RADIATION RESEARCH 182, 310–315 (2014) 0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13738.1 Leukemogenesis in Heterozygous PU.1 Knockout Mice Paula C. Genik, a,1 Irina Vyazunova, b,1 Leta S. Steffen, b Jeffery W. Bacher, b,c Helle Bielefeldt-Ohmann, d Scott McKercher, e Robert L. Ullrich, f Christina M. Fallgren, a Michael M. Weil a,2 and F. Andrew Ray a a Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado; b Promega Corporation, Madison, Wisconsin; c University of Wisconsin, Madison, Wisconsin; d School of Veterinary Science, University of Queensland, St. Lucia, Qld 4072, Australia; e Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford-Burnham Medical Research Institute, La Jolla, California; and f University of Texas Medical Branch Cancer Center, Galveston, Texas Genik, P. C., Vyazunova, I., Steffen, L. S., Bacher, J. W., Bielefeldt-Ohmann, H., McKirchner, S., Ullrich, R. L., Fallgren, C. M., Weil, M. M. and Ray, F. A. Leukemogenesis in Heterozygous PU.1 Knockout Mice. Radiat. Res. 182, 310– 315 (2014). Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic back- ground of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss. Ó 2014 by Radiation Research Society INTRODUCTION Radiation-induced acute myeloid leukemia (rAML) has been extensively studied in the mouse. In susceptible inbred strains such as CBA, the incidence of rAML increases to 15–25% with increasing radiation dose up to about 3 Gy and then decreases (1–3). Sfpi1, the mouse homologue of human PU.1, is a lineage-specific transcription factor involved in myeloid differentiation that behaves as a tumor suppressor gene in murine rAML. (Following common practice in the literature, we will use the human nomencla- ture for the mouse gene in this report.) Both copies of murine PU.1 are usually lost in radiation leukemogenesis, one through a large-scale chromosome 2 deletion and the other by point mutation, usually at a CpG dinucleotide in codon 235 (4, 5). The result is a block in myeloid differentiation. Chromosome 2 deletions encompassing PU.1 are evident in about 5% of bone marrow cells 24 h after 3 Gy gamma irradiation and are almost certainly the consequence of radiation exposure (6). The point mutation in PU.1 codon 235 is generally a C to T transition at a CpG dinucleotide, a mutation associated with spontaneous deamination of methylcytosine rather than with radiation exposure (7). rAML data from radiation leukemogenesis experiments fit well with a two-mutation carcinogenesis model, which is based on the assumption that there are two rate-limiting events in rAML. The sequential radiation-induced deletion of one PU.1 allele in an appropriate target cell followed by a spontaneous point mutation in the remaining allele are candidates for those events (4, 5, 8). Germline inactivation of both copies of the PU.1 gene in the mouse is embryonic or neonatal lethal (9, 10). However, conditional knockout of both alleles in adult mice leads to AML at high incidence (95%) with a short latency of 13–34 weeks (11), as does biallelic promoter knockdown that decreases expression levels to 20% (12). This suggests that once both copies of PU.1 are lost, additional events required for leukemogenesis occur frequently and spontaneously. It is curious then that spontaneous AML in PU.1 heterozygous knockout mice (PU.1 þ/– ) have not been reported in the literature, since one allele of PU.1 is already constitutionally inactive in all target cells in these animals as compared to 5% of cells carrying PU.1 deletions in 3 Gy irradiated mice. There are several possible explanations for this observation. 1 These two authors contributed equally. 2 Address for correspondence: Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80525; e-mail: michael.weil@colostate.edu. 310