Dephosphorylation of the pre-initiation complex is critical for origin firing.

In eukaryotes, cyclin-dependent kinase (CDK) ensures that the genome is duplicated exactly once by inhibiting helicase loading factors before activating origin firing. CDK activates origin firing by phosphorylating two substrates, Sld2 and Sld3, forming a transient and limiting intermediate-the pre-initiation complex (pre-IC). Here, we show in the budding yeast Saccharomyces cerevisiae that the CDK phosphorylations of Sld3 and Sld2 are rapidly turned over during S phase by the PP2A and PP4 phosphatases. PP2ARts1 targets Sld3 specifically through an Rts1-interaction motif, and this targeted dephosphorylation is important for origin firing genome-wide, for formation of the pre-IC at origins and for ensuring that Sld3 is dephosphorylated in G1 phase. PP2ARts1 promotes replication in vitro, and we show that targeted Sld3 dephosphorylation is critical for viability. Together, these studies demonstrate that phosphatases enforce the correct ordering of replication factor phosphorylation and in addition to kinases are also key drivers of replication initiation.

Dynamic Metabolons Using Stimuli-Responsive Protein Cages.

Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.

SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

ALKBH5 Drives Immune Suppression Via Targeting AXIN2 to Promote Colorectal Cancer and Is a Target for Boosting Immunotherapy.

BACKGROUND & AIMS: Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune checkpoint blockade efficacy is an unmet need. We found that AlkB homolog 5 (ALKBH5), an RNA N6-methyladenosine eraser, drives immunosuppression and is a molecular target to boost immune checkpoint blockade therapy in CRC. METHODS: Clinical significance of ALKBH5 was evaluated in human samples (n = 205). Function of ALKBH5 was investigated in allografts, CD34+ humanized mice, and Alkbh5 knockin mice. Immunity change was determined by means of flow cytometry, immunofluorescence, and functional investigation. Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to identify ALKBH5 targets. Vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA was constructed for targeting ALKBH5 in vivo. RESULTS: High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cell accumulation but reduced natural killer cells and cytotoxic CD8+ T cells to induce colorectal tumorigenesis in allografts, CD34+ humanized mice, and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 messenger RNA, which caused its dissociation from N6-methyladenosine reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/ß-catenin. Subsequently, Wnt/ß-catenin targets, including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited myeloid-derived suppressor cells to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 in vitro and in vivo. Finally, vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing antitumor immunity. CONCLUSIONS: This study identified an ALKBH5-N6-methyladenosine-AXIN2-Wnt-DKK1 axis in CRC, which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.

Photosynthetic mechanism of maize yield under fluctuating light environments in the field.

The photosynthetic mechanism of crop yields in fluctuating light environments in the field remains controversial. To further elucidate this mechanism, we conducted field and simulation experiments using maize (Zea mays) plants. Increased planting density enhanced the light fluctuation frequency and reduced the duration of daily high light, as well as the light-saturated photosynthetic rate, biomass, and yield per plant. Further analysis confirmed a highly significant positive correlation between biomass and yield per plant and the duration of photosynthesis related to daily high light. The simulation experiment indicated that the light-saturated photosynthetic rate of maize leaves decreased gradually and considerably when shortening the daily duration of high light. Under an identical duration of high light exposure, increasing the fluctuation frequency decreased the light-saturated photosynthetic rate slightly. Proteomic data also demonstrated that photosynthesis was mainly affected by the duration of high light and not by the light fluctuation frequency. Consequently, the current study proposes that an appropriate duration of daily high light under fluctuating light environments is the key factor for greatly improving photosynthesis. This is a promising mechanism by which the photosynthetic productivity and yield of maize can be enhanced under complex light environments in the field.

Enhancing the performance of AlGaN-based DUV-LEDs with multifocal laser stealth dicing.

In this study, AlGaN-based deep-ultraviolet light-emitting diodes (DUV-LEDs) processed via standard laser dicing (SLD) and multifocal laser stealth dicing (MFLSD) were investigated. Adopting the MFLSD technology would generate a roughing surface rather than the V-shaped grooves on the sidewall of 508 × 508 µm2 DUV-LEDs, which would reduce the forward operating voltage and increase the wall-plug efficiency, light output power, and far-field radiation patterns of these devices. In addition, the wavelength shift, far-field patterns, and light-tracing simulation results of the DUV-LEDs processed with SLD and MFLSD were clearly demonstrated and analyzed. Accordingly, it was observed that the MFLSD process provided more possibilities for photon escape to increase the light extraction efficiency (LEE) of DUV-LEDs, thus decreased the wavelength-redshift and junction temperature in DUV-LEDs. These results provide a reference for advanced nano-processing practices implemented during the fabrication of semiconductor devices.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Programmable living assembly of materials by bacterial adhesion.

