Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence.

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.

Subtle gene modification in mouse ES cells: evidence for incorporation of unmodified oligonucleotides without induction of DNA damage.

Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising tool for site-specific gene modification in mouse embryonic stem cells (ESCs). We have developed an ESC line carrying a mutant EGFP reporter gene to monitor gene correction events shortly after exposure to ssODNs. We used this system to compare the appearance and fate of cells corrected by sense or anti-sense ssODNs. The slower appearance of green fluorescent cells with sense ssODNs as compared to anti-sense ssODNs is consistent with physical incorporation of the ssODN into the genome. Thus, the supremacy of anti-sense ssODNs, previously reported by others, may be an artefact of early readout of the EGFP reporter. Importantly, gene correction by unmodified ssODNs only mildly affected the viability of targeted cells and did not induce genomic DNA double-stranded breaks (DSBs). In contrast, ssODNs that were end-protected by phosphorothioate (PTO) linkages caused increased H2AX phosphorylation and impaired cell cycle progression in both corrected and non-corrected cells due to induction of genomic DSBs. Our results demonstrate that the use of unmodified rather than PTO end-protected ssODNs allows stable gene modification without compromising the genomic integrity of the cell, which is crucial for application of ssODN-mediated gene targeting in (embryonic) stem cells.

3-deazaadenosine (3DA) alleviates senescence to promote cellular fitness and cell therapy efficiency in mice.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.

Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides.

Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.

Parameters of oligonucleotide-mediated gene modification in mouse ES cells.

Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. Here, we have studied the role of ssODN composition, transcription and replication of the target locus, and DNA repair pathways to gain more insight into the parameters governing ssODN-mediated gene targeting in mouse ES cells. We demonstrated that unmodified ssODNs of 35-40 nt were most efficient in correcting a chromosomally integrated mutant neomycin reporter gene. Addition of chemical modifications did not further enhance the efficacy of these ssODNs. The observed strand bias was not affected by transcriptional activity and may rather be caused by the different accessibility of the DNA strands during DNA replication. Consistently, targeting frequencies were enhanced when cells were treated with hydroxyurea to reduce the rate of replication fork progression. Transient down-regulation of various DNA repair genes by RNAi had no effect on the targeting frequency. Taken together, our data suggest that ssODN-mediated gene targeting occurs within the context of a replication fork. This implies that any given genomic sequence, irrespective of transcriptional status, should be amenable to ssODN-mediated gene targeting. The ability of ES cells to differentiate into various cell types after ssODN-mediated gene targeting may offer opportunities for future therapeutic applications.

Gene modification in embryonic stem cells by single-stranded DNA oligonucleotides.

Oligonucleotide-mediated gene targeting is an attractive alternative to current procedures to subtly modify the genome of mouse embryonic stem (ES) cells. However, oligonucleotide-directed substitution, insertion or deletion of a single or a few nucleotides was hampered by DNA mismatch repair (MMR). We have developed strategies to circumvent this problem based on findings that the central MMR protein MSH2 acts in two different mismatch recognition complexes: MSH2/MSH6, which mainly recognizes base substitutions; and MSH2/MSH3, which has more affinity for larger loops. We found that oligonucleotide-mediated base substitution could effectively be obtained upon transient suppression of MSH2 protein level, while base insertions were effective in ES cells deficient for MSH3. This method allows substitution of any codon of interest in the genome.

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells.

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.

Correction: Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

[This corrects the article DOI: 10.1371/journal.pone.0176578.].

Author Correction: P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development.

The original version of this article contained an error in the spelling of Juan Pedro Martinez-Barbera, which was incorrectly given as Juan Pedro Martinez Barbera. This error has now been corrected in both the PDF and HTML versions of the Article.

P53 and mTOR signalling determine fitness selection through cell competition during early mouse embryonic development.

Ensuring the fitness of the pluripotent cells that will contribute to future development is important both for the integrity of the germline and for proper embryogenesis. Consequently, it is becoming increasingly apparent that pluripotent cells can compare their fitness levels and signal the elimination of those cells that are less fit than their neighbours. In mammals the nature of the pathways that communicate fitness remain largely unknown. Here we identify that in the early mouse embryo and upon exit from naive pluripotency, the confrontation of cells with different fitness levels leads to an inhibition of mTOR signalling in the less fit cell type, causing its elimination. We show that during this process, p53 acts upstream of mTOR and is required to repress its activity. Finally, we demonstrate that during normal development around 35% of cells are eliminated by this pathway, highlighting the importance of this mechanism for embryonic development.

