Abstract 2000: ETV6 represses Pax5 in early B-cell development

Abstract

Abstract 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. Methods: B cell fractions- B cell progenitors from wild type mice were purified using flow cytometry. Gene Expression- RNA was collected and reverse transcriptase quantitative PCR was performed to quantitate Pax5, Etv6, Ebf1 and actin transcripts. Chromatin Immunoprecipitation- Cells were collected; proteins were cross-linked to DNA with formaldehyde, cell lysates were sonicated and immunoprecipitation was performed using 5μg indicated antibody or species-matched IgG as a negative control. DNA was recovered and assayed by quantitative PCR. Results: Recent studies have revealed a role for ETV6 germline mutations (P214L amino acid change) in the predisposition to Acute Lymphoblastic Leukemia (ALL). These mutations impair the transcriptional activity of ETV6 in a dominant negative fashion. Here, we demonstrate that Etv6 expression is inversely correlated to Pax5 and Ebf1 expression during B cell development (r2 = .9993; P = 0.0167). In a murine lymphoid progenitor line (Ba/F3), ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). In addition, Pax5 expression was increased by overexpression of EBF1, a known Pax5 transcriptional activator, in cells expressing the ETV6 P214L mutant (P≤0.05). This data suggests that loss of functional ETV6 is necessary for EBF1 to induce Pax5 expression. 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. Aberrant expression of Pax5 leads to myeloid lineage skewing and an increase in biphenotypic and myeloid leukemias. Patients with ETV6 germline mutations have a higher percentage of monocytes compared to unaffected family members, which we hypothesize, is due to aberrant PAX5 expression (P = 0.008). Conclusions: 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. Citation Format: Courtney L. Jones, Courtney J. Fleenor, Seth Welsh, Leila Noetzli, Susan Fosmire, Dmitry Baturin, Jorge Di Paola, James R. Hagman, Christopher C. Porter. ETV6 represses Pax5 in early B-cell development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2000.