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IMiDs block megakaryocytic maturation
of secondary malignancies such as myelodysplastic syn- drome and acute leukemia.13-15
Our laboratory has focused on exploring the effects of IMiDs on different hematopoietic lineages. We showed that IMiDs do not exhibit direct stem cell toxicity, but affect lineage commitment.16,17 Downregulation of GATA1 by IMiDs induces a shift into myeloid lineage commit- ment at the expense of erythroid commitment.16 The downregulation of SPI1 (PU.1), a critical transcription fac- tor for myeloid maturation, leads to maturational arrest with accumulation of immature myeloid precursors, resulting in neutropenia.17 Nevertheless, IMiD-induced thrombocytopenia, a major adverse side effect, is still not understood.
Here, we investigated the effect of IMiDs on megakary- opoiesis after thrombopoietin (TPO) stimulation. We showed that IMiDs induce self-renewal and proliferation of megakaryocytic progenitors by down-regulating GATA1 as a consequence of the degradation of its binding partner IKZF1. This is accompanied by decreased ZFPM1/FOG-1 and NFE2 expression, leading to inhibition of megakary- ocyte maturation. Our data further demonstrated that IMiD induced a decrease in CCND1/cyclin D1 accompa- nied by an increase in CDKN2A/p16, resulting in the mat- urational arrest of megakaryocytes (Mks). The effects of IMiDs on megakaryopoiesis could be abrogated by overex- pression of GATA1. This study provides for the first-time mechanistic insight into how IMiDs induce thrombocy- topenia and potentially contribute to secondary hematolog- ic malignancies by sustained cell proliferation.
Methods
CD34+ cells isolation and culture
Primary CD34+ cells were isolated from discarded peripheral
blood leukapheresis products after stem cell mobilization of con-
senting healthy individuals and MM patients. We tested the
CD34+ cells from MM patients or healthy individuals in cell pro-
liferation and colony assays and no difference was observed. Data
are not shown. The Institutional Review Boards (IRBs) of the
University of Pittsburgh, Pittsburgh, PA and Columbia University,
New York, NY approved all studies. Purified CD34+ cells were
grown in serum-free hematopoietic growth medium (HPGM)
(Lonza) supplemented with 10 ng/mL recombinant human throm-
bopoietin (rhTPO), 10 ng/mL recombinant human interleukin-3
(rhIL-3), 10 ng/mL recombinant human interleukin-6 (rhIL-6), and
50 ng/mL recombinant human stem cell factor (rhSCF). All
cytokines were purchased from PeproTech as described previous- ly.16, 17
LEN and POM (Sigma Aldrich) in DMSO were diluted in culture medium and added daily. Cell viability was measured by trypan blue exclusion, and cell proliferation was quantified by manual cell counting every 2 days during culture.
Megakaryocytic colony assays
Megakaryocytic colony forming unit (CFU-Mk) assays were generated using the MegaCultTM-C Staining Kit (StemCell Technologies) according to the manufacturer’s instructions. The number of CFU-Mk was determined using an anti-CD41 anti- body, an alkaline phosphatase detection system and by counter- staining with Evan’s Blue. The total numbers of colonies were counted on day 12 of culture. The colonies were subdivided by colony size: small (3-20 cells/colony), medium (21-49 cells/ colony), or large (≥ 50 cells/colony).
Colony-forming assay
Colony-forming assays were performed as described previous- ly.16, 17 For CD34+ cells self-renewal assessment, CD34+ cells were seeded in serum-free HPGM supplemented with rhIL-3, rhIL-6 and rhSCF as mentioned above and cultured in the presence of IMiDs or DMSO. After 14 days in culture, the CD34+ cells of each group (vehicle, LEN and POM) were purified using the CD34+ cell isolation kit and were plated in MethoCult H4434 medium (StemCell Technologies) for 14 days (without vehicle, LEN or POM).
Transmission electron microscopy
To identify megakaryocytic precursors by transmission electron microscopy (TEM), we labeled the precursors with CD61 magnet- ic beads (Miltenyi Biotec). Treated cells were fixed in 2.5% glu- taraldehyde in 0.1 M PBS, pH 7.4, for 1 h and post-fixed in aque- ous 1% OsO4, 1% K3Fe(CN)6 for 1 h. The pellet was dehydrated through a graded series of 30–100% ethanol, 100% propylene oxide and then infiltrated in 1:1 mixture of propylene oxide/Polybed 812 epoxy resin (Polysciences) for 1 h. Ultrathin (60 nm) sections were collected and counterstained with uranyl acetate and lead citrate and observed by using a JEOL JEM 1011 transmission electron microscope (JEOL) with a bottom mount AMT 2k digital camera (Advanced Microscopy Techniques).
Statistical analyses
Statistical significance of differences between group means (P<0.05) was established using Student's t test for two group com- parisons. Multiple comparisons were performed using one-way ANOVA with the Bonferroni step down correction of P. Error bars on graphs reflect standard error of the mean.
Results
IMiDs induce self-renewal and expansion of early myeloid and megakaryocytic progenitors by blocking apoptosis and enhancing proliferation
To determine the basis for thrombocytopenia after treatment with IMiDs, we analyzed the effect of POM on megakaryocytic colony formation of CD34+ cells. Under specific conditions allowing the development of CFU-Mk, POM significantly (P<0.001) increased the numbers of CFU-Mk in comparison to vehicle, with an increase from 53.2 (± 2.56) colonies (vehicle) to 144.8 (± 4.74) colonies (POM). The up-regulation of CFU-Mk was especially evi- dent (P<0.001) in medium/large CFU-Mk (34.4 ± 4.03 colonies in vehicle versus 113.8 ± 5.91 colonies in POM), which arose from more primitive progenitors (Figure 1A). We also studied the effects of LEN and POM on prolifera- tion and apoptosis of hematopoietic progenitors. Using CD34+ cells, IMiDs (LEN, P<0.05; POM, P<0.001) increased the absolute cell number in the cultures up to four-fold (LEN) and 10-fold (POM) after 2 weeks (Figure 1B). The cell expansion was not only the result of decreased apoptosis (PI+ cells, day 7: vehicle 58.0%, LEN 24.2%, and POM 6.4%; day 14: vehicle 68.0%, LEN 24.3%, and POM 8.3%; P<0.05), but also of increased mitosis according to the cell cycle analysis results. The proportion of cells in S phase increased (P<0.05) after CD34+ cells were treated with IMiDs on day 7: vehicle 8.3%, LEN 16.1%, POM 19.4% and day 14: vehicle 7.0%, LEN 14.0%, POM 15.8% (Figure 1C). Investigations of the effects of IMiDs on cell cycle regulation of progenitor cells revealed that S-phase cells were increased after
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