Abstract
CBFA2T3-GLIS2 is the most prevalent fusion oncogene in pediatric acute megakaryoblastic leukemia patients without Down syndrome and is associated with an event free survival of only 8%. A cryptic inversion event on chromosome 16 joins the three nervy homology regions (NHR) of CBFA2T3 to the five zinc fingers of GLIS2. This configuration enables the encoded chimeric transcription factor to bind GLIS2 consensus sequences throughout the genome and recruit transcriptional activators and repressors to alter gene expression and enhance self-renewal capability. Few cooperating mutations have been identified in patients harboring this fusion which suggests it is the sole oncogenic driver. The molecular mechanism by which CBFA2T3-GLIS2 drives leukemogenesis is not fully understood. Identification of components critical to the transcriptional complex and their role in gene regulation may reveal novel therapeutic targets to improve patient outcomes. Studies on the wild type CBFA2T3 and GLIS2 proteins have demonstrated interactions with the transcriptional regulators ETO and CtBP1 respectively. Further p300 has been shown to play a role in transcriptional regulation imparted by both transcription factors. We therefore hypothesize the fusion promotes transcriptional activation when the histone acetyltransferase p300 and the transcription factor ETO are recruited through NHR1 and NHR2, respectively. When the co-repressor CtBP1 is recruited through the PXDLS motif, located in the GLIS2 portion of the fusion, transcriptional repression predominates. Association of these co-factors with the fusion was confirmed through co-immunoprecipitations. Site-directed mutagenesis was then used to systematically delete NHR1 and NHR2 and mutate the PXDLS motif to evaluate the resultant effects on transcriptional regulation, self-renewal, and leukemogenesis imparted by the fusion. A luciferase reporter assay was used to assess transcriptional activation of the BMP2 promoter, a gene which is known to be upregulated by the CBFA2T3-GLIS2 fusion. Loss of NHR1, NHR2, or NHR3 did not alter the ability of the fusion to activate transcription. In contrast, loss of NHR1 and NHR2 in combination (NHR1-2Δ) and mutation of the PXDLS domain decreased transcriptional activation compared to the wild type fusion. The effect of the mutations on self-renewal capability was then evaluated through colony formation assays. Consistent with the luciferase reporter assay, NHR1-2Δ and PXDLS mutants decreased the number of colonies at week six compared to the unmanipulated fusion. Next, we investigated the effect of these mutations on leukemogenesis. Murine models harboring the CBFA2T3-GLIS2 fusion without cooperating mutations have been unsuccessful and patient-derived xenograft models are limited and difficult to manipulate. Therefore, we developed a novel in vivo model of CBFA2T3-GLIS2 driven leukemia. CD34+ stem cells were isolated from human cord blood and transduced with a lentivirus construct encoding the fusion and a GFP reporter. The cells were then differentiated to megakaryoblasts using human TPO and IL1-beta and sorted for purity prior to injection into immunodeficient NSG-SGM3 recipient mice. The fusion positive human primary megakaryoblasts induced a serially transplantable leukemia within 180 days that recapitulates CBFA2T3-GLIS2 positive patient specimens on a transcriptional and protein level. In contrast to our in vitro studies where NHR2 deletion alone did not alter transcriptional activation and self-renewal, the loss of this domain abrogated leukemogenesis in vivo, suggesting a dependency on the association of ETO with the fusion. Mice that received PXDLS mutant cells, however, developed leukemia at a normal latency suggesting that CtBP1 is not required. This study confirms the CBFA2T3-GLIS2 fusion is sufficient for oncogenic transformation of human CD34+ stem cells. We demonstrate that disruption of ETO, p300, and CtBP1 recruitment to the transcriptional complex decreases transcriptional regulation and self-renewal imparted by the fusion. The loss of ETO was the most detrimental to leukemogenesis in our murine model, uncovering a potential new pathway for the development of targeted therapies. Ongoing studies include CUT&RUN sequencing for the fusion, ETO, and CtBP1 to determine co-occupancy of target genes to further understand those that are critical in transformation.
Disclosures
Gruber: Kura Oncology: Consultancy.
Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA
