PF570 CARFILZOMIB TREATMENT CAUSES RETICULOCYTOSIS BY IMPAIRING TERMINAL ERYTHROID MATURATION IN PATIENTS WITH MULTIPLE MYELOMA

Background: The ubiquitin‐proteasome system (UPS) is crucial for cellular protein homeostasis by ensuring the degradation of redundant or misfolded proteins. In addition to its pleiotropic effects, the UPS also plays an important role in different steps of erythropoiesis. Proteasome inhibition constitutes one of the cornerstones of current multiple myeloma (MM) treatment, with three proteasome inhibitors (PI) approved for clinical use, including the reversible proteasome inhibitors (PI) bortezomib, ixazomib and irreversible PI carfilzomib. Based on a clinical observation of increased reticulocyte counts in patients with MM under treatment with carfilzomib, we hypothesized that PI can affect erythroid maturation. Aims: To address the effects of PIs on erythroid differentiation and maturation. Methods: We first retrospectively assessed the effect of carfilzomib treatment on the red cell parameters in 40 patients treated with a carfilzomib‐based regimen (carfilzomib‐dexamethasone (Kd) or carfilzomib‐lenalidomide‐dexamethasone (KRd)). Data were compared to matched cohorts of patients treated with bortezomib and the immunomodulatory drug (IMiD) lenalidomide. To address the effect of PI on erythroid differentiation in vitro , we differentiated CD34+ hematopoietic progenitor cells (HPC) into erythroid cells as previously described by the Douay group (Nature Biotechnology, 2002) in the presence of carfilzomib. To evaluate the effect of PI on terminal erythroid maturation, we purified reticulocytes from the peripheral blood of healthy volunteers, and performed ex vivo maturation in the presence the of PIs carfilzomib, bortezomib, ixazomib and MG‐132. Finally, we performed a quantitative proteomics analysis using liquid chromotrography‐mass spectrometry (LC‐MS/MS) on maturing reticulocytes to identify proteins involved in erythroid maturation. Results: We observed a specific and highly significant increase in the reticulocyte count ( p  < 0.001) during treatment with carfilzomib‐based regimens in patients with relapsed MM, an observation not made in a matched cohort of bortezomib‐ or lenalidomide‐treated patients. Surprisingly, this reticulocytosis was neither associated with improved hemoglobin levels nor with hemolysis. Moreover, carfilzomib treatment was not associated with increased EPO levels. Subsequent in vitro experiments demonstrated that carfilzomib did not alter the proportion of mature erythroid cells that were produced in vitro during differentiation experiments starting from CD34+ HPCs. In contrast, both continuous and pulse exposure to carfilzomib significantly impaired the terminal maturation of purified reticulocytes toward mature erythrocytes ( p  < 0.01). A similar effect could only be observed with reversible PI during continuous, but not during short‐term exposure. Subsequent quantitative proteomics analysis identified the RNA‐stabilizing protein PABPC1 as a candidate regulator of mRNA stabilization in terminal erythroid maturation. Summary/Conclusion: Our results indicate that proteasome inhibition with carfilzomib causes a delay of terminal erythroid maturation, which might be explained by increased mRNA stabilization through PABPC1 upregulation. This not only demonstrates the first pharmacologically induced delay in erythroid maturation but also provides new insights on physiological erythroid maturation. Moreover, delayed reticulocyte maturation should be considered as a differential diagnosis, aside from hemolytic anemia, in MM patients treated with carfilzomib.