We previously identified mutations of candidate genes including Flt3, Nras, Kras, Ptpn11, and Cbl. More recently, in collaboration with Dr. Paul Meltzer, we have used multiplex PCR and deep sequencing to identify mutations in 24 candidate genes in a set of 152 mouse leukemias and identified spontaneous, acquired mutations in Nras, Kras, Tp53, Notch1, Flt3, Ptpn11, Cbl, and Idh1; a manuscript describing these findings is in preparation. Again, in collaboration with Dr. Meltzer, we analyzed whole-exome deep sequence of the leukemias that develop in NUP98-PHF23 (NP23) mice and the PTCL that developed in Lin28b mice. Unexpectedly, we identified frequent mutations in progenitor B1 cell ALL in the Bcor and Jak1/2 genes. A manuscript describing these findings was published in FY2018. Furthermore, we have used CRISPR to introduce Bcor mutations in primary WT and NP23 BM cells; these cells have been transplanted into recipient mice to determine if we can verify collaboration between NP23 and Bcor in vivo. These experiments indicate that NP23 transgene will collaborate with CRISPR induced Bcor frameshift mutations. Additional in vivo genetic crosses, performed in collaboration with Dr. Donald Small and Dr. Trang Hoang have demonstrated that the NHD13 transgene can collaborate with a Flt3 ITD to induce a myeloid leukemia, and there is an in vivo interaction between SCL and c-Kit that is important for early hematopoietic differentiation. A manuscript describing the Flt3 and NHD13 interaction, as well as its potential clinical relevance, has recently been published. As mentioned above, spontaneous mutations of IDH2 were identified in NHD13 leukemias. These mutations occur at R140Q; homologous residues are mutated in human leukemia. We crossed IDH2 R140Q transgenic mice with NHD13 mice; the offspring develop a form of early T cell precursor (ETP) leukemia that resembles the human disease in terms of clinical presentation, immunophenotype, gene expression profile, and collaborative mutations. A manuscript describing these findings is in preparation. We previously generated mice that expressed either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment; both NP23 and NHD13 mice develop a wide variety of leukemia at 9-14 months of age. Surprisingly, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within 3 months. The leukemias were characterized by extraordinarily high WBC and replacement of the thymus with AML cells; the percent of malignant myeloid cells in the thymus was often higher than the bone marrow (BM). These findings led to the intriguing hypothesis that the AML in NP23-NHD13 mice arose in the thymus, as opposed to the BM. To investigate this possibility, we transplanted unfractionated cells or residual CD4-/CD8- double negative (DN) thymocytes from the thymus of a NP23-NHD13 mouse invaded by AML cells irradiated recipients. All mice developed AML within 26 days, indicating that the AML was aggressive and transplantable, and could be transmitted by DN thymocytes. To rule out the possibility that the leukemia was transmitted by rare, contaminant AML cells, we repeated the experiment, twice, using DN thymocytes from 4-5 wk old mice with no signs of leukemia. DN thymocytes again transmitted AML. Fractionating DN thymocytes into DN1-DN4 sub-populations revealed that AML initiating cells were found in the DN1 and DN2 compartments. Taken together, these results demonstrate that NP23-NHD13 thymic progenitors retain myeloid and erythroid potential and are potently leukemogenic, leading to the intriguing hypothesis that some human AML might originate in the thymus. Finally, in collaboration with Dr. Durum of CCR, we were able to demonstrate that IL7R mutants collaborated with Ras mutations to rapidly generate T-ALL in mice.
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