Leukemic transformation in mice expressing a NUP98-HOXD13 transgene is accompanied by spontaneous mutations in Nras, Kras, and Cbl

Abstract

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...

PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia

“…31,37 We previously showed that overexpression of BCL-2 with consequent blocking of apoptosis could have a similar effect in the presence of an established MDS-initiating cell. 16 The effects seen with loss of PUMA extend our previous work by identifying the BH3-only protein that is essential for initiating apoptosis in this setting.…”

“…Activating mutations in Nras and Kras are typical of AML in the NHD13 model. 16 The presence of self-renewing cells capable of generating MDS was demonstrated by transplantation of NHD13 bone marrow cells. 19 However, the phenotype of these cells was not defined beyond the absence of lineage markers; a population that includes both HSCs and committed progenitors.…”

“…Approximately one-third of mice progress to AML within 10-14 months and this is frequently associated with acquisition of Ras or Cbl mutations. 16 NHD13 mice also show evidence of increased apoptosis of bone marrow stem and progenitor cells. 17 In this model, we have demonstrated that apoptosis is driven by cell-intrinsic factors because it is effectively blocked by enforced expression of BCL2.…”

Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies

“…In a transgenic MDS NUP98/HOX13 mouse model, progression to sAML with acquisition of RAS and c-Cbl mutations occurs frequently. 18 We hypothesized that mutations inactivating oncogene degradation pathways may constitute a new class of molecular lesions in myeloid malignancies modifying current paradigms of leukemogenesis. We therefore investigated the presence of mutations in the Cbl family of E3 ubiqutin ligases in selected subtypes of malignant myeloid disorders and determined the phenotypic and functional features as well as clinical outcomes.…”

Inhibition of apoptosis by BCL2 prevents leukemic transformation of a murine myelodysplastic syndrome

“…8 The NHD13 model recapitulates many of the features of human MDS, with an early preleukemic phase of cytopenias and increased apoptosis in the bone marrow (BM) followed by the development of AML harboring mutations in genes such as N-Ras. 9 Here, we used this MDS model to investigate the role of apoptosis in cytopenias and leukemic transformation.…”