Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

A Versatile Strategy for Screening Custom-Designed Warhead-Armed Cyclic Peptide Inhibitors.

Demand for peptide-based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide-based drug discovery, we developed a general scheme for cell-based screening of cyclic peptide inhibitors armed with a user-designed warhead. We combined unnatural amino acid incorporation and split intein-mediated peptide cyclization techniques and integrated a yeast-based colorimetric screening assay to generate a new scheme that we call the custom-designed warhead-armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5-6Q, that targets human histone deacetylase 8 (HDAC8) with a KD value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.

Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently.

One of the hallmarks of cancer cells is their ability to evade apoptosis, which confers survival advantages and resistance to anti-cancer drugs. Cancers often exhibit overexpression of anti-apoptotic BCL-2 proteins, the loss of which triggers apoptosis. In particular, the inhibition of both BCL-xL and MCL-1, but neither one individually, synergistically enhances apoptotic cell death. Here, we report computational design to produce a protein that inhibits both BCL-xL and MCL-1 simultaneously. To a reported artificial three-helix bundle whose second helix was designed to bind MCL-1, we added a fourth helix and designed it to bind BCL-xL. After structural validation of the design and further structure-based sequence design, we produced a dual-binding protein that interacts with both BCL-xL and MCL-1 with apparent dissociation constants of 820 pM and 196 pM, respectively. Expression of this dual binder in a subset of cancer cells induced apoptotic cell death at levels significantly higher than those induced by the pro-apoptotic BIM protein. With a genetic fusion of a mitochondria-targeting sequence or the BH3 sequence of BIM, the activity of the dual binder was enhanced even further. These data suggest that targeted delivery of this dual binder alone or as a part of a modular protein to cancers in the form of protein, mRNA, or DNA may be an effective way to induce cancer cell apoptosis.

The effect of autocrine motility factor alone and in combination with methyl jasmonate on liver cancer cell growth.

Neoplastic cells secrete autocrine motility factor (AMF) to stimulate the motility of cancer cells. In this study, AMF secreted from HT-29 colorectal cancer cells selectively suppressed liver cancer cells by downregulating pAKT and ß-catenin. In addition, HT-29 AMF significantly augmented the activity of methyl jasmonate against liver cancer cells and is a promising alternative for liver cancer therapy.

Synergistic effects of autocrine motility factor and methyl jasmonate on human breast cancer cells.

Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option.

Autocrine motility factor secreted by HeLa cells inhibits the growth of many cancer cells by regulating AKT/ERK signaling.

In cell competition, a secreted death signal can determine cell fate. However, the nature of such a signal remains unclear. In this study, conditioned medium from HeLa cells (HeLa CM) inhibited growth of A549 and MCF-7 cells. Through HeLa CM fractionation, glucose 6-phosphate isomerase/autocrine motility factor (GPI/AMF) was identified as the main growth inhibitor. Previously, AMF was known for its mitogenic, motogenic, and differentiation functions and was implicated in tumor progression and metastasis. HeLa CM lost its growth inhibitory property after treatment with erythrose-4-phosphate (E4P) or anti-GPI antibody. Purified HeLa recombinant AMF (rAMF) proteins inhibited the growth of A549, MDA-MB-232, MCF-7, AsPC-1, DU145, Hep-2, Hep G2, and HT-29 cells. However, growth of HL-60, SKOV3, U-87 MG, SNU-484, U-87 MG, and 3T3-L1 cells was little affected. In a Transwell assay, HeLa rAMF effectively reduced A549 cell migration and invasion. HeLa rAMF effectively induced apoptosis in A549 cells, apparently by reducing the levels of Bcl-2, GPI, and poly(ADP-ribose) polymerase (PARP)14 and activating caspase-3 and p53. HeLa rAMF antagonized HER2 and the AMF receptor (AMFR or GP78) in relation to the AKT/EKT signaling pathway. These results suggest that HeLa AMF could act as a diffusible death signal that could induce cancer cell-selective growth inhibition and apoptosis.

