The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo. This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.
Epigenetic therapies are emerging strategies to prime host immune system to immunotherapies. Here we show that depletion of H3K4 demethylase KDM5B in melanoma cells induces anti-tumor T cell immunity through up-regulation of retroelements, which activates cytosolic nucleic acid sensing pathways and subsequent type I interferon responses. Mechanistically, KDM5B recruits H3K9 methyltransferase SETDB1 to the chromatin to repress these retroelements. Ablation of KDM5B enhances responses of poorly immunogenic melanoma tumors to anti PD-1 treatment. Our studies suggest that therapies targeting KDM5B are a novel approach to enhance tumor immunogenicity and overcome immunotherapy resistance. MainKDM5B expression is negatively correlated with anti-tumor immunity and response to immune checkpoint blockade. To explore the roles of KDM5B in melanoma, we performed Gorilla Gene Ontology analysis of 9,336 genes negatively correlated with KDM5B expression in TCGA melanoma dataset. The top 5 gene ontology categories anti-correlated with KDM5B expression are all related to immune system processes (Fig. 1a). To further examine the relationship of KDM5B with anti-tumor immune responses, we correlated KDM5B mRNA levels with T cell markers, antigen presentation and cytokine genes in TCGA melanoma dataset. Remarkably, KDM5B expression is inversely associated with all the genes examined, including IFNG (IFN-γ) and TNF (TNF-α), two crucial effector cytokines in anti-tumor immunity 1 (Extended Data Table 1). These results suggest that KDM5B expression is inversely correlated with intra-tumoral in ammation. Consistently, patients with higher KDM5B expression have lower expression of markers for CD8 + T cells in the tumors in TCGA melanoma dataset (Extended Data Fig. 1a). Lack of T cell in ltration in pre-treatment biopsies normally correlates with resistance to immune checkpoint blockade (ICB) 2 . To evaluate whether KDM5B levels predict response to immune checkpoint blockade in melanoma, we analyzed RNA-seq data of pre-treatment specimens from tumors sampled prior to anti-PD-1 therapy 3 . We found that KDM5B expression levels are signi cantly lower in patients with complete response than those in patients with progressive disease (Fig. 1b and Extended Data Table 2). Furthermore, we evaluated KDM5B protein levels in a tissue microarray (TMA) containing samples from patients treated with ICB.Patients who responded to ICB had a very low intensity of KDM5B staining and lacked detectable KDM5B high melanoma cells, while the non-responders had a major subset of KDM5B high melanoma cells (Extended Data Fig. 1b). These data raised the possibility that KDM5B could be a biomarker for poor response to ICB and a KDM5B targeting therapy might overcome resistance to ICB. KDM5B loss induces anti-tumor immunity in a murine model of melanomaTo test whether ablation of KDM5B in melanoma induces anti-tumor immunity, we assessed the effects of Kdm5b deletion in the immunogenic YUMMER1.7 mouse melanoma cells 4 on their tumor growth in syngeneic C57BL/6J mice ...
Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 , represents the greatest public health threat in a century. More than 7,500,000 people have been infected with more than 420,000 deaths globally (1). Novel therapeutics and vaccines are desperately needed. Coronaviruses are enveloped, positive-sense RNA viruses with genomes of approximately 30 kb that exhibit broad host-range among birds and mammals and are typically transmitted via the respiratory route (2, 3). There are four circulating seasonal coronaviruses in humans (NL63, OC43, 229E, and HKU1) and three highly pathogenic zoonotic coronaviruses (SARS-CoV, MERS, and SARS-CoV-2), none of which have effective antiviral drugs or vaccines (4-7).Viral entry, the first stage of the SARS-CoV-2 life cycle, is mediated by the viral spike protein. The receptor binding domain of spike binds to the cell surface receptor angiotensinconverting enzyme 2 (ACE2), a major determinant of host range and cell tropism (8,9). The coronavirus spike protein requires two proteolytic processing steps prior to entry. The first cleavage event occurs at the interface of the S1 and S2 domains of the spike protein (10, 11). This can occur in the producer cell, the extracellular environment, or in the endosome and can be mediated by several proteases including furin and the plasma membrane protease TMPRSS2 (12)(13)(14). A second proteolytic event is required within S2 to expose the viral fusion peptide and enable membrane fusion. This second cleavage event can occur at the target cell plasma membrane by TMPRSS2 or in the endosome by Cathepsin L (14,15). Upon viral membrane fusion, the viral RNA is released into the cytoplasm where it is translated and establishes viral replication and transcription complexes before assembling and budding (16)(17)(18). The host genes that mediate these processes largely remain elusive.Identification of host factors essential for infection is critical to inform mechanisms of COVID-19 pathogenesis, reveal variation in host susceptibility, and identify novel host-directed therapies, which may have efficacy against current and future pand...
Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34 þ p75 À (CD34 þ) and CD34 À p75 À (CD34 À) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34 þ and CD34 À subpopulations carrying the BRAF V600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34 À state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor-sensitive CD34 þ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.
◥ KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer.Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110a and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110a/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer.Significance: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.
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