Abstract B9: Myc represses Ebf1 and Pax5 to impair early B-cell differentiation and drive progenitor B lymphomagenesis

Abstract

Abstract Activation of Myc oncogenic transcription factors is a hallmark of rapidly dividing malignancies. The Eμ-Myc transgenic mouse is a validated model of MYC activation due to MYC/Ig translocations seen in human Burkitt lymphoma (BL). Normally, these transgenics develop pre-B cell (fractions C-E) stage lymphoma with a mean latency of 3–4 months. Here we report that Myc represses the transcription of Pax5 and Ebf1, which are master transcriptional regulators of B cell fate commitment, and that Pax5 and Ebf1 function as tumor suppressors that impair the tumorigenic potential of Myc-driven lymphoma. Expression analyses of wild-type and premalignant Eμ-Myc fractions A0 through F bone marrow and splenic B cells showed that Pax5 and Ebf1 expression is normally induced in fraction A2 progenitors, but that this response is markedly suppressed in Myc-expressing B cells. In addition, activation of c-Myc in P493-6 B lymphoid cells, which harbor a Tet-regulated c-Myc transgene, repressed Pax5 and Ebf1 mRNA levels, and ChIP analyses demonstrated this was associated with direct binding of c-Myc to the promoter-regulatory regions of Pax5 and Ebf1. Notably, FACS analyses demonstrated marked increases in the numbers of Fraction A1 cells (AA4.1 +B220+CD19−CD4+) and corresponding reductions in Fraction A2 (AA4.1+B220+CD19-CD4-) progenitors in Eμ-Myc versus wild-type mice. Thus, Myc impairs the A1-to-A2 developmental transition of B cells and this is associated with Myc-mediated repression of Pax5 and Ebf1. Collectively these findings, and those of others showing that Pax5 loss promotes the development of progenitor B cell tumors and that inactivating mutations in PAX5 and EBF1 are found in human B-ALL, supported the hypothesis that Ebf1 and Pax5 function as tumor suppressors. To test this, we transduced Eμ-Myc lymphomas harboring inactivating mutations in either Arf or p53 with retroviruses expressing Ebf1 or Pax5 together with a GFP marker, or with control GFP-only virus. Mixtures of GFP+ (transduced) and GFP− (nontransduced) lymphomas were then either cultured ex vivo in medium with IL7, or were directly transplanted into either Nude or NOD/SCID mice. Notably, Ebf1-expressing lymphoma cells underwent rapid growth arrest when cultured ex vivo, indicating Ebf1 cancels Myc's proliferative response. More importantly, enforced expression of Pax5 and especially Ebf1 markedly impaired the tumorigenic potential of Myc-driven lymphoma, irrespective of mutations in Arf or p53, where there was a profound selection against Pax5+GFP+ or Ebf1+GFP+ lymphoma cells in tumor-bearing recipients compared to recipients transplanted with GFP-only expressing lymphoma cells. To assess the effects of Ebf1 and Pax5 in Eμ-expressing (stages A1-F) B cells, as well as their ability to impair the development of lymphomas, we have generated Eμ-Ebf1 and Eμ-Pax5 transgenic mice. These are being crossed to both Eμ-Myc and Eμ-Myc/?μ-Bcl2 transgenics to assess whether Pax5 or Ebf1 will also block lymphoma onset. We predict that Pax5 and Ebf1 will impair lymphomagenesis and override the A1-to-A2 developmental block manifest in Eμ-MycB cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B9.