IMiDs Cause Thrombocytopenia By the Inhibition of Proplatelet Formation, through Blocking Estradiol Autocrine Loop in Human Megakaryocytes

Introduction:

Immunomodulatory drugs (IMiDs), lenalidomide (LEN) and pomalidomide (POM), are key agents for hematologic malignancies including multiple myeloma and 5q^- syndrome. Thrombocytopenia is one of main side effects of IMiDs, which sometimes interrupts treatment. We here investigated the mechanism of IMiDs-mediated thrombocytopenia.

Materials and methods:

Colony-forming and differentiation activities of normal human hematopoietic stem/progenitor cells (HSPCs) were evaluated in the presence or absence of 10 μM LEN and POM. The alteration of gene expression profile in megakaryocytes (MKs) exposed to LEN was assessed by DNA microarray. To evaluate the stability of the protein responsible for the IMiDs-mediated inhibition of proplatelet formation (PPF), Western blot and co-immunoprecipitation analyses were performed.

Results:

We first tested whether IMiDs affect colony-forming potential and lineage readout of human HSPCs. Unexpectedly, the addition of LEN and POM to the culture of HSPCs resulted in a dramatic increase in number of megakaryocytic colonies (CFU-MK and CFU-MegE), whereas the number of erythroid colonies (BFU-E) was decreased. HSPCs preferentially differentiated into CD41⁺CD105^- MK lineage cells in the presence of IMiDs, suggesting that IMiDs enhanced commitment into the MK lineage in human hematopoiesis at the bipotent megakaryocytic/erythroid stage. In clinical setting, however, IMiDs do not cause thrombocytosis, but often lead to thrombocytopenia. We therefore examined whether IMiDs inhibit MK maturation at a later stage of their development. Treatment with IMiDs did not result in morphological changes of MKs in terms of cell size and nuclear ploidy. However, in the presence of LEN (5.7 fold, p < 0.01) and POM (4.1 fold, p < 0.01), MKs did not display PPF, a mechanically indispensable step for platelet production. Transcriptome analysis of MKs suggested that estradiol (E2) signaling was attenuated by LEN (NES = -2.56, P < 0.01). Consistent with a previous finding that the E2 autocrine signaling triggers PPF, we found that the addition of 10nM E2 in the medium effectively recovered PPF of MKs in the presence of IMiDs. Thus, it is suggested that IMiDs-mediated blockade of endogenous E2 signaling impairs PPF. IMiDs modulate proteolysis by binding cereblon (CRBN), the substrate adaptor for ubiquitin ligase complex. Western blot analysis revealed that IMiDs reduced the protein level of aromatase (AROM), a key enzyme for a final step of E2 biosynthesis, without affecting mRNA level of AROM in MKs. We also found that AROM was co-immunoprecipitated with IMiDs-bound CRBN. These results collectively suggest that AROM is the novel substrate of CRBN, and that in the presence of IMiDs, AROM is degradated by binding to IMIDs-bound CRBN, which hampers E2 synthesis to impair PPF in MKs.

Conclusions:

IMiDs do not inhibit, but rather promote the megakaryocytic lineage commitment of human HSPCs at an early stage of hematopoiesis. IMiDs, however, inhibited PPF through the blockade of E2 autocrine signaling as a consequence of degradation of AROM in MKs, which may be the main cause for thrombocytopenia in IMiDs-treated patients. Thus, development of new IMiDs having low affinity to AROM may be a key to resolve unwanted thrombocytopenia in IMiDs treatment.