In collaboration with Dr. Donald Small (Johns Hopkins University), we have crossed mice that express a NUP98-HOXD13 (NHD13) transgene and develop myelodysplastic syndrome (MDS) with mice that express a FLT3 internal tandem duplication and develop a myeloproliferative disesase (MPD). Both of these abnormalities were initially identified in patients with acute myeloid leukemia (AML), underscoring the clinical relevance of this experiment. Although very few of the NHD13 or FLT3 only mice developed AML, 100% of the mice with both NHD13 and FLT3 alleles devleoped AML, demonstrating that these two genetic lesions collaboarted with one another quite powerfully. Retroviral insertional mutagenesis (RIM) has proven to be a valuable whole-genome screen for the identification of genes involved in malignant transformation. Common insertion sites (CIS) are regions that have retroviral integrations in more than one leukemic sample, have been biologically selected as dominant clones, and are thought to harbor genes important for malignant transformation. We previously described the use of RIM to identify a number of genes which collaborate with a NUP98-HOXD13 to produce a leukemic clone. We have used the same approach to identify genes that collaborate with a CALM-AF10 fusion to produce acute leukemia. We identified 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. Of note, over 25% of the mice had retroviral integrations near Zeb2, a transcriptional co-repressor in the TGF-beta signaling pathway, leading to over-expression of the Zeb2-transcript. 91% of mice with Zeb2 insertions developed B-lineage ALL suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. Over half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2 and Ras pathway mutations. We then searched for Nras, Kras, and Ptpn11 point mutations in this series of CALM-AF10 leukemic mice infected with the replication competent retrovirus . Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10. The prior experiments used either gene targeting or RIM to experimentally induce mutations that we suspected might be leukemogenic in combination with an NHD13 or CALM-AF10 transgene. As a complementary approach, we searched for spontaneous (ie, not induced by RIM or gene targeting) mutations that might collaborate with the NHD13 or CALM-AF10 fusions. We searched for mutations of Runx1, Npm1, Tp53, Flt3, Kit, Nras, Kras, and Cbl. We thought this was an important study to help validate murine AML models, because there are no examples of spontaneous N/Kras mutations associated with murine AML. We studied 22-26 mice with each transgene. We found no mutations of Runx1, Npm1, Tp53, or Kit. 25-30 % of both NHD13 and CALM-AF10 mice had Nras or Kras mutations. Intriguingly, over 20% of the CALM-AF10 mice had Flt3 mutations, whereas none of the NHD13 mice had Flt3 mutations. One potential explanation for the lack of Flt3 mutations in the NHD13 mice is as follows. Both the NHD13 and CALM-AF10 fusions lead to overexpression of HOXA-cluster genes, specifically HOXA7/9/10, in the bone marrow of clinically healthy (pre-leukemic) mice. However, one distinction is that the CALM-AF10 mice overexpress Meis1, whereas the NHD13 mice downregulate Meis1. Therefore, the lack of Flt3 mutations in the NHD13 mice may be due to the lack of Meis1 expression in the NHD13 mice, as Meis1 is reported to drive Flt3 expression. Given that Nras, Kras, and Flt3 mutations have been shown to enhance proliferation, these results support a working hypothesis that predicts AML cells have one mutation which impairs differentiation (such as NHD13 or CALM-AF10), and a second, complementary mutation which enhances proliferation or inhibit apoptosis. Ongoing and planned experiments include using gene expression arrays to identify genes and pathways that are upregulated in bone marrow from CALM-AF10 mice compared to bone marrow from clinically healthy CALM-AF10 mice. We will infect IL3-dependent, non-malignant NHD13 cell lines with a lentiviral siRNA library, and determine if down regulation of specific gene(s) results in cells that are IL3-independent and malignant.
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