasmid expressing eIF5A(K50R). SNS01 exhibited significant antitumoral activity in both KAS-6/1 (95% inhibition; P < 0.05) and RPMI 8226 (59% inhibition; P < 0.05) multiple myeloma xenograft models following systemic administration. These results highlight the potential of using this approach as a new therapeutic strategy for multiple myeloma.

Abstract A196: Characterization of stability and biological activity of the cancer gene therapy biologic SNS01 following storage at ambient and freezing temperatures

Introduction: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post‐translational addition of a hypusine residue. Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identified as markers of neoplastic growth and metastasis. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro‐apoptotic and thus functionally distinct from hypusine‐modified eIF5A. In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and overexpression of a non‐hypusinable mutant of eIF5A potently induces apoptosis in multiple cancer cell types. SNS01 is a nanoparticle specifically designed for the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B‐cell‐specific (B29) promoter driving expression of a pro‐apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI). Methods: Ethidium‐bromide agarose gel electrophoresis was used to detect heparin‐mediated release of nucleic acids from nanoparticles. Dynamic light scattering (DLS) was used to assess the short‐term stability of SNS01 by monitoring changes in size distribution, polydispersity, and zeta potential. In vitro biological activity of SNS01 was assessed using RT‐qPCR to measure eIF5A and eIF5AK50R transgene expression as well as FACS detection of AnnexinV‐FITC labeled apoptotic cells. Anti‐tumoral activity of SNS01 was evaluated using a subcutaneous human myeloma model (KAS‐6/1 cells) in SCID mice with twice‐weekly intra‐venous injections of SNS01. Results: SNS01 nanoparticles were found to be relatively stable for as long as 48 hours at room temperature with no significant loss in nucleic acid integrity. The nanoparticles had an average diameter of 146.2 nm, a polydispersity of 0.287 and an average zeta potential of 35.3+0.7 mV. These parameters were quite stable over 48 hours. SNS01 could also be frozen at − 80°C for at least one month with no observable loss in biological activity. Twice‐weekly intra‐venous injections into myeloma tumor‐bearing mice of a frozen SNS01 preparation was found to have anti‐tumoral activity comparable to freshly prepared SNS01 (frozen SNS01: 89 % tumor inhibition, p = 0.00158; SNS01: 84 % tumor inhibition, p = 0.00003). Conclusions: SNS01 is relatively stable at room temperature and was also found to be stable and biologically active following freezing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A196.