Efficacy of three proton-pump inhibitor therapeutic strategies on laryngopharyngeal reflux disease; a prospective randomized double-blind study.

OBJECTIVES: Proton-pump inhibitor (PPI) prescribing practices in laryngopharyngeal reflux disease (LPR) differ among physicians. We assessed the improvement in reflux symptom index (RSI) and reflux finding score (RFS) after treating LPR with three different regimens. DESIGN: A prospective, double-blind, randomized clinical trial. SETTING: Chungnam national university hospital in Korea. PARTICIPANTS: From July 2015 to July 2017, 100 patients with LPR included in the study. The patients were prescribed one of the following regimens for 3 months: group A, ilaprazole 10 mg, once a day (QD), n = 29; group B, ilaprazole 10 mg, twice a day (BID), n = 27; and group C, ilaprazole 10 mg BID plus mosapride citrate 5 mg three times a day (TID), n = 44. MAIN OUTCOME MEASURES: The total RSI and RFS scores and each subitems in RSI and FRS of the patients were evaluated. RESULTS: Total RFS and RSI scores improved significantly at the 3-month follow-up in all groups, and the improvements were of similar magnitudes. Regarding the RFS, the degrees of improvement in vocal cord oedema (P = 0.002) and diffuse laryngeal oedema (P = 0.003) scores differed significantly among the three groups. Moreover, overweight or obese patients in group C showed the greatest improvement in RFS. However, age had no effect on treatment efficacy. CONCLUSION: Three PPI therapeutic strategies showed similar efficacies against LPR according to total RFS and RSI scores. The addition of a prokinetic resulted in improvements in specific endoscopic findings, such as vocal cord oedema and diffuse laryngeal oedema. Furthermore, the addition of a prokinetic to PPI therapy was particularly beneficial for overweight or obese patients.

MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression.

Mitogen- and stress-activated kinase 1 (MSK1) is a chromatin kinase that facilitates activator-dependent transcription by altering chromatin structure through histone H3 phosphorylation. The kinase activity of MSK1 is activated by intramolecular autophosphorylation, which is initially triggered by the activation of upstream mitogen-activated protein kinases (MAPKs), such as p38 and ERK1/2. MSK1 has been implicated in the expression of p21, a p53 target gene; however, the precise connection between MSK1 and p53 has not been clearly elucidated. Here, using in vitro and cell-based transcription assays, we show that MSK1 functions as a transcriptional coactivator of p53 in p21 expression, an action associated with MAPK-dependent phosphorylation of MSK1 and elevated kinase activity. Of special significance, we show that MSK1 directly interacts with p53 and is recruited to the p21 promoter, where it phosphorylates histone H3 in a p53-dependent manner. In addition, phosphomimetic mutant analysis demonstrated that negative charges in the hydrophobic motif are critical for serine 212 phosphorylation in the N-terminal kinase domain, which renders MSK1 competent for histone kinase activity. These studies suggest that MSK1 acts through a direct interaction with p53 to function as a transcriptional coactivator and that MSK1 activation by upstream MAPK signaling is important for efficient p21 gene expression.

Radiological parameters related to success of the round window approach in cochlear implantation: A retrospective study.

OBJECTIVES: To evaluate the radiologic parameters related to success of round window (RW) approach for cochlear implantation (CI). DESIGN: A retrospective cohort study. SETTING: Academic-tertiary centre. PARTICIPANTS: Eighty-four consecutive patients without inner ear anomaly who underwent CI with the intent of the RW approach were included. The RW approach was performed through the facial recess after posterior tympanotomy (RW group). When the RW approach was not possible despite maximum effort to expose the RW, promontory cochleostomy (PC) was performed (PC group). MAIN OUTCOME MEASURES: The following radiologic parameters were compared between the two groups: (a) Width of the facial recess, (b) oblique distance between the cochlear basal turn (CBT) and facial nerve (FN), (c) anteroposterior distance between the posterior margin of the RW and FN and (d) angle between the EAC and CBT. RESULTS: Seventy patients (83.3%) were implanted using the RW approach, and 14 patients (16.7%) underwent the PC approach for CI. The anteroposterior distance between the posterior margin of the RW and FN and the angle between the EAC and CBT in the RW group were significantly longer and wider than those in the PC group (P < 0.001 and P = 0.001, respectively). Multivariate analysis revealed that these two parameters were independent parameters for success of the RW approach. CONCLUSIONS: The distance between the posterior margin of the RW and FN and the angle between the EAC and CBT are associated with success of RW approach. Therefore, preoperative radiologic analysis of the two parameters might help CI surgeons to select RW approach.

