Molecular Analyses Of The Effects Induced By Orally Administered Bortezomib In Drosophila Flies: A Novel In Vivo Experimental Platform To Screen For The Tissue- and Age-Dependent Effects Of Proteasome Inhibitors

Organisms require efficient surveillance of proteome quality to prevent disruption of proteostasis (homeostasis of the proteome). Central to the proteostasis ensuring mechanisms is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Proteome quality control also depends on the activity of the Nrf2/Keap1 signaling pathway which upon increased oxidative stress stimulates the expression of phase II and antioxidant enzymes. Recent findings indicate that over-activation of the proteostasis ensuring mechanisms (e.g. the proteasome) represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. This approach is effectively applied in multiple myeloma (MM) that represents the second most common hematological malignancy. Bortezomib is the first-in-class proteasome inhibitor that is used in the clinic for the treatment of MM, both as a single agent and as part of combination regimens. Nevertheless, the impact of the in vivo impaired proteasome functionality in tissues of higher metazoans (which maybe related to adverse effects in the clinic) remains poorly understood.

To address this issue we harnessed the power of Drosophila genetics and developed a novel in vivo model of specific dose-dependent pharmacological inhibition of proteasome in adult flies. Drosophila is well-suited to this line of investigation, due to its powerful genetics and its similarities in key metabolic and aging pathways with mammals; the fact that its proteasome resemble those from mammals and finally, because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cell lineages. We have found that feeding of bortezomib to young flies causes dose-dependent decrease of proteasome activities in the hemolymph and the somatic tissues, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Further molecular analyses showed that proteasome dysfunction is signaled to the proteostasis network of the young (but not the aged) somatic tissues by reactive oxygen species that originated from damaged mitochondria and downstream activated the Nrf2/Keap1 signaling pathway. Nrf2 activation was essential for stimulation of the genomic antioxidant response elements and the upregulation of the proteasome subunits in order to restore normal proteasome proteolysis rates. Interestingly, the reproductive tissues of the flies were more resistant than somatic tissues to proteasome inhibition triggering (in an age-independent manner) a more intense upregulation of proteasome components after bortezomib-mediated proteasome dysfunction. Additional observations indicated that the toxicity of the bortezomib may also relates to the type of diet and that aged flies are extremely sensitive (compared to young organisms) to proteasome inhibition, while even short term exposures of young flies to bortezomib still affected their overall longevity. Finally, our studies showed that the lower threshold of proteasome activities that can support life is ∼30-40% of the physiological basal activities. Taken together, our findings establish that impaired proteasome function triggers the activation of a tissue- and age-dependent regulatory circuit aiming to adjust the actual cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis regulatory circuit has significant detrimental effects and accelerates aging.

These studies at the in vivo setting of fruit flies add new knowledge on the proteasome inhibitors effects in higher metazoans. Also, as research in this area of high biomedical interest has been developing fast they will, most likely, be of interest to a broader scientific community from distinct disciplines and they have the potential to enter the important, yet challenging, arena of translational medicine. To this end we have started translating findings from our Drosophila model in the clinical setting in order to demonstrate that our Drosophila pharmacological model fit in the spectrum of bench to bedside research.