Correction: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

[This corrects the article DOI: 10.1371/journal.pone.0149099.].

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.

Functional Genetic Screen Identifies Increased Sensitivity to WEE1 Inhibition in Cells with Defects in Fanconi Anemia and HR Pathways.

WEE1 kinase regulates CDK1 and CDK2 activity to facilitate DNA replication during S-phase and to prevent unscheduled entry into mitosis. WEE1 inhibitors synergize with DNA-damaging agents that arrest cells in S-phase by triggering direct mitotic entry without completing DNA synthesis, resulting in catastrophic chromosome fragmentation and apoptosis. Here, we investigated how WEE1 inhibition could be best exploited for cancer therapy by performing a functional genetic screen to identify novel determinants of sensitivity to WEE1 inhibition. Inhibition of kinases that regulate CDK activity, CHK1 and MYT1, synergized with WEE1 inhibition through both increased replication stress and forced mitotic entry of S-phase cells. Loss of multiple components of the Fanconi anemia (FA) and homologous recombination (HR) pathways, in particular DNA helicases, sensitized to WEE1 inhibition. Silencing of FA/HR genes resulted in excessive replication stress and nucleotide depletion following WEE1 inhibition, which ultimately led to increased unscheduled mitotic entry. Our results suggest that cancers with defects in FA and HR pathways may be targeted by WEE1 inhibition, providing a basis for a novel synthetic lethal strategy for cancers harboring FA/HR defects.

Effects of bariatric surgery on inspiratory muscle strength.

BACKGROUND: The respiratory function is affected by obesity due to an increased deposition of fat on the chest wall. The objective of this study was to investigate the strength of the inspiratory respiratory muscles of obese individuals and the possible influence of bariatric surgery on it by measuring the maximum inspiratory pressure (MIP). METHODS: Patients referred to a bariatric centre between the 3rd of October 2011 and the 3rd of May 2012 were screened preoperatively by a multidisciplinary team. Their MIP was measured at screening and 3, 6 and 9 months postoperative. In case of a preoperative MIP lower than 70% of predicted pressure training was provided supervised by a physiotherapist. RESULTS: The mean age of 124 included patients was 42.9 ± 11.0 years and mean BMI was 43.1 ± 5.2 kg/m(2). The mean predicted MIP preoperatively was 127 ± 31 in cm H2O and the mean measured MIP was 102 ± 24 in cm H2O. Three patients (2.4%) received training. Three months after surgery the MIP was 76 ± 26 cm H2O, after 6 months 82 ± 28 cm H2O and after 9 months 86 ± 28 cm H2O. All postoperative measurements were significant lower than preoperatively (P < 0.05). The only influencing factor for the preoperative MIP was age (p = 0.014). CONCLUSION: The preoperative MIP values were significantly lower than the predicted MIP values, probably due to altered respiratory mechanics.

Tumour selective targeting of cell cycle kinases for cancer treatment.

The deregulation of the cell cycle and checkpoint machinery in cancer presents a highly attractive therapeutic strategy. We review here the strategies used to exploit the dysregulated cell cycle, both through targeting kinases required for cell cycle progression, and checkpoint kinases to inappropriately force cells through the cell cycle. Appropriate control of the cell cycle is critical for proliferating normal cells, and we discuss the importance of defining tumour specific vulnerabilities that could be targeted with cell cycle kinase inhibitors. Recent studies have shown that ER-positive breast cancers rely on CDK4 to promote proliferation. TP53 mutant cancer cell lines are sensitive to WEE1 and CHK1 inhibitors in combination with chemotherapy, while PTEN-deficient aneuploid cancer cell lines are sensitive to TTK inhibitors.

Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1.

Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase-arrested cells directly into mitosis without completing DNA synthesis, resulting in highly abnormal mitoses characterized by dispersed chromosomes and disorganized bipolar spindles, ultimately resulting in mitotic exit with gross micronuclei formation and apoptosis. This mechanism of cell death is shared by CHK1 inhibitors, and combined WEE1 and CHK1 inhibition forces mitotic entry from S-phase in the absence of chemotherapy. We show that p53/p21 inactivation combined with high expression of mitotic cyclins and EZH2 predispose to mitotic entry during S-phase with cells reliant on WEE1 to prevent premature cyclin-dependent kinase (CDK)1 activation. These features are characteristic of aggressive breast, and other, cancers for which WEE1 inhibitor combinations represent a promising targeted therapy.

