Identification of Oncogenic Collaborators Providing Insights into the Tumor Suppressing Function of the Eed Polycomb Group (PcG) Gene.

Polycomb Group (PcG) proteins are key regulators of normal and leukemic hematopoiesis (Lessard and Sauvageau, Nature 2003; Lessard and al., Genes Dev 1999). The eed gene is part of a PcG complex together with Ezh2 and Suz12. Evidence from our laboratory indicates that eed plays a crucial role in the negative regulation of the proliferative capacity of lymphoid and myeloid progenitors, suggesting that this PcG gene is a tumor suppressor. Interestingly, the human eed locus is in a region of chromosome 11 (11q14.2-22.3) which is associated with recurrent deletions in several reported cases of B-CLL, mantle cell lymphoma (MCL) and T-PLL. We now show that when exposed to ionizing radiation at 5 weeks of age, both eed^hypo/hypo and eed^null/+ mutant mice developed mono- or oligoclonal B and T cell lymphomas with much shorter latencies than observed in littermate controls (50% lethality at 16 wks; 27 wks and 34 wks post-irradiation for eed^hypo/hypo, eed^null/+ and controls, respectively). We reasoned that new pathways involved in tumorigenesis could be at play in this eed sensitized background, potentially allowing for the identification of new oncogenic collaborators. Therefore, a proviral insertional mutagenesis screen was conducted. Neonatal MMLV infection of eed mutant mice dramatically accelerated the occurrence of B and T cell lymphomas (50% lethality at 16 wks; 22 wks and 26 wks post-infection for eed^hypo/hypo,eed^hypo/+ and controls, respectively). To characterize retroviral insertions inverse-PCR (IPCR) was performed using genomic DNA isolated from 32 eed^hypo/hypo and 27 wild-type tumors. More than 780 IPCR products were recovered and purified. To date, at least 140 retroviral insertion sites (RIS) have been cloned, sequenced and mapped against the mouse genome. Of these, 7 insertion targeting gene loci known to be cancer-related, like Pim1 (n= 7 tumors), Notch1 (n= 14 tumors) and c-Myc (n= 3 tumors) have been identified. In addition, 8 new common insertion sites (CIS), such as Socs1 (n= 6 tumors), Gadd45g (n= 5 tumors) and Fgfr3 (n= 6 tumors) have also been identified. Rearrangements of the Pim1 locus were found in more than 22% of the eed^1989/1989 mutant mice whereas none could be found in wild-type littermates. Moreover, RT-PCR analysis indicates that the Pim1 gene is clearly overexpressed in 6 out of 16 eed^1989/1989 tumors analyzed compared to wild-type, further supporting a key role for the Pim1 pathway in eed sensitized tumor cells. Thus, the data presented in this study provide evidence that eed has tumor suppressing function. It also points to a novel mechanism of tumor suppression by a PcG protein. Based on these results, it will be interesting to study the status of eed and of the newly identified CISs in human diseases associated with deletions of 11q14.2-22.3.