We established Wt and two Mt
We established Wt- and two Mt- (p.R308P and p.A350_N351ins8)-GATA2-expressing 32D Lomustine to analyze the cellular effects of Mt-GATA2 (Fig. 3A). Immunofluorescence analysis revealed that Mt-GATA2 showed similar localization in the nucleus as Wt-GATA2 (Fig. 3B). Both Wt- and mutant-GATA2-expressing 32D cells did not show autonomous proliferation without the presence of IL3. Furthermore, there was no difference of proliferation abilities at lower concentrations of IL3 between them, indicating that mutant GATA2 did not provide a growth advantage (Fig. 3C). Since mutant GATA2 did not affect the proliferation ability, we examined the G-CSF-mediated granulocytic differentiation in Wt- and Mt-GATA2-expressing 32D cells. Mock- and Wt-GATA2-expressing 32D cells were differentiated to mature neutrophils in the culture with G-CSF for 10 days, while Wt-GATA2 overexpression moderately impaired the granulocytic differentiation. In p.R308P-GATA2-expressing 32D cells, mature neutrophil counts and promyelocyte to neutrophil counts were significantly lower than those of Wt-GATA2-expressing 32D cells after the 10-day culture (P=0.020 and P=0.003, respectively), while promyelocyte to neutrophil counts were the same after the 14-day culture. However, in p.A350_N351ins8-GATA2-expressing 32D cells, mature neutrophil counts and promyelocyte to neutrophil counts were significantly lower than those of Wt-GATA2-expressing 32D cells both after the 10-day and the 14-day cultures (P=0.016 and P=0.0005 after 10 days, and P=0.009 and P=0.007 after 14 days, respectively), indicating that p.A350_N351ins8-GATA2 impaired the G-CSF-mediated granulocytic differentiation (Fig. 4A and B). These morphological results were confirmed by the surface expression of CD11b after G-CSF stimulation (Fig. 4C). CD11b expression levels of p.R308P-GATA2-expressing 32D cells were the same as Wt-GATA2-expressing 32D cells, while that of p.A350_N351ins8-GATA2-expressing 32D cells was significantly lower than those of Wt-GATA2-expressing 32D cells on day 14 (P=0.04).
Discussion In this study, we identified two novel GATA2 gene mutations (p.R308P in the ZF-1 domain and p.A350_N351ins8 in the ZF-2 domain) in adult de novo AML patients. Most mutations in the ZF-2 domain of the GATA2 gene are reportedly missense mutations. Although three types of in-frame deletion mutations were reported, p.A350_N351ins8 mutation was firstly identified as an in-frame insertion mutation in the ZF-2 domain. L359V mutation, which was recurrently identified in CML-BC, increased transactivation activity and inhibited myelomonocytic differentiation and proliferation. In contrast, T354M and T355del mutations, which were identified in families with hereditary MDS/AML, dominant-negatively reduced transactivation activity over Wt-GATA2. However, it is notable that T354M-GATA2 inhibited the all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, but T355del-GATA2 did not. Consistent with the T354M mutation, p.A350_N351ins8 mutation reduced DNA-binding and transcriptional activities and impaired G-CSF-induced granulocytic differentiation of 32D cells. However, in contrast to T354M-GATA2, p.A350_N351ins8-GATA2 did not show a dominant-negative effect over Wt-GATA2 by transcriptional assay. Since p.A350_N351ins8 mutation was heterozygous in the clinical sample, this result raised the question of how this mutation is involved in leukemogenesis, particularly in the impairment of granulocytic differentiation. In 32D cells, endogenous GATA2 expression was very faint by western blot and immunohistochemical analyses. Furthermore, Wt-GATA2 transcript was expressed little in the primary AML cells harboring p.A350_N351ins8-GATA2 mutation. These results suggested that p.A350_N351ins8-GATA2 might act as a loss of function of GATA2 in the absence of Wt-GATA2, resulting in the impairment of granulocytic differentiation. Recently, several kinds of missense mutations within the ZF-1 domain were reported in AML. Although transcriptional activities of GATA2 ZF-1 mutants were different among the mutation types, all of them reduced the capacity to enhance CEBPA-dependent activation of transcription. p.R308P-GATA2 did not show significant reduction of DNA-binding and transcriptional activities, while p.R308P-GATA2-expressing 32D cells revealed the delay of G-CSF-induced granulocytic differentiation, suggesting a possible effect on maturation machinery through the reduction of CEBPA-dependent transcriptional activity.