Clinical significance of extrathyroidal extension according to primary tumor size in papillary thyroid carcinoma.

BACKGROUND: Extrathyroidal extension (ETE) is a risk factor for poor papillary thyroid carcinoma (PTC) outcomes. However, the clinical significance of ETE according to primary tumor size has not been well-established. The purpose of this study was to compare differences in clinical outcomes, according to the presence and extent of ETE, between different primary tumor size groups. METHODS: In total, 381 patients with PTC underwent total thyroidectomy with or without lymph node (LN) dissection from 2004 to 2010. We divided the patients into two groups according to primary tumor size: ≤ 1 cm or >1 cm. Each group was further divided into subgroups according to the presence of ETE (ETE vs. no ETE) and degree of ETE (microscopic ETE vs. macroscopic ETE). The clinicopathological features and rate of recurrence during follow-up were compared among groups. RESULTS: Among the PTC patients with primary tumors >1 cm, patients with ETE had a higher recurrence rate than those without ETE, and only macroscopic ETE affected recurrence in patients with PTC > 1 cm (P < 0.05). However, there was no significant difference in recurrence rates between those without ETE and those with microscopic ETE (P = 0.100). When the primary tumor size was less than 1 cm, there were no difference in recurrence rates between the groups with or without ETE, or between the groups with microscopic and macroscopic ETE (P > 0.05). CONCLUSIONS: Our data suggests that the presence and degree of ETE may be associated with PTC outcome based on primary tumor size.

EpCAM peptide-primed dendritic cell vaccination confers significant anti-tumor immunity in hepatocellular carcinoma cells.

Cancer stem-like cells (CSCs) may play a key role in tumor initiation, self-renewal, differentiation, and resistance to current treatments. Dendritic cells (DCs) play a vital role in host immune reactions as well as antigen presentation. In this study, we explored the suitability of using CSC peptides as antigen sources for DC vaccination against human breast cancer and hepatocellular carcinoma (HCC) with the aim of achieving CSC targeting and enhancing anti-tumor immunity. CD44 is used as a CSC marker for breast cancer and EpCAM is used as a CSC marker for HCC. We selected CD44 and EpCAM peptides that bind to HLA-A2 molecules on the basis of their binding affinity, as determined by a peptide-T2 binding assay. Our data showed that CSCs express high levels of tumor-associated antigens (TAAs) as well as major histocompatibility complex (MHC) molecules. Pulsing DCs with CD44 and EpCAM peptides resulted in the efficient generation of mature DCs (mDCs), thus enhancing T cell stimulation and generating potent cytotoxic T lymphocytes (CTLs). The activation of CSC peptide-specific immune responses by the DC vaccine in combination with standard chemotherapy may provide better clinical outcomes in advanced carcinomas.

Attomolar detection of extracellular microRNAs released from living prostate cancer cells by a plasmonic nanowire interstice sensor.

Prostate cancer (PC) is the second leading cause of cancer death for men worldwide. The serum prostate-specific antigen level test has been widely used to screen for PC. This method, however, exhibits a high false-positive rate, leading to over-diagnosis and over-treatment of PC patients. Extracellular microRNAs (miRNAs) recently provided valuable information including the site and the status of the cancers and thus emerged as new biomarkers for several cancers. Among them, miR141 and miR375 are the most pronounced biomarkers for the diagnosis of high-risk PC. Herein, we report an attomolar detection of miR141 and miR375 released from living PC cells by using a plasmonic nanowire interstice (PNI) sensor. This sensor showed a very low detection limit of 100 aM as well as a wide dynamic range from 100 aM to 100 pM for all target miRNAs. In addition, the PNI sensor could discriminate perfectly the diverse single-base mismatches in the miRNAs. More importantly, the PNI sensor successfully detected the extracellular miR141 and miR375 released from living PC cell lines (LNCaP and PC-3), proving the diagnostic ability of the sensor for PC. We anticipate that the present PNI sensor can hold great promise for the precise diagnosis and prognosis of various cancer patients as well as PC patients.

