Search

Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.

Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle. the IHU Fondation ARC (French Cancer Agency) TheraHCC program IHU201301187 and IHU201901299, the Institut Universitaire de France and the Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R03AI131066. C.C.C. acknowledges fellowships from the Canadian Institutes of Health Research (201411MFE-338606-245517) and the Canadian Network on Hepatitis C.E.R.V. acknowledges fellowship from ANRS (ECTZ50121).

04-14; International audience

PURPOSE: Novel targeted therapeutics have transformed the care of subsets of patients with cancer. In pediatric malignancies, however, with simple tumor genomes and infrequent targetable mutations, there have been few new FDA-approved targeted drugs. The cyclin-dependent kinase (CDK)4/6 pathway recently emerged as a dependency in Ewing sarcoma. Given the heightened efficacy of this class with targeted drug combinations in other cancers, as well as the propensity of resistance to emerge with single agents, we aimed to identify genes mediating resistance to CDK4/6 inhibitors and biologically relevant combinations for use with CDK4/6 inhibitors in Ewing. EXPERIMENTAL DESIGN: We performed a genome-scale open reading frame (ORF) screen in 2 Ewing cell lines sensitive to CDK4/6 inhibitors to identify genes conferring resistance. Concurrently, we established resistance to a CDK4/6 inhibitor in a Ewing cell line. RESULTS: The ORF screen revealed IGF1R as a gene whose overexpression promoted drug escape. We also found elevated levels of phospho-IGF1R in our resistant Ewing cell line, supporting the relevance of IGF1R signaling to acquired resistance. In a small-molecule screen, an IGF1R inhibitor scored as synergistic with CDK4/6 inhibitor treatment. The combination of CDK4/6 inhibitors and IGF1R inhibitors was synergistic in vitro and active in mouse models. Mechanistically, this combination more profoundly repressed cell cycle and PI3K/mTOR signaling than either single drug perturbation. CONCLUSIONS: Taken together, these results suggest that IGF1R inhibitors activation is an escape mechanism to CDK4/6 inhibitors in Ewing sarcoma and that dual targeting of CDK4/6 inhibitors and IGF1R inhibitors provides a candidate synergistic combination for clinical application in this disease.

Mutations in the human parkin (PARK2) gene cause autosomal recessive-juvenile Parkinson's disease (AR-JP). In Drosophila melanogaster, mutant parkin alleles display a broad range of phenotypic alterations, including female infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E (eIF4E) activity specifically rescues the female sterile phenotypes associated with the parkinP23 mutant allele. Additional defects, including reduction of pupal viability and body size, are also entirely recovered in both male and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein (4EBP) allele counteracts the in vivo effects produced, in a parkinP23 mutant background, by the reduction of functional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior end of developing oocytes. Finally, we show that eIF4E is over-expressed in parkinP23 mutant ovaries as compared to wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a common pathway, likely modulating cap-dependent translation initiation events

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties