The NUP98-HOXD13 (NHD13) fusion gene occurs in patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). We reported that transgenic mice expressing NHD13 develop MDS, and that more than half of these mice eventually progress to acute leukemia. The latency period suggests a requirement for at least 1 complementary event before leukemic transformation. We conducted a candidate gene search for complementary events focused on genes that are frequently mutated in human myeloid leukemia. We investigated 22 ANLL samples and found a high frequency of Nras and Kras mutations, an absence of Npm1, p53, Runx1, Kit and Flt3 mutations, and a single Cbl mutation. Our findings support a working hypothesis that predicts that ANLL cases have one mutation which inhibits differentiation, and a complementary mutation which enhances proliferation or inhibit apoptosis. In addition, we provide the first evidence for spontaneous collaborating mutations in a IntroductionWe previously reported a transgenic mouse model for myelodysplastic syndrome (MDS), in which NUP98-HOXD13 (NHD13) mice develop MDS at an early age, and progress to acute leukemia between 4 and 14 months of age. 1 This latency period is likely due to a requirement for additional genetic events before leukemic transformation. Many studies have investigated the nature of such secondary events through experimental induction of complementary events, such as retroviral insertional mutagenesis 2-4 or ENUinduced mutagenesis. 5,6 To our knowledge, no study has investigated the nature of complementary mutations that occur spontaneously. Therefore, we evaluated NHD13 ANLL samples for the presence of mutations commonly seen in patients with ANLL. Methods DNA and RNA isolationAll animal experiments were conducted with the approval of the NIH Intramural Animal Care and Use Committee. Peripheral blood complete blood counts were obtained, bone marrow was harvested for cytospins, and paraffin-embedded spleen and liver were stained with hematoxylin and eosin. Routine immunohistochemical stains included F4/80, CD3, B220, and myeloperoxidase (MPO), and ANLL diagnosis was based on the Bethesda proposals for hematopoietic neoplasms in mice. 7,8 Effaced spleen tissue from NHD13 mice with acute leukemia was snap frozen on dry ice. DNA and RNA were prepared by standard techniques. RT-PCR and PCRReverse transcription (RT) was performed using Superscript II (Invitrogen, Carlsbad, CA). Genomic-and RT-polymerase chain reaction (PCR) were performed using either Supermix (Invitrogen) or Taq DNA Polymerase (Invitrogen). Primers, thermal cycling profiles, and regions amplified are listed in Tables S1 and S2 (available on the Blood website; see the Supplemental Materials link at the top of the online article). PCR products were purified using Qiagen (Valencia, CA) protocols, and were directly sequenced (Retrogen, San Diego, CA). Sequence chromatograms were manually inspected to detect mutations ( Figure S1).Reference sequences (NCBI accession numbers 9 ) used were as follows...

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.