Leukemic Transformation in Mice Expressing a NUP98-HOXD13 Transgene Is Accompanied by Spontaneous Mutations in Receptor Tyrosine Kinase Pathway Genes.

The NUP98-HOXD13 (NHD13) fusion gene, formed by the t(2;11)(q31;p15), has been identified in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We recently reported that mice which express an NHD13 transgene develop MDS; over half of these mice eventually develop acute leukemia between six and fourteen months. An emerging paradigm holds that at least one event from each of two classes is required for leukemic transformation: Class I (proliferation or survival promoting events) and Class II (differentiation blocking events). The NHD13 fusion protein has previously been shown to fit the criteria of a Class II event, as it impairs differentiation of haematopoietic cells. Therefore, we used a candidate gene approach that focused on Class I mutations to search for secondary genetic events that might collaborate with the NHD13 transgene. Since the most frequently mutated Class I genes in human myeloid leukemia are FLT3 (24%), NRAS (16%), KIT (5%) and KRAS (4%), we sequenced relevant regions of these genes (Flt3, exons 14, 15 and 20; Kit, exons 8 and 17; Nras, exons 2 and 3; and Kras, exons 2 and 3) in a cohort of 26 mice with AML. We found no Flt3 or Kit mutations in this cohort; however, we identified 3 (12%) Nras and 3 (12%) Kras mutations. All six of the ras mutations identified in the AML cohort occurred within codon 12, which is predicted to result in a constitutively active ras protein. We also screened a smaller sample set of NHD13 mice that developed pre-T lymphoblastic leukemia/lymphoma and found 0/8 Nras mutations but 1/8 (13%) Kras mutation; this mutation occurred in codon 13. CBL is a ubiquitin ligase that is responsible for targeting degradation of selected receptor tyrosine kinases. Intriguingly, CBL mutations that lead to FLT3 activation have recently been identified in AML patients. Therefore, we screened the NHD13 AML cohort for mutations in Cbl and identified a deletion of 297 bp that results in loss of the exon 8 splice acceptor site. RT-PCR demonstrated an aberrant Cbl cDNA which lacks the RING finger domain encoded by exon 8. We have previously reported the derivation of an IL-3 dependent haematopoietic cell line from embryonic stem (ES) cells that expressed an NHD13 “knock-in” allele. Preliminary results indicate that the activated ras mutations are capable of transforming the cell line to IL-3 independence, supporting the contention that the ras mutations (Class I) complement the NHD13 fusion protein (Class II). We are currently investigating the potential of the Cbl mutant to transform the IL3-dependent cell line. Although the current paradigm for AML predicts these results, this is the first report of spontaneous Class I (Cbl or ras) mutations that complement a defined, genetically engineered Class II mutation in an animal model of AML. These results have general implications for other oncogenic mouse models, as well as specific implications for the nature of NHD13-mediated leukemogenesis.