Clinical implications of the extent of BRAFV600E alleles in patients with papillary thyroid carcinoma.

OBJECTIVE: There are many conflicting reports about the clinical implications of BRAFV600E in papillary thyroid cancer (PTC). We investigated the associations between the extent of BRAFV600E alleles and both clinico-pathological features and recurrence of PTC. MATERIALS AND METHODS: Carcinoma tissues from 60 patients with PTC were genotyped for BRAFV600E using pyrosequencing, and the clinico-pathological factors and disease outcomes of the patients were examined. The associations between the extent of mutant BRAF alleles and both clinico-pathological parameters and recurrence-free survival (RFS) were analyzed. RESULTS: The BRAFV600E mutation was detected in 66.7% (40/60) of our PTC patients. When we defined four groups on the basis of the extent of BRAFV600E alleles by pyrosequencing-negative (less than 5%), low (5 - less than 15%), intermediate (15 - less than 25%), and high (25% or greater)- the four groups showed statistically significant differences regarding lymph node (LN) metastasis and recurrence (P<0.05). However, age, gender, tumor size, multicentricity, capsular invasion, and lymphovascular invasion were not significantly different among the groups. The 10-year RFS rates in PTC patients with greater than 25% and less than 25% mutated BRAF alleles were 74% and 100%, respectively. This difference was significant (P=0.043). CONCLUSIONS: A high extent more than 25% of BRAFV600E alleles may be associated with disease outcome in PTC patients. We need more data to verify a hypothesis that the extent of BRAF mutations may be clinically informative in the management of PTC, such as by tailoring proper surgical and radioactive iodine treatments and determining appropriate management during follow-up.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

The resveratrol analog HS-1793 enhances radiosensitivity of mouse-derived breast cancer cells under hypoxic conditions.

Tumor hypoxia is associated with treatment resistance, cell proliferation, and metastatic potential, all of which contribute to a poor prognosis. Resveratrol [RES (trans-3,4',5-trihydroxystilbene)], a naturally occurring polyphenol, is enriched in grapes and red wine. This study investigated whether the resveratrol analog HS-1793 modulates the hypoxic status and the level of perfusion in mouse breast cancer FM3A cells. Our data show that HS-1793 decreased the levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor protein under hypoxic conditions in FM3A cells. HS-1793 improved perfusion and hypoxic status in tumor tissues and inhibited angiogenesis through HIF-1α suppression in mice. Moreover, HS-1793 inhibited hypoxia-induced cancer stem cell properties and enhanced ionizing radiation-induced apoptosis in hypoxic FM3A cells. Collectively, the resveratrol analog HS-1793 might act as a potent radiosensitizer and be a useful adjuvant agent against radiotherapy-resistant hypoxic cells in solid tumors.

Truncated SRA RNA derivatives inhibit estrogen receptor-α-mediated transcription.

The steroid receptor RNA activator (SRA) is a long non-coding RNA (lncRNA) that acts as a putative coactivator for steroid receptor-mediated transcription. A recent study showed that SRA RNA can be structurally dissected into four domains comprising various secondary structures, but the contribution of each domain to the coactivation ability of SRA RNA was previously unknown. Here, we assessed the functional contributions of the various domains of SRA. We examined the effects of each domain on the coactivation of estrogen receptor-α (ERα)-mediated transcription of a luciferase reporter gene in HeLa cells. Then the detailed domain analysis was focused on domain III (D3) not only with the reporter gene in HeLa cells, but also with ERα-responsive genes in MCF7 breast cancer cells. Domain deletion analysis showed that the deletion of any domain decreased the luciferase activity, and that deletion of D3 caused the largest decrease. This D3 deletion effect was not recovered by co-expression of D3 alone; moreover, the expression of D3 fragments (particularly helices H15-H18, which are highly conserved across vertebrates) inhibited luciferase expression in HeLa cells. Moreover, a fragment containing helices H15-H18 reduced ERα-responsive gene expression in MCF7 breast cancer cells. Our findings indicate that D3 inhibited ERα-mediated transcription of a reporter gene in HeLa cells and that helices H15-H18, as a core element responsible for the D3-driven inhibition, reduced expression of ERα-responsive genes in breast cancer cells.

