Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway.

Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action. A loss-of-function screen for genes required for BRD4 degradation revealed strong dependence on the E2 and E3 ubiquitin ligases as well as for members of the COP9 signalosome complex for both cereblon- and Von Hippel-Lindau-engaging BRD4 degraders. Cancer cell lines raised to resist BRD4 degraders manifested a degrader-specific mechanism of resistance, resulting from the loss of components of the ubiquitin proteasome system. In addition, degrader profiling in a cancer cell line panel revealed a differential pattern of activity of Von Hippel-Lindau- and cereblon-based degraders, highlighting the need for the identification of degradation-predictive biomarkers enabling effective patient stratification.

Characterizing responsive and refractory orthotopic mouse models of hepatocellular carcinoma in cancer immunotherapy.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients. Mouse models are commonly used to evaluate new therapeutic agents or regimens. However, to make clinical translation more successful, better characterized preclinical models are required. We therefore extensively investigated two immune-competent orthotopic HCC mouse models, namely transplanted Hep-55.1c and transgenic iAST, with respect to morphological, immunological and genetic traits and evaluated both models' responsiveness to immunotherapies. Hep-55.1c tumors were characterized by rich fibrous stroma, high mutational load and pronounced immune cell infiltrates, all of which are features of immune-responsive tumors. These characteristics were less distinct in iAST tumors, though these were highly vascularized. Cell depletion revealed that CD8+ T cells from iAST mice do not affect tumor growth and are tumor tolerant. This corresponds to the failure of single and combined ICB targeting PD-1 and CTLA-4. In contrast, combining anti-PD-1 and anti-CTLA-4 showed significant antitumor efficacy in the Hep-55.1c mouse model. Collectively, our data comprehensively characterize two immune-competent HCC mouse models representing ICB responsive and refractory characteristics. Our characterization confirms these models to be suitable for preclinical investigation of novel cancer immunotherapy approaches that aim to either deepen preexisting immune responses or generate de novo immunity against the tumor.

Contribution of epigenetic variation to adaptation in Arabidopsis.

In plants, transgenerational inheritance of some epialleles has been demonstrated but it remains controversial whether epigenetic variation is subject to selection and contributes to adaptation. Simulating selection in a rapidly changing environment, we compare phenotypic traits and epigenetic variation between Arabidopsis thaliana populations grown for five generations under selection and their genetically nearly identical ancestors. Selected populations of two distinct genotypes show significant differences in flowering time and plant architecture, which are maintained for at least 2-3 generations in the absence of selection. While we cannot detect consistent genetic changes, we observe a reduction of epigenetic diversity and changes in the methylation state of about 50,000 cytosines, some of which are associated with phenotypic changes. Thus, we propose that epigenetic variation is subject to selection and can contribute to rapid adaptive responses, although the extent to which epigenetics plays a role in adaptation is still unclear.

A genome-wide resource of cell cycle and cell shape genes of fission yeast.

To identify near complete sets of genes required for the cell cycle and cell shape, we have visually screened a genome-wide gene deletion library of 4843 fission yeast deletion mutants (95.7% of total protein encoding genes) for their effects on these processes. A total of 513 genes have been identified as being required for cell cycle progression, 276 of which have not been previously described as cell cycle genes. Deletions of a further 333 genes lead to specific alterations in cell shape and another 524 genes result in generally misshapen cells. Here, we provide the first eukaryotic resource of gene deletions, which describes a near genome-wide set of genes required for the cell cycle and cell shape.

The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact to repress autonomous endosperm development in Arabidopsis.

In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization.

Natural enemies drive geographic variation in plant defenses.

Plants defend themselves against attack by natural enemies, and these defenses vary widely across populations. However, whether communities of natural enemies are a sufficiently potent force to maintain polymorphisms in defensive traits is largely unknown. Here, we exploit the genetic resources of Arabidopsis thaliana, coupled with 39 years of field data on aphid abundance, to (i) demonstrate that geographic patterns in a polymorphic defense locus (GS-ELONG) are strongly correlated with changes in the relative abundance of two specialist aphids; and (ii) demonstrate differential selection by the two aphids on GS-ELONG, using a multigeneration selection experiment. We thereby show a causal link between variation in abundance of the two specialist aphids and the geographic pattern at GS-ELONG, which highlights the potency of natural enemies as selective forces.

Adaptation and extinction in experimentally fragmented landscapes.

Competition and disturbance are potent ecological forces that shape evolutionary trajectories. These forces typically work in opposition: when disturbance is infrequent, densities are high and competition is intense. In contrast, frequent disturbance creates a low-density environment in which competition is weak and good dispersal essential. We exploited recent advances in genomic research to quantify the response to selection by these powerful ecological forces at the phenotypic and molecular genetic level in experimental landscapes. We grew the annual plant Arabidopsis thaliana in discrete patches embedded in a hostile matrix and varied the number and size of patches and the intensity of disturbance, by creating both static and dynamic landscapes. In static landscapes all patches were undisturbed, whereas in dynamic landscapes all patches were destroyed in each generation, forcing seeds to disperse to new locations. We measured the resulting changes in phenotypic, genetic, and genotypic diversity after five generations of selection. Simulations revealed that the observed loss of genetic diversity dwarfed that expected under drift, with dramatic diversity loss, particularly from dynamic landscapes. In line with ecological theory, static landscapes favored good competitors; however, competitive ability was linked to growth rate and not, as expected, to seed mass. In dynamic landscapes, there was strong selection for increased dispersal ability in the form of increased inflorescence height and reduced seed mass. The most competitive genotypes were almost eliminated from highly disturbed landscapes, raising concern over the impact of increased levels of human-induced disturbance in natural landscapes.

Specific replication origins promote DNA amplification in fission yeast.

To ensure equal replication of the genome in every eukaryotic cell cycle, replication origins fire only once each S phase and do not fire after passive replication. Failure in these controls can lead to local amplification, contributing to genome instability and the development of cancer. To identify features of replication origins important for such amplification, we have investigated origin firing and local genome amplification in the presence of excess helicase loaders Cdc18 and Cdt1 in fission yeast. We find that S phase controls are attenuated and coordination of origin firing is lost, resulting in local amplification. Specific origins are necessary for amplification but act only within a permissive chromosomal context. Origins associated with amplification are highly AT-rich, fire efficiently and early during mitotic S phase, and are located in large intergenic regions. We propose that these features predispose replication origins to re-fire within a single S phase, or to remain active after passive replication.

Cyclin-dependent kinase inhibits reinitiation of a normal S-phase program during G2 in fission yeast.

To achieve faithful replication of the genome once in each cell cycle, reinitiation of S phase is prevented in G(2) and origins are restricted from refiring within S phase. We have investigated the block to rereplication during G(2) in fission yeast. The DNA synthesis that occurs when G(2)/M cyclin-dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S phase without mitosis, but this has not been demonstrated to be the case. We show here that on G(2)/M CDK depletion in G(2), repeated S phases are induced, which are correlated with normal G(1)/S transcription and attainment of doublings in cell size. Mostly normal mitotic S-phase origins are utilized, although at different efficiencies, and replication is essentially equal across the genome. We conclude that CDK inhibits reinitiation of S phase during G(2), and if G(2)/M CDK is depleted, replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.

Genome-wide characterization of fission yeast DNA replication origins.

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.