To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the non-essential genome. Using these signatures, we generated a catalogue of 297 functional modules, and assigned function to 144 previously uncharacterised genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional cross-talk between biological processes, suggesting general design principles of genetic interactomes.

BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancerpreventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly

S U M M A R YCell proliferation is tightly coordinated with cell growth. The oncosuppressor proteins pRb and p53 may exert a key role in coupling growth and proliferation by controlling both ribosome biogenesis and cell cycle progression. In the present study we evaluated the relationship between the pRb and p53 status and rRNA transcriptional activity in histological sections of 343 human primary breast carcinomas. Ribosomal biogenesis was quantified by morphometric analysis of silver-stained interphase nucleolar organizer regions (AgNORs). pRb and p53 status was assessed by immunohistochemistry. Twenty-four tumors were considered to be pRb deleted, 260 tumors showed a phosphorylated-pRb labeling index (LI) up to 25%, and 55 tumors an LI Ͼ 25%. Tumors with deleted pRb or phosphorylated-pRb-LI Ն 25% were characterized by significantly greater mean AgNOR area values than those with unaltered pRb ( p Ͻ 0.001). In the 71 tumors with mutated p53 the NOR area mean value was greater than in the 272 tumors with normal p53 ( p Ͻ 0.001). Our results demonstrate, for the first time in vivo, that pRb and p53 status is related to the ribosome biogenesis rate and suggest that in tumors with altered pRb and p53 function the up-regulation of rRNA synthesis may always assure an adequate growth to cancer cells with uncontrolled cell cycle progression.

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38␥ has been implicated in the G 2 phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38␥. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G 2 checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr 68 by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38␥ but had no effect on p38␣ activation, indicating that MRK is a specific activator of p38␥ after IR. Inhibition of p38␥ by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38␥ MAPK pathway.

Cell growth is closely related to cell proliferation and an adequate ribosome biogenesis appears to be necessary for cell duplication. In the present study, we have investigated the relationship between rRNA synthesis and cell cycle progression. For this purpose, in a first set of experiments, we evaluated the effect of rRNA synthesis variation on cycle duration in asynchronously growing H4-II-E-C3 rat hepatoma cells. Cells were either treated with insulin or insulin plus actinomycin D (AMD). The hormone stimulated ribosome biogenesis, which was later followed by an increased synthesis of DNA and a shortening of cell doubling time (DT). Bivariate flow cytometry indicated that the reduced length of the cell cycle was mainly due to the shorter G1-phase. AMD, at the concentration of 0.04 microg/ml, hindered ribosome biogenesis without affecting heterogeneous RNA production. A 12-h reduction in ribosome biogenesis level by AMD caused a lowering of DNA synthesis and a lengthening of cell DT with a longer G1-phase. In a second set of experiments, we analyzed the cell content variations of 28S and 18S rRNA transcripts during G1 phase in H4-II-E-C3 cells, synchronized by serum deprivation, and then stimulated by serum, serum plus insulin, and serum plus insulin and AMD. In control cells, a progressive increase in rRNA content occurred until the highest value of rRNA content was reached 21 h after serum stimulation. In insulin-treated cells, the highest rRNA value was reached at 12 h whereas in AMD-treated cells, the rRNA quantity was constantly low until 18 h and then sharply increased at 21 h. In the three experimental conditions, the highest values of rRNA amount were reached at the end of G1 phase and were quite similar to one another. We also evaluated, by real-time RT-PCR, cyclin E mRNA expression, which appeared to sharply increase at those times in which the maximum increase in the rRNA content was observed. Our results indicated that the achievement of an appropriate amount of rRNA allows G1/S phase transition, probably by modulating the expression of cyclin E mRNA.