Abstract
Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia.
To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction.
To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression.
To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression.
In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis.
Disclosures
No relevant conflicts of interest to declare.
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