Effects of triamcinolone-impregnated nasal dressing on subjective and objective outcomes following endoscopic sinus surgery.

The aim of this study was to compare the effects of triamcinolone (TA)- and saline-soaked biodegradable nasal dressing on subjective symptoms, wound healing and improvement of olfactory dysfunction in patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) after undergoing endoscopic sinus surgery (ESS). The study was a prospective, randomized, double-blinded, placebo-controlled study. A total of 80 patients undergoing bilateral ESS for CRSwNP were enrolled and randomly assigned to two groups. Nasal dressing was impregnated with normal saline in the control group, while patients received triamcinolone-impregnated dressing in the TA group. Sino-Nasal Outcome Test 20 (SNOT-20) and Korean Version of the Sniffin' Stick (KVSS) II test were used to assess the patients' condition preoperatively and at postoperative 1 and 3 months. Lund-Kennedy (L-K) and perioperative sinus endoscopy (POSE) scores were assessed on postoperative months 1, 2, and 3. There were significant differences between the control group and the TA group in terms of postoperative L-K scores and POSE scores at 1 and 2 months. The postoperative endoscopic scores were significantly decreased in the TA group compared to the control at 1 month. Olfactory functions were significantly improved at postoperative 3 months (p = 0.0099) compared to the preoperative score in the TA group. Significant improvement in the olfactory functions among anosmic and hyposmic patients at postoperative 1 month (p = 0.0475) and 3 months (p = 0.0019) compared to their preoperative olfactory function score was observed only in the TA group. TA-impregnated dressing had a significant advantage over saline-soaked dressing with regard to postoperative wound healing and improvement of olfactory function.

Carboxyl-Terminal Modulator Protein Positively Acts as an Oncogenic Driver in Head and Neck Squamous Cell Carcinoma via Regulating Akt phosphorylation.

The exact regulatory mechanisms of carboxyl-terminal modulator protein (CTMP) and its downstream pathways in cancer have been controversial and are not completely understood. Here, we report a new mechanism of regulation of Akt serine/threonine kinase, one of the most important dysregulated signals in head and neck squamous cell carcinoma (HNSCC) by the CTMP pathway and its clinical implications. We find that HNSCC tumor tissues and cell lines had relatively high levels of CTMP expression. Clinical data indicate that CTMP expression was significantly associated with positive lymph node metastasis (OR = 3.8, P = 0.033) and correlated with poor prognosis in patients with HNSCC. CTMP was also positively correlated with Akt/GSK-3ß phosphorylation, Snail up-regulation and E-cadherin down-regulation, which lead to increased proliferation and epithelial-to-mesenchymal transition, suggesting that CTMP expression results in enhanced tumorigenic and metastatic properties of HNSCC cells. Moreover, CTMP suppression restores sensitivity to cisplatin chemotherapy. Intriguingly, all the molecular responses to CTMP regulation are identical regardless of p53 status in HNSCC cells. We conclude that CTMP promotes Akt phosphorylation and functions as an oncogenic driver and prognostic marker in HNSCC irrespective of p53.

Development of background-free tame fluorescent probes for intracellular live cell imaging.

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as 'tame' probes, and novel tools for live cell intracellular imaging.

Akt Kinase-Mediated Checkpoint of cGAS DNA Sensing Pathway.

Upon DNA stimulation, cyclic GMP-AMP synthase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-ß (IFN-ß) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-ß production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-ß production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-ß production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts' immune responses to DNA stimulation.