gene encodes a zinc finger transcription factor that is essential for normal erythroid and megakaryocytic differentiation.
Acquired somatic mutations in exon 2 of GATA1
gene have been consistently detected in nearly all DS TMD and AMkL cases, whereas none were detected in non-DS AML and non-AMkL DS leukemia except for rare cases,
highlighting their critical roles in leukemogenesis in DS. The net effect of these mutations is the introduction of stop codons and synthesis of GATA1s (40 kDa), initiated from a downstream initiation site and distinguishable from GATA1 (50 kDa).
Both GATA1s and GATA1 show similar DNA binding abilities and interact with partner proteins, such as FOG1, although GATA1s exhibits altered transactivation capacity due to the loss of the N
-terminal activation domain.
Lineage selective knockdown of GATA1
in megakaryocytes has been reported to cause increased megakaryocyte proliferation coupled with impaired maturation in mice.
It is believed that the presence of constitutional chromosome 21 disturbs fetal hematopoiesis promoting the acquisition of GATA1
As a consequence, GATA1
mutations would promote accumulation of poorly differentiated megakaryocytic precursors and would represent initiating or early “genetic hits” in a multi-step process of leukemogenesis initiated prenatally.
One mechanism by which those effects can be explained is that GATA1s fails to repress certain genes normally regulated by GATA1.
However, the possibility that expression of GATA1s in DS AMkL may itself be causal, due to its unique function in regulating specific genes, cannot be excluded. To date, the possibly unique role of GATA1s in promoting leukemogenesis and chemotherapy sensitivity in a human DS AMkL model has not been considered.
In this report, we established that GATA1s has novel biological functions in relation to DS AMkL leukemogenesis and responses to therapy. Our approach involved lentiviral shRNA knockdown of the GATA1
gene in a human DS AMkL cell line, CMK, which harbors a mutated GATA1
gene and expresses only GATA1s. Interestingly, down-regulation of GATA1s in CMK cells resulted in impaired cell proliferation and increased differentiation toward the megakaryocytic lineage to extents that paralleled the decreased levels of GATA1s seen in the stable clones ( and ). The increase in megakaryocytic differentiation of the CMK-5a and CMK-5b cells was further supported by the substantially induced expression of proplatelet formation genes, such as PF4
(). In addition, down-regulation of GATA1s was accompanied by both up- and down-regulation of a large number of genes, including genes related to cell proliferation and cell differentiation, such as IL1A.
Previous studies have established that interleukin 1 enhances proliferation of megakaryocytic progenitors.
Our finding that GATA1s promotes cell proliferation and represses differentiation toward megakaryocytic lineage in DS suggests that expression of GATA1s in itself could be a causal factor in leukemogenesis in DS.
EFS rates for DS patients with AMkL are significantly higher than non-DS AML patients, and in particular, non-DS AMkL patients,
suggesting that the presence of GATA1s may contribute to the high EFS rates of DS AMkL patients. Our previous study suggested that loss of GATA1 may contribute to the enhanced chemotherapy responses of DS AMkL compared to non-DS AML.
However, it is not clear whether expression of GATA1s itself contributes to the increased chemotherapy sensitivities, as seen in DS AMkL patients, compared to non-DS AML patients. In the present study, we found that down-regulation of GATA1s in CMK cells resulted in significantly increased in vitro
sensitivities to ara-C, daunorubicin, and VP-16 compared to that in the negative control, suggesting that expression of GATA1s can confer resistance to chemotherapy in DS AMkL.
Accordingly, expression of GATA1s by itself is unlikely
to explain the enhanced chemotherapy responses in DS AMkL compared to non-DS AML. Rather, expression levels of GATA1s may represent an important biomarker related to chemotherapy sensitivity or resistance in DS AMkL. Loss of GATA1 may represent a significant contributing factor accounting for the increased chemotherapy sensitivity of DS AMkL. The role of GATA1s in chemosensitivity only applies to the DS AML group itself (based on the lack of expression in non-DS AML) and its relative impact may be modest. Nonetheless, assessing the role of GATA1s expression in DS AMkL may identify a small subset of DS patients with refractory or relapsed disease.
A study of DS patients with TMD reported that differences in GATA1s levels were associated with the risk of progression to myeloid leukemias, supporting the notion that GATA1s may impact the clinical features of DS leukemia cases. 
What are the molecular mechanism(s) responsible for the effects of GATA1s on ara-C sensitivity? Our real-time RT-PCR assays showed no significant differences in dCK, CDA, and hENT-1 transcript levels among the stable clones (data not shown). However, significantly increased basal apoptosis in the CMK-5a and -5b clones was detected compared to that in the CMK-neg cells. This was accompanied by decreased levels of Bcl-2 protein, which was certainly responsible for the increased basal apoptosis and increased ara-C sensitivities in the CMK-5a and -5b clones, as demonstrated by our lentiviral shRNA knockdown experiments. Surprisingly, down-regulation of GATA1s resulted in increased Bcl-2 transcripts in the GATA1 shRNA stable clones compared to the CMK-neg cells, suggesting that GATA1s must confer some means of posttranscriptional regulation of Bcl-2 expression in CMK cells. Current studies are underway to determine the mechanisms underlying the regulation of Bcl-2 expression by GATA1s in CMK cells.
To summarize, our findings suggest that GATA1s has unique functions in DS AMkL. GATA1s appears likely to facilitate DS leukemogenesis and to confer resistance to chemotherapy by promoting proliferation and survival, and by repressing differentiation towards the megakaryocytic lineage, potentially by regulating expression of Bcl-2 and other relevant genes.