<i>KMT2C</i> Mutations Co-Operate with <i>KMT2A-MLLT1</i> Fusion to Inhibit Hematopoietic Differentiation in a Zebrafish Model of Infant Leukemia

Infant leukemia (IL) is an aggressive form of leukemia that occurs in children less than one year of age. It is characterized by high white blood cell counts, hepatosplenomegaly, central nervous system involvement and skin infiltration. Compared to leukemia in older children, IL has increased relapse rates and higher treatment-related morbidities. Fusions involving the KMT2A gene, termed ‘KMT2A-r‘ are seen in 70-80% of infant acute lymphoblastic leukemia and in 50% of infant acute myeloid leukemia patients. However, KMT2A-r alone is not sufficient to induce rapid-onset leukemia, suggesting the presence of additional cooperative mutations. Heterozygous germline mutations in the KMT2C gene were found to be enriched in IL patients with KMT2A-r. Further, the neonatal microenvironment strongly influences KMT2A-r-driven leukemia phenotypes. A highly conserved hematopoietic system and access to early embryonic progenitors make the zebrafish (Danio rerio) an ideal preclinical model for IL. We established a loss-of-function kmt2ca zebrafish mutant using CRISPR-Cas9 mutagenesis. Characterization of hematopoiesis by in situ hybridization showed reduced primitive and definitive erythropoiesis and myelopoiesis at 24- and 48-hours post-fertilization (hpf), respectively, in kmt2ca-/- embryos compared to wildtype. While we observed no changes in hematopoietic stem and progenitor cell specification, cebpa+ myeloid progenitors were upregulated in kmt2ca-/- embryos. Similarly, in adult kmt2ca-/- fish, we observed a significant expansion of myeloid cells accompanied by reduced erythrocytes and lymphocytes. Our findings suggest that kmt2ca loss inhibits hematopoietic differentiation in zebrafish. To model the co-operativity between KMT2C mutations and the KMT2A-MLLT1 fusion, we expressed the human KMT2A-MLLT1 fusion under the zebrafish lmo2 promoter, which drives expression in early hematopoietic progenitors. Using in situ hybridization, we found that zebrafish harboring both the kmt2ca-/- mutation and the KMT2A-MLLT1 fusion exhibited severely reduced expression of hbbe3+ erythrocytes and lcp1+ leukocytes, compared with zebrafish harboring kmt2ca mutation alone, fusion alone or wildtype. KMT2A-r fusions are known to disrupt hematopoietic differentiation via overexpression of HOX genes, regulated by the MEN1-MEIS1 complex. Moreover, enforced co-overexpression of Hoxa9 and Meis1 in mice promotes leukemia with short latency. At 24 hpf, using quantitative PCR, we observed an upregulation of meis1b and hoxa9a genes in kmt2ca-/-:KMT2A-MLLT1 embryos compared with zebrafish harboring kmt2ca mutation alone, fusion alone or wildtype. The inherent overexpression of these pro-leukemia genes may accelerate leukemia development, a phenotype currently under investigation. In conclusion, our preliminary findings suggest that kmt2ca mutations cooperate with the KMT2A-MLLT1 fusion to activate leukemogenic pathways. This in turn may inhibit hematopoietic differentiation and promote the expansion of immature myeloid progenitors, potentially resulting in rapid-onset leukemia. Our zebrafish mutant and transgenic lines thus represent high fidelity in vivo models of IL that can be leveraged for therapeutic screening.

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