The number of known monogenic rare diseases (~7000) exceeds the number of effective treatments (~500) by more than an order of magnitude underlining the pressing need for generalizable therapeutic approaches for this class of conditions. In this regard, the majority of recessive and x-linked recessive disorders are caused by missense mutations encoding proteins that frequently have residual function but are rapidly degraded by the 26S proteasome. Bortezomib is a small molecule that inhibits the 26S proteasome and has been approved for use in patients for an unrelated condition; multiple myeloma. Previous work has shown that, for a small number of disorders, bortezomib can inhibit the degradation of the mutant protein, thereby increasing the protein level and activity, holding out the promise of a beneficial therapeutic effect by the repurposing of this agent. We present here a high level western blot based survey of nine recessive disorders to characterize the general effectiveness of such an approach. Thirteen patient fibroblast cell lines comprising 9 different diseases with 19 known mutations were selected on the basis of missense mutations protein expression data when available. The cell lines were incubated with bortezomib (10 nM and 50 nM; 24 hrs) and levels of the mutated protein were quantified by western blot. Unfortunately, no consistent, appreciable increase was observed for any of the conditions tested. The general therapeutic value of re-purposing bortezomib for recessive and x-linked diseases appears limited at best. The few reported cases of bortezomib successfully working in increasing mutated protein levels appear to be the exceptions and not the norm. Moreover successes are more often limited to cell lines carrying a transgene expressing the mutated protein rather than endogenous mutated protein in patient cell lines.

Anti-tumor therapeutic strategies based on combinations of chemotherapeutic agents with a death inducing ligand such as TNF-α Related Apoptosis Inducing Ligand (TRAIL), are directed towards selective and effective cancer cell apoptosis and enhanced therapeutic response. We previously demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of initiator caspase-8. The present study investigated the functional link between caspase-8 and the proteasome, by analyzing the impact of caspase-8 ubiquitination and proteasomal degradation on the outcomes of the extrinsic apoptosis pathway in cancer cells. Caspase-8 ubiquitination status was assessed by polyubiquitin immunoprecipitation (IP) and fluorescent microscopy. Apoptosis induction in response to death receptor stimuli or proteasome inhibitor was evaluated using the Annexin V/Propidium iodide staining (PI). To determine the consequences of proteasome inhibition on caspase-8 stability, trafficking, and activity following death receptor activation, we used the TRAIL-resistant human prostate cancer LNCaP cells, and the caspase-8 deficient Neuroblastoma 7 (NB7) cells, as cellular models for reconstituting the non-cleavable mutant forms of caspase-8. Our findings demonstrate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and GST-TRAIL. Furthermore, caspase-8 processing into its active subunits preceded caspase-8 polyubiquitination, implicating caspase-8 processing as a potential regulatory mechanism, rather than a requirement for caspase-8 activation in apoptosis induction. The mechanistic control of caspase-8 by ubiquitination in cancer cells may have significant significance in bypassing mechanisms of therapeutic resistance in human tumors and optimization of anti-cancer treatment strategies in human tumors and optimization of anti-cancer treatment strategies.

The farnesyl transferase inhibitor R115777 (Zarnestra, Tipifarnib) has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341 (Bortezomib, Velcade). Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Identification and characterization of the 8226/R5 cell line helped us evaluate and confirm that the Akt tumor survival pathway plays an important role in Tipifarnib induced apoptosis and resistance in myeloma cells. Additionally, 8226/R5 cells helped to evaluate other molecules exhibiting synergistic cell death. In this study, we investigated the activity of R115777 combined with Bortezomib in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to Tipifarnib, combined activity was maintained. Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Our study provides the preclinical rationale for trials testing the Tipifarnib and Bortezomib combination in patients with multiple myeloma and AML.