The HIV Protease Inhibitor Nelfinavir: A Unique Oral Drug That Inhibits the Proteasome and AKT-Phosphorylation, Induces ER Stress and Sensitizes Bortezomib-Refractory Primary Myeloma Cells and Primary AML Cells towards Bortezomib

HIV protease inhibitors (HIV-PI: Ritonavir, Lopinavir, Saquinavir, Nelfinavir, Amprenavir, Indinavir, Atazanavir, Tipranavir and Darunavir) are oral drugs approved for HIV treatment. Although designed to inhibit the HIV protease, HIV-PI likely have additional, yet unidentified, mammalian targets. Individual HIV-PI have been reported to inhibit the proteasome as well as AKT phosphorylation, induce ER stress and exert synergistic cytotoxicity with bortezomib in solid tumors, suggesting a therapeutic potential of HIV-PI especially in myeloma and AML. However, testing and comparing the effects of the different HIV-PI in AML or myloma cells, a prerequisite to identify the most promising HIV-PI to enter clinical testing, have not yet been performed.

We systematically compared and analysed the effects of all currently approved HIV-PI on proteasome inhibition, cytotoxicity, induction or ER-stress, inhibition of p-AKT, and synergism with bortezomib in human myeloma and AML cell lines and primary cells in vitro.

HIV-PI can be divided into two subclasses, based on their activity on myeloma and AML cells: Lopinavir, Nelfinavir, Ritonavir and Saquinavir (LNRS-PI) showed biological and molecular activity at concentrations within or near to therapeutic drug levels (10-20 μ M). All remaining HIV-PI were inactive in this concentration range. The LNRS-PI induced ER stress in a very similar fashion (concentration-dependent increase in expression of the chaperones BIP and PDI at 10 μ M). This translated into a uniform pattern of ER stress-induced apoptosis, as deferred from increased expression of CHOP and cleaved PARP, leading to cell death. Likewise, the concentration-dependent inhibition of AKT-phosphorylation was similarly observed for all LNRS-PI, starting at 10 μ M. The LNRS-PI also showed a uniform synergistic cytotoxic activity with bortezomib at therapeutic drug levels, as observed with myeloma cell lines, as well as AML cell lines and primary cells in vitro. By contrast, all remaining HIV-PI did not show a synergistic effect with bortezomib. To assess the effects of the different HIV-PI on proteasome activity, we used activity-based proteasome-specific probes that visualize the activities of the proteasome β2 and β1/β5 active sites in intact, viable cells. Nelfinavir stood out as the only HIV-PI with proteasome-inhibiting activity at therapeutic drug levels. Nelfinavir led to a dose-dependent decrease in active proteasome β1/β5 as well as β2 species in the concentration range of 10–40 μ M. By contrast, low doses of Lopinavir, Ritonavir or Saquinavir induced proteasome β2 and β1/β5 activity, which then decreased to baseline activity levels upon higher drug concentrations (up to 80 μ M). All remaining HIV-PI had no effect on proteasome activity. When cells were pre-treated with bortezomib, which preferentially inhibits the proteasome β1/β5 subunits, and were then exposed to the different HIV-PI, only Nelfinavir led to an additional inhibition of the proteasomal β1/5 and also β2-activity, while all other HIV-PI did not have such an effect. Strikingly, primary myeloma cells from a patient who was refractory to bortezomib-lenalidomide combination therapy, and which were refractory to Bortezomib 10 nM also in vitro, showed robust (> 90%) cytotoxicity when bortezomib was combined with therapeutic drug levels of Nelfinavir (10 μ M).

Nelfinavir is a unique drug in the class of HIV-PI, which leads not only to the induction of ER stress and inhibition of AKT-phosphorylation, but also to proteasome inhibition of all active subunits in intact cells at therapeutic drug levels. It re-sensitizes bortezomib-refractory myeloma cells towards bortezomib treatment. Nelfinavir may therefore be an active drug warranting clinical testing in hematologic malignancies, such as myeloma, mantle cell lymphoma or AML.

No relevant conflicts of interest to declare.