Abstract 4867: Identification of an inv(16)-encoded CBFA2T3-GLIS2 fusion protein in 34% of non-infant acute megkaryoblastic leukemias: A report from the Pediatric Cancer Genome Project

Abstract 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.AM2012-486

The genomic landscape of core-binding factor acute myeloid leukemias

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes

An Inv(16)(p13.3q24.3)-Encoded CBFA2T3-GLIS2 Fusion Protein Defines an Aggressive Subtype of Pediatric Acute Megakaryoblastic Leukemia

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis. ► CBFA2T3-GLIS2 is a recurrent fusion gene in pediatric AMKL ► CBFA2T3-GLIS2 AMKL has a distinct expression profile and an inferior outcome ► CBFA2T3-GLIS2 induces BMP signaling and enhanced self-renewal of progenitor cell

The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL