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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...