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IMiDs block megakaryocytic maturation
(Figure 3A). The decrease of GATA1 by IMiDs was con- firmed by immunofluorescence of megakaryocytic pro- genitors (Figure 3B) and Western blot analysis (Figure 3C). We have shown that IMiDs induce cereblone-ubiq- uitination with subsequent IKZF1 degradation in CD34+ cells8. ZFPM1 (FOG-1), similar to its interacting partner, GATA1, is required for normal differentiation of ery- throid precursors and Mks.20 Moreover, most GATA1- regulated events require GATA1 to bind to ZFPM1.21,22 Subsequently, we asked if IMiDs also modulate the expression of ZFPM1. Figure 3D shows that in IMiD- treated CD34+ cells, ZFPM1 is almost completely abro- gated after 9 days of treatment with kinetics similar to those of GATA1. GATA1 and ZFPM1 synergistically acti- vate the p45 NFE2 promoter,23,24 which is essential for
late maturation of Mks and platelet formation. Accordingly, we found that expression of NFE2 were down-regulated in IMiD-treated cells (Figure 3D). It is known that polyploidy formation in Mks depends on the expression of cyclin D isotypes, and that cyclin D1 is a direct target of GATA1.25 In accordance with this, we found that IMiD-induced downregulation of GATA1 was associated with decreased cyclin D1 (Figure 4D), con- tributing to the inhibition of maturation. The CDK inhibitor, p16, potently inhibits endomitosis of Mks25 and is decreased upon differentiation of Mks.26 Analysis of p16 by Western blot revealed that p16 was up-regulated by IMiD treatment (Figure 4E), suggesting that the loss of GATA1 induced a cascade of inhibitors of megakaryocyt- ic maturation.
AB
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D
P<0.05
P<0.05
Figure 4. IKZF1 mediates the IMiD-induced inhi- bition of megakaryocytic maturation. (A) IKZF1 shRNA #1 (shIKZF1-1), #2 (shIKZF1-2) or control shRNA (shCNTL) transduced CD34+cell lysates were analyzed by western blotting to compare the levels of IKZF1, CRBN and GATA1. The result shown here is one representative experiment of triplicates. (B) CD34+ cells were transduced using a lentivirus carrying the control shRNA (shCNTL), IKZF1-shRNA #1 (shIKZF1-1), or IKZF1-shRNA #2 (shIKZF1-2) sequence and cultured in serum-free HPGM hematopoietic growth medium with TPO to induce megakaryopoiesis. At 6 days after trans- duction, the cells were sorted with GFP and subse- quently lysed and analyzed for IKZF1 and GATA1 mRNA levels using qRT-PCR. The result shown here is one representative experiment of tripli- cates. (C) CD34+ cells were transduced using a lentivirus carrying the control shRNA (shCNTL), IKZF1-shRNA #1 (shIKZF1-1), or IKZF1-shRNA #2 (shIKZF1-2) sequence. CD34+ cells were cultured in serum-free HPGM hematopoietic growth medi- um with TPO to induce megakaryopoiesis. After 10 days, flow cytometry analyzed the cells gated with GFP and showed an increase of immature Mks (CD41a+/CD42b–) with IKZF1 knockdown. The result shown here is one representative experi- ment of triplicates. (D) CD34+ cells were treated with DMSO (0.01%) or POM (1 mM) for 24 h and cell lysates were analyzed by chromatin immuno- precipitation (CHIP) using IKZF1 antibody. Control IgG was used as a negative control. The precipitat- ed DNA fragments were subjected to qRT-PCR analysis with primers amplifying the GATA1 pro- moter. The result shown here is representative of two independent experiments.
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