Immunomodulatory Derivatives of Thalidomide (IMiD)-Induced Neutropenia Is Associated with PU.1 Downregulation and Myeloid Maturation Arrest

Abstract

Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop.In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged.In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.