In search of novel working mechanisms of proteasome inhibitors in multiple myeloma

Proteasome inhibitors (PIs) constitute one of the cornerstones of the treatment of multiple myeloma (MM), with bortezomib, carfilzomib and ixazomib approved for clinical use.  However, MM still remains incurable, and almost all patients will eventually develop treatment-refractory disease.  Moreover, there are currently no validated biomarkers to predict treatment response to PIs.  Additionally, due to the relatively recent introduction of PIs to clinical practice, many aspects of the pleiotropic effects of PIs still remain unexplored, particularly for the second-generation PIs carfilzomib and ixazomib. 

One of the main parts of this project evaluates proteasome activity as a potential biomarker for PI drug sensitivity.  For this purpose, we measured proteasome activity in primary myeloma cells, purified from the bone marrow of patients with MM.  However, the baseline proteasome activity was not significantly different in myeloma cells derived from treatment-responsive or –refractory MM patients.  Moreover, the degree of proteasome inhibition by PIs was similar in both groups.  As a result, the clinical applicability of proteasome activity as a biomarker for drug sensitivity in MM currently remains limited.  Nevertheless, these data also suggest that drug sensitivity to PIs is in part proteasome-independent, indicating that our understanding of PI drug resistance is suboptimal. 

During the follow-up of MM patients treated with PIs in the first part of this project, we observed a consistent and highly significant increase in the reticulocyte count during treatment with carfilzomib-based regimens in patients with relapsed multiple myeloma.  This observation was not made in a matched cohort of bortezomib-treated patients.  As this increased reticulocytosis was neither associated with elevated hemoglobin levels nor with hemolysis, we subsequently performed ex vivo experiments to unravel the underlying mechanisms of this clinical observation in this second part of the project.  While carfilzomib did not affect erythroid differentiation of CD34+ hematopoietic progenitor cells, both continuous and pulse exposure to carfilzomib significantly impaired terminal maturation of purified primary reticulocytes towards erythrocytes.  These results indicate that carfilzomib significantly impairs terminal erythroid maturation, independent of erythroid commitment, expansion or differentiation.  Quantitative proteomics on purified reticulocytes undergoing maturation demonstrated that carfilzomib treatment impaired the degradation of various proteins involved in terminal erythroid maturation. Our results report the first pharmacologically induced delay in erythroid maturation as a mechanism for carfilzomib-induced reticulocytosis in patients with multiple myeloma.

The present project has aimed to connect in vitro to clinical findings, in regard to off-target activity of PIs, but also in the search for a predictive biomarker of PI drug sensitivity.  On one hand, it showed that clinical and in vitro data on drug sensitivity to PIs do not always correlate, and exemplifies that the current models need to be further optimized to push the myeloma field further forward.  In this context, we have started a follow-up project where we will use genome-wide CRISPR/Cas9 screens to study drug resistance to PIs.  On the other hand, this work resulted in explaining clinical reticulocytosis in carfilzomib-treated patients by demonstrating decreased terminal erythroid maturation upon carfilzomib exposure.  This phenomenon could therefore lead to new therapeutic applications for carfilzomib in disorders of mature erythrocytes, such as sickle cell anemia.  Additionally, these results highlight the importance of gaining a mechanistic understanding of clinical phenotypes associated with drug administration.  As novel compounds are continuously being introduced in clinical practice, similar findings might equally lead to novel and sometimes unexpected insights in off-target drug activity.