Differential donor‐, cell type‐ and drug‐specific molecular responses to therapeutic proteasome inhibitors in Multiple Myeloma patients and cells

Introduction: Purpose of the study: Proteasome over‐activation represents a hallmark of several advanced tumors and thus, its selective inhibition provides a promising anti‐tumor therapy. Consistently, proteasome inhibitors (PIs) have demonstrated clinical efficacy in the treatment of multiple myeloma (MM) and mantle cell lymphoma and are evaluated for the treatment of other malignancies. Herein we analyzed the molecular responses induced in red blood‐ (RBCs) cells and peripheral blood mononucleated‐ (PBMCs) cells of MM patients treated with therapeutic PIs; given also that disease relapse usually refers to MM cells that survive therapy, we also investigated the molecular responses induced in MM cells after non‐lethal proteasome inhibition.

Case Presentation: Summarized description of the project: We used isolated RBCs and PBMCs and performed several molecular‐cellular biology assays, as well as proteasome activity measurements in various time points of the MM patients’ therapeutic protocol. Also, the molecular readouts after exposing MM cells to non‐lethal (IC10) doses of BTZ, Epoxomicin (EPOX; a CFZ‐like PI) and three PIs that selectively target each of the three proteasome peptidases were recorded.

Description: Results: In healthy donors we found that PBMCs express higher, as compared to RBCs, basal proteasome activities, as well as that proteasome activities decline during aging. Studies in cells isolated from MM patients treated with PIs revealed that in most (but, interestingly enough, not all) patients, proteasome activities decline in both cell types during therapy. RBCs were particularly sensitive to the inhibitor, and their proteasome activities remained low during the entire course of treatment, while PBMCs were characterized by phases of rebound proteasome activities during the periods of no drug administration; these phases correlated with upregulation of proteasome genes expression. Also, the expression levels of antioxidant, chaperone and aggresomes removal/autophagy genes were found to inversely associate with patients' survival. Interestingly, phosphoproteomics profiling after treatment of MM cells with non‐lethal (IC10) doses of PIs revealed inhibitor‐ and cell type‐specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokines/chemokines profiling revealed the increased secretion of immunosuppressive pro‐tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T‐cell chemoattractant chemokines (CXCL10).

Conclusions: Despite donor‐, cell type‐ and drug‐dependent molecular responses to therapeutic PIs in MM patients, our findings indicate the upregulation of several compensatory proteostatic (including proteasome genes) mechanisms in human cells upon effective proteasome inhibition. Also, MM cells that survive treatment with therapeutic PIs likely shape a pro‐tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse. Our ongoing studies aim to contribute to a more detailed understanding of the molecular effects induced in human cells by therapeutic PIs and consequently to a more personalized clinical therapeutic approach in hematological malignancies.