RB constrains lineage fidelity and multiple stages of tumour progression and metastasis.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1-3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4-6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7-9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy.

PDS5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis.

Cohesin cofactors regulate the loading, maintenance, and release of cohesin complexes from chromosomes during mitosis but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists as two paralogs in somatic and germline cells, PDS5A and PDS5B, with unclear functions. Here, we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous excision of Pds5A and Pds5B results in severe defects during early prophase I while their individual depletion does not, suggesting their functional redundancy. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after the strong depletion of PDS5A/B proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I.

PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.

Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes.

The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1ß. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.

Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres.

Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential for cohesion establishment by allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and binding of Sororin. While both proteins contribute to telomere and arm cohesion, Pds5B is specifically required for centromeric cohesion. Furthermore, reduced accumulation of Aurora B at the inner centromere region in cells lacking Pds5B impairs its error correction function, promoting chromosome mis-segregation and aneuploidy. Our work supports a model in which the composition and function of cohesin complexes differs between different chromosomal regions.

Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

PP2A-B55 phosphatase counteracts Ki-67-dependent chromosome individualization during mitosis.

Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.

p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism.

The p53 tumor suppressor regulates multiple context-dependent tumor suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here, using a mouse model of SCLC in which endogenous p53 expression can be conditionally and temporally regulated, we show that SCLC tumors maintain a requirement for p53 inactivation. However, we identify tumor subtype heterogeneity between SCLC tumors such that p53 reactivation induces senescence in a subset of tumors, while in others, p53 induces necrosis. We pinpoint cyclophilins as critical determinants of a p53-induced transcriptional program that is specific to SCLC tumors and cell lines poised to undergo p53-mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53 transcriptional output without impacting p53 chromatin binding. Our study demonstrates that intertumoral heterogeneity in SCLC influences the biological response to p53 restoration, describes a cyclophilin-dependent mechanism of p53-regulated cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant tumor type.

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin.

BACKGROUND: The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood. RESULTS: The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast. CONCLUSION: Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.