Characterizing the Role of KMT2C Mutations in Rapid-Onset Infant Leukemia

Infant leukemia (IL) presents in children less than one year of age with aggressive features such as high white blood cell counts, hepatosplenomegaly, central nervous system involvement and skin infiltration. Chromosomal rearrangements involving the KMT2A gene, termed 'KMT2A-r’ are seen in 80% of IL patients. However, KMT2A-r alone is not sufficient to induce rapid-onset leukemia, suggesting that mutations in other leukemia-related genes are required. Heterozygous germline mutations in the KMT2C gene were found to be enriched in IL patients. As the neonatal microenvironment strongly influences KMT2A-r driven leukemia phenotypes, embryonic models are required. Given their highly conserved hematopoiesis and access to early hematopoietic progenitors, we used zebrafish (Danio rerio) to model KMT2C mutations in IL.

We generated novel loss-of function germline mutations in the KMT2C zebrafish homologues, kmt2ca and kmt2cb. Characterization using whole-mount in situ hybridization showed reduced differentiation of erythrocytes and leukocytes in kmt2ca mutant embryos. In contrast, kmt2cb loss did not affect hematopoiesis. Flow cytometry of whole kidney marrows (WKMs, equivalent to human bone marrow) from kmt2ca mutant adult fish revealed an expansion of myeloid and progenitor cells accompanied by a decrease in erythrocytes and lymphocytes, suggesting myeloproliferative neoplasm. kmt2ca mutant fish had a mean survival of 11 months. Between 7 and 13 months of age, mutant fish exhibited 'dropsy', a pathological bloating condition characterized by distention, sloughing of scales, protruding mouth, bulging eyes, as well as difficulty breathing and maintaining buoyancy, indicating infections in major organs. To elucidate the transcriptional programs regulated by kmt2ca, we performed RNA sequencing of whole embryos at 24 hours post-fertilization. We found a total of 928 downregulated genes and 379 upregulated genes. Erythrocyte differentiation, heme biosynthesis and immune-related processes were downregulated whereas tyrosine and fatty acid metabolism were upregulated. In conclusion, we have generated a novel kmt2ca mutant zebrafish line that exhibits anemia and limited leukocyte specification. We have now introduced human KMT2A-MLLT3 or KMT2A-MLLT1 fusion genes in lmo2⁺ early hematopoietic progenitors to evaluate how KMT2C loss contributes to KMT2A-r-mediated IL. We are in the process of characterizing hematopoietic lineage specification in these compound fish.

In summary, our zebrafish models recapitulate the germline nature of KMT2C mutations and the tissue-specific nature of KMT2A fusions seen in patients. These models can be leveraged as in vivo screening platforms to identify novel therapies which will improve outcomes for this incredibly vulnerable patient population.

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