Inverse Correlation between Flt3 and PU.1 Expression in Acute Myeloblastic Leukemias.
Authors: Takahashi Shinichiro , Inomata Mitsue , Harigae Hideo , Kameoka Junichi , Kaku Mitsuo , Sasaki Takeshi 
Published: April 2020

Abstract
Flt3 is a member of receptor tyrosine kinase. Mutations in the flt3 gene have been demonstrated to be the most common genetic alteration in acute myeloblastic leukemia (AML). Flt3 receptor phosphorylation/activation results in the activation of downstream kinase pathways, like Ras/mitogen-activated protein kinase (MAPK), leading to the abnormal cell growth and aberrant gene regulation. Notably, more than 2/3 of AML patients have phosphorylation of Flt3 receptor even in the absence of activating mutations. Moreover, increased levels of Flt3 transcript are observed in a large number of AML specimens (Blood2004; 1901–1908). This increased expression also contributes to the phosphorylation of Flt3, activation of this pathway and playing a role in leukemogenesis. PU.1 is a member of the Ets transcription factors and is expressed in granulocyte and B-lymphoid cells. Recently reported that mice carrying hypomorphic PU.1 alleles that reduce PU.1 expression, to 20% of normal levels, developed AML (Nat Genet2004; 624–630). These suggest that aberrantly reduced expression of PU.1 is playing an important role in leukemogenesis. Therefore, we speculated that there is an inverse relationship between these two factors. To clarify this, we measured the mRNA expression level of Flt3 and PU.1 in 31 primary AML specimens by real time RT-PCR. As a result, there is a significant negative correlation between Flt3 and PU.1 (r= −0.41, p=0.023). Next we speculated that whether flt3 promoter activity is affected by the PU.1 expression. We amplified the 5′-upstream region of the flt3 gene up to approximately 1kb from the transcription start site. TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html) program predicted three G/C boxes, one Myc site, one AML1 and one Ets site in this region. No TATA or CAAT boxes were found. The flt3 promoter showed 30 to 40 fold activity compared to promoterless PGL3 basic plasmid in Flt3 highly expressed THP-1 cells. In contrast, there was up to 10 fold activity in Flt3 low expressing K562, 293, Jurkat cells, suggesting that this 5′ region of flt3 gene is functionally active, has a tissue specific activity and serve as a promoter. Finally, we revealed that over-expression of PU.1 significantly represses flt3 promoter activity, shown by transient co-transfection assays. Proximal region from −226 to transcriptional start site is important for this repression of PU.1. PU.1 is known to repress promoter activity when promoters have no PU.1 binding sequences, whereas PU.1 transactivates the promoter with functional PU.1 binding site(s). Although there is one Ets site in −95/−90 of flt3 promoter, our functional promoter assays revealed that PU.1 does not bind to this site. PU.1 interact with transcriptional co-repressors, mSin3A and histone deacetylase (HDAC) that modify chromatin structure, associates with various transcription factors maintain basal transcription like Sp1 or TFIID (Blood1997; 4135–4143)(Oncogene2001; 6039–6047). These suggest that the repression effect of PU.1 to flt3 promoter is not only single site specific but rather affects to the transcription factor complex forming proximal region of this promoter. In summary, this is a first report demonstrating a negative correlation between PU.1 and Flt3 in AML specimens and showed that PU.1 is a potential repressor of flt3 gene.