Using retroviral transduced bone marrow, several labs have shown that NUP98-HOXA9, NUP98-HHEX, NUP98-NSD1, and NUP98-TOP1 fusions are leukemogenic, and NUP98-HOXA9 and NHD13 fusions have been shown to be been shown to be leukemogenic in genetically engineered mice. However, a NUP98-TOP1 fusion was at best weakly oncogenic when expressed from a Vav promoter in transgenic mice. In order to better understand the leukemogenicity of NUP98 fused to non-HOX genes, we generated mice that express either a NUP98-RAP1GDS (NRG) or NUP98-PHF23 (NP23) fusion in hematopoietic cells using Vav regulatory elements to direct expression in the hematopoietic compartment. NRG mice were generated and transmitted the transgene in expected Mendellian ratio, and were documented to express the transgene. However, we could discern no hematopoietic abnormalities in these mice. NP23 mice have been generated, and we have followed a large cohort of offspring from two founders. Almost 100% of these mice develop leukemia within 1 year of life. Interestingly, the leukemic phenotype is very broad, including T and B cell leukemias, myeloid leukemias, and erythroid leukemias. These leukemias are clonal, and frequently activate a cluster of genes within the Hoxa locus, including Hoxa7,9,10,11, and Meis1. In addition, we have identified novel genes, including Gm525, that are overexpressed in the NP23 leukemias. Chromatin immunoprecipitation/sequencing (ChIP-Seq) experiments demonstrate the presence of active histone marks (H3K4Me3), and the absence of inactive histone marks (H3K27Me2) at the Hoxa cluster; moreover, additional ChIP-seq experiments deomnstrate binding of the NP23 fusion protein at the H3K4Me3 sites, suggesting a mechanism for the leukemic transformation. This work has been presented in abstract form, and a manuscript is in preparation. We have also published a comprehensive review article on NUP98 fusion proteins in the prestigious journal Blood. Recent studies have demonstrated that HOXA9 is important for hematopoietic stem cell self-renewal and is one of the most differentially expressed genes in patients with AML and MDS. Indeed, Hoxa cluster genes, especially Hoxa7/9/10, were among the most differentially expressed genes in the NHD13, NP23, and CALM-AF10 mice described in previous projects, suggesting that Hoxa9 is an important target for leukemic transformation. Therefore, we have generated mice that express Hoxa9 in hematopoietic cells, using Vav regulatory elements to enable us to compare these mice to the NHD13 mice. Some of the potential founders did not transmit the transgene, despite having &gt;30 pups genotyped, suggesting that the transgene may have been embryonic lethal in these mice. We have euthanized mice with timed pregnancies, and were able to document embryos that were transgenic, further supporting the possibility that the transgene was embryonic lethal in some founders. Two founders were able to transmit the transgene; however, they only expressed levels of the Hoxa9 transgene that were only slightly higher than wild-type controls. Despite this low level of expression, the Hoxa9 mice have developed a leukemic phenotype. At approximately 12 months of age, approximately 25% of the Hoxa9 mice have developed a precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL). These pre-T LBLs are clonal and are typically accompanied by spontaneous Notch1 mutations. We are currently characterizing additional tumors, and assaying the Hoxa9 mice for evidence of disordered T cell differentiation prior to the onset of the pre-T LBL. A manuscript describing these findings is currently in preparation. We noted that Lin28b, a gene involved in microRNA metabolism and stem cell pluripotency, was overexpressed in the NHD13 mice. To determine if this overexpression was related to malignant transformation, we used Vav regulatory elements to express Lin28b in hematopoietic cells. Lin28b transgenic mice develop an aggressive, clonal, lethal peripheral T cell lymphoma, which is associated with release of inflammatory cytokines. The cell of origin shows immunophenotypic and gene expression features consistent with those of T follicular helper (TFH) cells. A manuscript describing these features was recently published, and we have shipped these mice to several collaborators. Deep sequencing studies have recently identified recurrent point mutations in ASXL1 and U2AF1. We have generated transgenic constructs to express the mutant proteins in the hematopoietic cells of mice. With respect to ASXL1, despite injections of &gt;150 mouse eggs, and the birth of &gt;150 pups, no transgenic mice were identified, leading to the suspicion that expression of the mutant ASXL1 protein was embryonic lethal. We have now generated several founder mice with a revised, conditional ASXL1 mutant protein. We have recently crossed these mice to Cre-expressing mice, in order to activate the transgene. With respect to U2AF1, we have recently generated 5 founders that have transmitted the mutant protein. The mutant protein is expressed in hematopoietic cells, albeit at levels 20% that of the endogenous, wild-type U2AF1 protein. Although it is possible that expression of this level of protein will be oncogenic, we are developing a strategy to cross the U2AF1 transgenic mice to either hemizygous or homozygous U2AF1 knockout mice, in order to increase the ratio of mutant to wild-type U2AF1 protein.