IMiD® Compounds Affect the Hematopoiesis Via CRBN Dependent Degradation of IKZF1 Protein in CD34⁺ Cells

Introduction: Lenalidomide (LEN) and pomalidomide (POM) are derivatives of thalidomide (IMiD® compounds) and currently used to treat multiple myeloma (MM) and B-cell malignancies. We have shown before that IMiD® compounds shift lineage commitment of CD34⁺ cells towards myeloid development by affecting critical transcription factors such as GATA1 and PU.1 with concomitant inhibition of cell maturation resulting in anemia and neutropenia. Nonetheless the underlying pathomechanism is still unknown. Recently, IMiD® compounds were shown to bind to cereblon (CRBN) in MM cells, which is the substrate recognition component of cullin-dependent ubiquitin ligase and LEN leads to ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IZKF3 by the CRBN-CRL4 ubiquitin ligase. We investigated here the role of CRBN, IKZF1 and IKZF3 in IMiD®-induced effects on lineage commitment and maturation of CD34⁺ cells.

Methods and Results: By western blot analysis we found that CRBN and IKZF1, but not IKZF3 are expressed in CD34⁺ cells. Treatment of CD34⁺ cells with LEN and POM for only 1h almost completely decreased the expression of IKZF1 without affecting CRBN protein expression. By using a thalidomide analog bead assay, we found that IMiD® compounds directly bind CRBN in CD34⁺ cells. In contrast to our protein studies, IKZF1-mRNA level was not altered in real-time PCR, suggesting that IMiD® compounds regulate IKZF1 at post-transcriptional level. Treatment with proteasome inhibitors MG132, PS341 or MLN4924 which function as cullin-dependent ubiquitin ligase inhibitors blocked LEN and POM induced IKZF1 degradation, confirming that IMiD® compounds induce ubiquitination and subsequent protein degradation of endogenous IKZF1. Next we generated lentiviral constructs to knockdown the expression of CRBN in CD34⁺ cells. Knockdown of CRBN in CD34⁺ cells induced resistance to POM-induced IKZF1 downregulation and subsequently reversed the POM-induced lineage shift in colony-formation assays, suggesting that POM-induced degradation of IKZF1 in HSC requires CRBN.Knowing that PU.1 and GATA-1 are critically involved in the IMID-induced lineage shift in CD34⁺ cells we demonstrated in chromatin immunoprecipitation assays that IKZF1 binds to promoter regions of PU.1 and GATA-1, suggesting that PU.1 and GATA-1 are direct downstream targets of IKZF1 in CD34⁺ cells .

Conclusion: Our findings show that CRBN and IKZF1 mediate the effects of IMiD® compounds on hematopoietic progenitors. IMiD® compounds promote CRBN dependent degradation of IKZF1 protein in CD34⁺ cells that subsequently decreases transcription factors such as GATA1 and PU.1 which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Our findings that IMiD® compounds mediate their effects via CRBN and IKZF1 provide for the first time the pathomechanism how LEN and POM affect hematopoiesis and induce neutropenia, thrombocytopenia as well as anemia.