Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with Down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS < 40%. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases, we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. GLIS2 is a member of the GLI family of transcription factors that mediate sonic hedgehog (SHH) signaling and has been demonstrated to play a role in regulating expression of GLI target genes. Evaluation of a recurrency cohort of 52 samples including 24 additional pediatric cases and 28 adult cases revealed 6 additional pediatric samples carrying the fusion for an overall frequency of 34% in pediatric AMKL. To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Cells transduced with a mCherry expressing retrovirus failed to form colonies after the 2nd replating. By contrast, expression of either wild-type GLIS2 or CBFA2T3-GLIS2 resulted in a marked increase in the self-renewal capacity, with colony formation persisting through 12 replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. GLI transcription factors modulate expression of multiple downstream targets including components of BMP, WNT, and SHH pathways. To interrogate these pathways as potential contributors to the enhanced self-renewal capacity, we conducted luciferase reporter assays and found that CBFA2T3-GLIS2 functioned as a strong activator of the BMP responsive element. Furthermore, expression of CBFA2T3-GLIS2 in Drosophila resulted in ectopic expression of endogenous dpp, the fly homolog of BMP4, and conferred a dpp gain of function phenotype. Taken together these data identify a novel inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in ∼35% of non-infant pediatric non-DS-AMKLs. The alteration of a key transcriptional regulator within the SHH signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway or downstream activated pathways may have a therapeutic benefit in this aggressive form of AML.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4867. doi:1538-7445.AM2012-4867