Pomalidomide Induces Mir-125b with Subsequent Downregulation of P53 in CD34⁺cells Conferring Decreased Apoptosis: A Potential Mechanism of Secondary Primary Malignancies

Introduction:IMiD® compounds such as lenalidomide (LEN) and pomalidomide (POM) represent a novel class of agents in the treatment of multiple myeloma. Increasing concerns about its potential toxicity on stem cells resulting in an increase of secondary malignancies primarily affecting the myeloid cells have been reported. However, the exact mechanism is unclear. MicroRNAs (miRNAs) are emerging as important regulators in stem cell development. Therefore, the present study investigated the effect of IMiD® compounds on stem cell renewal/proliferation and the role of miRNAs involved in this process.

Methods and Results: When human CD34⁺ cells were cultured with POM or DMSO under conditions favoring myeloid development, POM significantly increased the percentage of CD34⁺cells in S phase in cell cycle analysis with concomitant dramatic decrease of apoptotic cells. This resulted in a strong and significant proliferation of CD34⁺cells as well as induction of colony formation. Systematic screening of miRNAs expression profile by microarray showed that POM significantly enhanced the expression of miR-125b. Upregulation of miR-125b was further confirmed by realtime PCR. Interestingly, recently miR-125b was shown to function as an oncomir by inducing tumorigenesis in myeloid cells by So et al., Blood 2014. In accordance with that we found that overexpression of miR-125b in CD34⁺ cells exhibited similar effects as POM on CD34⁺ cells, such as increased cell number in S-phase, decreased cell apoptosis resulting in significant induction of colony formation and the absolute number of hematopoietic progenitors. Bioinformatics prediction models revealed a potential binding site of miR-125b on 3'UTR of P53. Indeed subsequent experiments showed that P53 was downregulated when miR-125b was overexpressed in CD34⁺cells or when treated with POM. Further analysis of the P53 down stream targets revealed that POM decreased the expression of PUMA and BAK1 all critical for control of cell proliferation and apoptosis. The POM-induced upregulation of mir-125b was independent of the CRBN/IKZF1 induced protein degradation.

Conclusion: Based on our results we postulate that POM upregulates the oncomir miR-125b in CD34⁺cells. Mir-125b controls proliferation and apoptosis by downregulating P53 and genes involved in the P53 pathway including BAK1 and PUMA. Thus, our findings on the effects of POM on the miR-125b/P53 axle provide for the first time a potential mechanism for the development of secondary hematologic malignancies in patients treated with IMiD® compounds.