Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

Golgi organization is regulated by proteasomal degradation.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.