The Skn7 response regulator of Cryptococcus neoformans is involved in oxidative stress signalling and augments intracellular survival in endothelium.

Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis. There is accumulating evidence that C. neoformans is a facultative intracellular pathogen, residing in macrophages and endothelium. The molecular mechanism conferring resistance to phagolysosomal killing in these cells is a key unresolved issue. To gain insight into the fungal adaptive strategies, serial analysis of gene expression was used to map genes differentially expressed in an intraphagocytic environment. By comparing transcript profiles of C. neoformans serotype D B3501 cells recovered from endothelial cells with those from free-grown cryptococci, we identified the cryptococcal homologue of the SKN7 two-component stress response regulator gene from Saccharomyces cerevisiae. Studies with C. neoformans cells disrupted for SKN7 revealed an increased susceptibility to t-butyl hydroperoxide (100% lethality at 0.7 mM, vs. 1.0 mM for wild type) and significantly lower survival rates in endothelial infection experiments. Mice experiments revealed that SKN7 disruption strongly attenuates cryptococcal virulence in vivo. We propose that Skn7 (co-)regulates the fungal adaptive strategy, allowing intraphagocytic survival by conferring resistance to phagolysosomal killing in endothelial cells.

Microarray-based CGH of sporadic and syndrome-related pheochromocytomas using a 0.1-0.2 Mb bacterial artificial chromosome array spanning chromosome arm 1p.

Pheochromocytomas (PCC) are relatively rare neuroendocrine tumors, mainly of the adrenal medulla. They arise sporadically or occur secondary to inherited cancer syndromes, such as multiple endocrine neoplasia type II (MEN2), von Hippel-Lindau disease (VHL), or neurofibromatosis type I (NF1). Loss of 1p is the most frequently encountered genetic alteration, especially in MEN2-related and sporadic PCC. Previous studies have revealed three regions of common somatic loss on chromosome arm 1p, using chromosome-based comparative genomic hybridization (CGH) and LOH analysis. To investigate these chromosomal aberrations with a higher resolution and sensitivity, we performed microarray-based CGH with 13 sporadic and 11 syndrome-related (10 MEN2A-related and 1 NF1-related) tumors. The array consisted of 642 overlapping bacterial artificial chromosome (BAC) clones mapped to 1p11.2-p36.33. Chromosomal deletions on 1p were detected in 18 of 24 cases (75%). Among 9 tumors with partial 1p loss, the deleted region was restricted to 1cen-1p32.3 in six cases (25%), indicating a region of genetic instability. The consensus regions of deletion in this study involved 1cen-1p21.1, 1p21.3-1p31.3, and 1p34.3-1p36.33. In conclusion, these data strongly suggest that chromosome arm 1p is the site for multiple tumor suppressor genes, although the potential candidate genes CDKN2C and PTPRF/LAR are not included in these regions.

Intrathecal production and secretion of vascular endothelial growth factor during Cryptococcal Meningitis.

BACKGROUND: Patients with cryptococcal meningitis (CM) show elevated intracranial pressure (ICP) and blood-brain barrier (BBB) disruption in most cases. Elevated ICP is an important contributor to mortality. Vascular endothelial growth factor (VEGF) might be the mediator of BBB disruption during CM. METHODS: We measured VEGF levels in serum, plasma, and cerebrospinal fluid (CSF) of 95 patients and 63 control subjects, and we analyzed the required trigger and cellular source of VEGF secretion in vitro. RESULTS: Cryptococcus neoformans and its capsular antigens dose-dependently induced VEGF secretion by polymorphonuclear neutrophils, monocytes, and peripheral blood mononuclear cells (PBMCs). VEGF production by PBMCs induced by antigens strongly exceeded production by monocytes (P<.001). The addition of major histocompatibility complex class II antibody inhibited this production of VEGF (P=.005). Confirming the in vitro data, patients with CM showed significantly elevated VEGF levels in CSF (P<.001), plasma (P=.028), and serum (P<.001), compared with healthy control subjects. Calculated VEGF indices demonstrated that VEGF was produced intrathecally. CONCLUSIONS: Our findings suggest that VEGF plays a role in the pathophysiology of CM. We propose that CD4(+) T lymphocytes--stimulated by monocytes acting as antigen-presenting cells--are the cells that produce VEGF in response to cryptococcal antigens.