mass of multiple small nodules in the mediastine with little or no associated spleen or peripheral lymph node enlargement. Despite their unusual appearance, the atypical tumors that were tested demonstrated Igh gene rearrangement confirming their B lymphoid origin. Overall, eleven E2f42/2 tumors, including examples of standard, late, and atypical types, were assessed for clonality and all proved to be monoclonal. In summary, along with the general delay in tumor onset there was also a difference in the predominant site of lymphomagenesis and gross morphological appearance of tumors in the E2f42/2 Em-myc mice. Distinct types of B lineage lymphoma that are influenced by the presence of E2F2 or E2F4 As a further basis for exploring the effects of E2F loss of function on the development of Myc-induced tumors, we have made use of genome-scale gene expression profiles to characterize the tumors arising in the E2f2-null and E2f4-null Em-myc mice. Our recent work has identified expression profiles that distinguish different tumor types within the Em-myc mice including a cluster characterized by generally early onset and pre-B markers as well as three distinct clusters characterized by late onset and different sets of more differentiated B lineage markers [41]. Examples of wild type tumors exhibiting this early and late onset pattern are shown in Figure 7A. Analysis of the tumors from the E2f22/2 mice indicated that they were relatively homogeneous with respect to their expression profiles and reflected the characteristics of the ‘‘early’’ category of wild type tumors. In contrast, the tumor types from the E2f42/2 mice were heterogeneous with a distribution across both broad categories of the wild type tumors. The distribution of the E2f42/2 tumors corresponded with their dissection phenotypes - standard, late and atypical - described above. Three early-onset E2f42/2 tumors, all with the standard morphological phenotype, clustered with the ‘‘early’’ wild type and E2f22/2 tumors. Three more E2f42/2 tumors, all with the late morphological phenotype, clustered alongside a group of wild type tumors that overexpress genes characteristic of plasmacytomas. These particular E2f42/2 tumors shared marginally decreased myc mRNA and low Myc protein compared to other E2f42/2 tumors (data not shown). The final six E2f42/2 tumors were all of the atypical phenotype and segregated in the ‘‘early’’ category despite being late onset chronologically. These tumors shared qualities with the ‘‘early-standard’’ tumors in that they featured high levels of myc mRNA and Myc protein (data not shown). Notably, these tumors fell at the extreme end of the early cluster and beside a rare group of wild type tumors that had similarly modest spleen enlargement and late chronological onset. In fact, these tumors highlight a significant subgroup within the ‘‘early’’ category that we have designated ‘‘early-atypical’’. This tumor subgroup was notable for a high incidence of p53 deletion or mutation (67% of tumors assessed versus 18% of other wild type tumors assessed; by Fisher’s exact test P = 0.0061). Among the genes that characterized each tumor cluster, increased expression of number of genes distinguished this subgroup from both ‘‘early-standard’’ and ‘‘late’’ clusters (Figure S10). Cdkn2a, the locus that encodes the two tumor suppressors p16(INK4a) and p19(ARF), was preferentially ex- pressed in these tumors. Given that most of these tumors were mutant for p53, the increased expression may be a consequence of a role for p53 in negatively regulating the expression of ARF [42]. Other genes that were particularly highly expressed in the ‘‘early- atypical’’ tumors included Dlk1, a member of the epidermal growth factor-like family that influences B lineage differentiation [43,44] and Fzd6, a receptor for Wnt signaling and a frizzled family member [45]. E2F and Myc-Induced Lymphoma To further characterize the distinctions in the Em-myc tumors, we have made use of signatures of various cell signaling pathways that have previously been shown to distinguish human Burkitt lymphoma (BL) from diffuse large cell B lymphoma (DLBCL) [46]. These include signatures for Myc pathway activity, the expression of a subgroup of germinal-center B-cell genes, the expression of MHC class I genes, and NFkB pathway activity. An analysis of tumors from the E2f2-null and E2f4-null mice using these pathway signatures is shown in Figure 7B. Consistent with the analysis of whole genome gene expression data in Figure 7A that revealed distinct types of B lymphoma, the analysis using pathway signatures also revealed that the tumors from the E2f2-null mice exhibited a pattern similar to the ‘‘early’’ tumors, characterized by high Myc and germinal center signatures, whereas the tumors from E2f4-null mice were heterogeneous with respect to these patterns. The atypical E2f42/2 tumors did not feature the usual ‘‘late’’ characteristics but instead had elevated Myc and germinal center signatures and low MHC Class I and NF-KB signatures. These tumors highlight the existence of certain tumors of late chronological onset, whether E2F wild type or E2f42/2, that were unusual in their behavior. Discussion A dominant characteristic of the oncogenic process is complexity – the realization that the development of a tumor results from a complex array of genetic alterations that ultimately combine to contribute to the oncogenic phenotype. While transgenic mouse models reduce this complexity by fixing one event, various studies have shown a clear heterogeneity in the tumors that develop reflecting the acquisition of additional alterations. Based on the studies we describe here, we suggest that there are at least two distinct types of B lineage lymphoma that develop in the Em-myc mice. In mice with wild type E2F function, the predominant tumor arising with an early onset likely reflects the relative abundance of the target cell population. We suggest that the emergence of these tumors reflects a stochastic acquisition of additional mutations. Since this is a probabilistic event, there is then an opportunity for tumors to emerge from other populations of cells with characteristics distinct from the ‘‘early-standard’’ tumors. We propose that the late-onset tumors develop more slowly because the target cell populations are less abundant. It is also possible that these late-onset tumors are genetically more complex, requiring more mutations than the predominant form of tumors. The work we present here provides clear evidence for an interaction between Myc and two E2Fs as seen by the effect on timing of tumor onset and the different characteristics of the lymphomas that arise in the absence of E2F2 or E2F4. The results suggest distinct roles for the E2F2 and E2F4 proteins in the process of B lineage development that then impacts Myc-mediated oncogenesis. A role for E2F4 in cell differentiation is consistent with past work that documented abnormalities in hematopoietic lineage development as well as the development of the gut and nasal epithelia with loss of E2F4 activity [19,40,47–49]. This past work revealed a deficiency of various mature hematopoietic cell types and defects in the differentiation of immature cells in the absence of E2F4. Taken together, these observations suggest a critical role for E2F4 activity in controlling the maturation of hematopoietic lineage cells, including the B lymphoid compart- ment that generates the target cells for Em-myc-induced lympho- magenesis. A role for E2F2 appears to be more complex. E2F2 has generally been characterized as one of the E2Fs involved in the PLoS Genetics | www.plosgenetics.org 10 September 2009 | Volume 5 | Issue 9 | e1000640