E2F and Myc-Induced Lymphoma Figure 1. Myc-induced lymphomagenesis is influenced by E2F status. (A) Kaplan-Meier survival analysis showing the percentage of tumor-free Em-myc mice with the indicated E2F1 genotypes (E2f1+/+, E2f1+/2, and E2f12/2 sibling mice in the E2F1 cohort) plotted against the onset of disease. Onset of disease did not vary with E2F1 status. (B) Same as (A) but for E2f3+/+, E2f3+/2, and E2f32/2 sibling mice in the E2F3 cohort. Onset of disease did not vary with E2F3 status. (C) Same as (A) but for E2f2+/+, E2f2+/2, and E2f22/2 sibling mice in the E2F2 cohort. Disease onset was faster in E2f22/2 Em-myc mice (median age 63 days) than in E2f2+/+ Em-myc mice (median age 126 days), and the onset curves were significantly different (P,0.0001). E2f2+/2 Em-myc mice exhibited an intermediate onset of disease (median age 92 days), and the onset curve was significantly different from that of the E2f2+/+ Em- myc mice (P,0.0001) and the E2f22/2 Em-myc mice (P,0.0001). (D) Same as (A) but for E2f4+/+, E2f4+/2, and E2f42/2 sibling mice in the E2F4 cohort. Disease onset was slower in E2f42/2 Em-myc mice (median age 375 days) than in E2f4+/+ Em-myc mice (median age 110 days), and the onset curves were significantly different (P,0.0001). Relative to the E2f4+/+ Em-myc mice, E2f4+/2 Em-myc mice may have a slightly delayed disease onset (P,0.0465). doi:10.1371/journal.pgen.1000640.g001 appearance of each of the wild type curves was similar, with some mice succumbing early and others succumbing late. As shown in Figure 1A, the loss of E2F1 function did not alter the timing of lymphoma appearance; there was no statistical difference between tumor onset curves when comparing E2f1+/+ (n = 37), E2f1+/2 (n = 91) and E2f12/2 (n = 40) mice. The failure of E2F1 status to influence lymphomagenesis conflicts with the earlier finding that E2F1 deficiency delays lymphoma develop- ment in Em-myc mice [23]. That study attributed the delay to a defect in p27Kip1 degradation and reduced Myc-induced prolifer- ation when E2F1 is reduced or absent. In our assessments the level of p27Kip1 protein in splenic B lineage cells did not vary according E2F1 status but rather with progression to disease: p27Kip1 was highest in cells isolated from non-transgenic siblings, reduced in healthy Em-myc transgenics, and lower still in very sick mice and tumors (Figure S3A). In addition, the accelerated proliferation induced by expression of the Em-myc transgene [24] was unaffected by E2F1 deficiency: splenic B lineage cells isolated from E2f1 wild type, heterozygous and Em-myc transgenic mice all exhibited the same dramatically higher proliferative index when compared to that of cells isolated from non-transgenic siblings (Figure S3B). We note that, analogous to our results, E2F1 deficiency did not alter Myc-induced T cell lymphomagenesis [25]. Given that the timing of Em-myc-driven tumor development can be influenced by strain background [26] and breeding strategy, we can only surmise that these potential differences or the specific E2f1-null allele [27,28] used in our studies versus that of Baudino and colleagues are sufficient to account for the discrepant effects of E2F1 deficiency. As shown in Figure 1B, lymphoma onset was also not appreciably influenced by E2F3 status. While the number of E2f3-null animals was low in this study, reflecting the low number of E2f3-null mice born, there is nevertheless no suggestion that E2F3 loss was protective as several E2f3-null mice died before the average age of onset for their wild type siblings. In contrast to the results seen with the E2f1 and E2f3 knockout animals, a deficiency of E2F2 dramatically accelerated the appearance of lymphoma (Figure 1C). The E2f22/2 mice were prone to early tumor onset with tumors appearing on average 60 days earlier than in their wild-type siblings and there was a significant difference (p,0.0001) between the E2f2+/+ and E2f22/2 tumor curves. Notably, the E2f2+/2 mice exhibited a median tumor-free span of 92 days and a significantly accelerated course of disease compared to wild-type siblings (p,0.0001). The PLoS Genetics | www.plosgenetics.org 3 September 2009 | Volume 5 | Issue 9 | e1000640