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A. Liu et al.
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Figure 5. Overexpression of GATA1 reversed the effects of IMiDs on megakaryopoiesis. (A) Schematic diagram of the structure of GATA1pLenti V5 topo expression constructs. CF and NF represent C- and N-fingers of GATA1; R1, R2, R3 represent three regions of the transactivation domain of GATA1 (modified from reference45). (B) The expression of GATA1 in either EV or GATA1 transfected CD34+ cells were analyzed by Western blotting. CD34+ cells transfected with EV or GATA1 were cultured in serum-free HPGM hematopoietic growth medium containing 10 ng/mL TPO with either vehicle, LEN or POM for 6 days. b-actin was used as the loading control. Data are representative of three independent experiments. (C) Purified CD34+ cells were transfected with GATA1 or EV and cultured in serum-free HPGM hematopoi- etic growth medium containing 10 ng/mL TPO to induce megakaryocytic development with LEN, POM or DMSO as vehicle control for 6 days. The expression of the indicated proteins was analyzed by Western blotting. b-actin was used as the loading control. Data are representative of three independent experiments. (D) EV and GATA1 transfected CD34+ cells were subjected to colony formation assays using standard MethoCult assays with and without IMiDs. Overexpression of GATA1 abro- gated the IMiD-induced upregulation of myeloid lineage commitment and rescued the development of BFU-E. Data shown are mean ± SEM from triplicates; **P<0.001, compared with Vehicle group.
IKZF1 mediates the IMiD-induced inhibition of megakaryocytic maturation
Previous studies have demonstrated that lenalidomide induces binding of IKZF1 and IKZF3 to CRBN and pro- motes their ubiquitination and degradation.27,28 Since IKZF1 is required for the development of the erythroid lin- eage,29 we were interested in the effects of IMiDs on the interaction between IKZF1 and GATA1 in CD34+cells. So, we examined the role of IKZF1 in POM-induced inhibi- tion of megakaryocytic maturation. First, we confirmed that IKZF1 regulates GATA1 expression in CD34+ cells by knockdown of IKZF1 in CD34+ cells. Knockdown of IKZF1 resulted in decreased GATA-1 protein expression, further suggesting that GATA1 is under IKZF1 regulation (Figure 4A). In IKZF1 knockdown cells, the mRNA level of GATA-1 was significantly decreased, suggesting that GATA-1 expression is regulated by IKZF1 (Figure 4B). To examine the effects of IKZF1 in megakaryopoiesis, we performed flow cytometric analyses. In IKZF1 knock- down cells, the proportion of immature CD41a+/CD42b– cells was significantly increased, suggesting that IKZF1 downregulation is critical for the POM-induced matura- tional arrest (Figure 4C). To find how GATA1 is regulated
at a transcriptional level, we analyzed GATA-1 promoter sequences and several potential IKZF1 binding sites (Figure 4D upper). Indeed, In CHIP assays, we confirmed that IKZF1 binds directly to the GATA1 promoter area. Treatment of CD34+ cells with POM completely inhibited IKZF1 binding to the GATA1 promotor due to the decreased IKZF1 levels (Figure 4D bottom). Our findings showed that IMiDs promote CRBN-dependent degrada- tion of IKZF1 protein in CD34+ cells and decrease GATA1.
Overexpression of GATA1 abrogated the effects of IMiDs on megakaryopoiesis
To further confirm the critical role of GATA1 in inhibit- ing maturation of Mks by IMiDs, we stably overexpressed GATA1 in human CD34+ cells using the pLenti V5 GATA1 expression vector system (Figure 5A). Overexpression (OE) of GATA1 prevented its downregulation by IMiDs compared to control cells transfected with empty vector (EV) alone (Figure 5B). Concomitant with the overexpres- sion of GATA1, GATA1 co-factors and targets, such as ZFPM1, NFE2 and cyclin D1, remained stably expressed despite IMiDs treatment (Figure 5C). Thus, GATA1 appeared to play a critical role in the IMiDs-induced inhi-
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*P<0.001