AMKL chimeric transcription factors are potent inducers of leukemia

Abstract

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.

Figure 1. AMKL Fusion Genes Lead to a Highly Penetrant Leukemia

(A) Murine bone marrow was transduced with a control retroviral construct (MIC) or one of four fusion genes (CBFA2T3-GLIS2, NIPBL-HOXB9, GATA2-HOXA9, or MN1-FLI1) followed by injection into lethally irradiated recipients. Leukemia free survival is shown. (B) Bone marrow sections from a representative mouse in each of the cohorts are shown. White arrows: neoplastic cells with megakaryocytic features, Yellow arrows: immature megakaryocytes, Red arrows: dysplastic megakaryocytes.

Figure 2. Cooperative Mutations in Fusion Driven Leukemias

(A) Cumulative SNVs/Indels as determined by WES in each of the subgroups. Total number of non-silent mutations are shown, in addition to those whose gene products are expressed as determined by RNAseq. Mutations were analyzed by PROVEAN prediction algorithm to assess their functional consequence. (B) Total number of cooperating mutations per tumor subtype as determined by aCGH and WES.

Figure 3. Hematopoietic Regulators Invoke Megakaryocytic Gene Expression Signatures

(A) Expression levels in murine leukemias of five key transcription factors in megakaryocytes: RUNX1, FLI1, TAL1, GATA1, and GATA2. MEP: megakaryocyte erythroid progenitor; PRE: proerythroblast; BPE: basophilic erythroblast; OCE: orthochromic erythroblast; MkP: megakaryocyte progenitor; iMk: immature megakaryocyte; MMk: mature megakaryocyte (B) Single sample GSEA was done for leukemias induced by each of the three fusions for target genes of the 6 occupancy patterns in megakaryoblasts as described by Tijssen and colleagues (21, 45).

Figure 4. Overexpression of Fusion Gene Partners is Insufficient to Induce a Highly Penetrant Leukemia with the Exception of MN1

(A) MN1, FLI1, GATA2, HOXA9, NIPBL^e1–6, and HOXB9 transduced bone marrow was injected into lethally irradiated recipients. Leukemia free survival is shown. Due to size restraints, the NIPBL^e1–6 retroviral construct carries NIPBL exons 1–6, the portion of the gene involved in the fusion.