The field of engineered living materials aims to construct functional materials with desirable properties of natural living systems. A recent study demonstrated the programmed self-assembly of bacterial populations by engineered adhesion. Here we use this strategy to engineer self-healing living materials with versatile functions. Bacteria displaying outer membrane-anchored nanobody-antigen pairs are cultured separately and, when mixed, adhere to each other to enable processing into functional materials, which we term living assembled material by bacterial adhesion (LAMBA). LAMBA is programmable and can be functionalized with extracellular moieties up to 545 amino acids. Notably, the adhesion between nanobody-antigen pairs in LAMBA leads to fast recovery under stretching or bending. By exploiting this feature, we fabricated wearable LAMBA sensors that can detect bioelectrical or biomechanical signals. Our work establishes a scalable approach to produce genetically editable and self-healable living functional materials that can be applied in biomanufacturing, bioremediation and soft bioelectronics assembly.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Single-domain antibodies against SARS-CoV-2 RBD from a two-stage phage screening of universal and focused synthetic libraries.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobodies, as well as other single-domain antibodies (sdAbs), have been recognized as a potential diagnostic and therapeutic tool for infectious diseases. High-throughput screening techniques such as phage display have been developed as an alternative to in vivo immunization for the discovery of antibody-like target-specific binders. METHODS: We designed and constructed a highly diverse synthetic phage library sdAb-U (single-domain Antibody - Universal library ) based on a human framework. The SARS-CoV-2 receptor-binding domain (RBD) was expressed and purified. The universal library sdAb-U was panned against the RBD protein target for two rounds, followed by monoclonal phage ELISA (enzyme-linked immunosorbent assay) to identify RBD-specific binders (the first stage). High-affinity binders were sequenced and the obtained CDR1 and CDR2 sequences were combined with fully randomized CDR3 to construct a targeted (focused) phage library sdAb-RBD, for subsequent second-stage phage panning (also two rounds) and screening. Then, sequences with high single-to-background ratios in phage ELISA were selected for expression. The binding affinities of sdAbs to RBD were measured by an ELISA-based method. In addition, we conducted competition ELISA (using ACE2 ectodomain S19-D615) and SARS-CoV-2 pseudovirus neutralization assays for the high-affinity RBD-binding sdAb39. RESULTS: Significant enrichments were observed in both the first-stage (universal library) and the second-stage (focused library) phage panning. Five RBD-specific binders were identified in the first stage with high ELISA signal-to-background ratios. In the second stage, we observed a much higher possibility of finding RBD-specific clones in phage ELISA. Among 45 selected RBD-positive sequences, we found eight sdAbs can be well expressed, and five of them show high-affinity to RBD (EC50 < 100nM). We finally found that sdAb39 (EC50 ~ 4nM) can compete with ACE2 for binding to RBD. CONCLUSION: Overall, this two-stage strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD sdAb with potential therapeutic activity, and this two-stage strategy can potentially be used for rapid discovery of sdAbs against other targets.

Minimizing Computation and Communication Costs of Two-Sided Secure Distributed Matrix Multiplication under Arbitrary Collusion Pattern.

This paper studies the problem of minimizing the total cost, including computation cost and communication cost, in the system of two-sided secure distributed matrix multiplication (SDMM) under an arbitrary collusion pattern. In order to perform SDMM, the two input matrices are split into some blocks, blocks of random matrices are appended to protect the security of the two input matrices, and encoded copies of the blocks are distributed to all computing nodes for matrix multiplication calculation. Our aim is to minimize the total cost, overall matrix splitting factors, number of appended random matrices, and distribution vector, while satisfying the security constraint of the two input matrices, the decodability constraint of the desired result of the multiplication, the storage capacity of the computing nodes, and the delay constraint. First, a strategy of appending zeros to the input matrices is proposed to overcome the divisibility problem of matrix splitting. Next, the optimization problem is divided into two subproblems with the aid of alternating optimization (AO), where a feasible solution can be obtained. In addition, some necessary conditions for the problem to be feasible are provided. Simulation results demonstrate the superiority of our proposed scheme compared to the scheme without appending zeros and the scheme with no alternating optimization.

Fundamental Limits of Coded Caching in Request-Robust D2D Communication Networks.

D2D coded caching, originally introduced by Ji, Caire, and Molisch, significantly improves communication efficiency by applying the multi-cast technology proposed by Maddah-Ali and Niesen to the D2D network. Most prior works on D2D coded caching are based on the assumption that all users will request content at the beginning of the delivery phase. However, in practice, this is often not the case. Motivated by this consideration, this paper formulates a new problem called request-robust D2D coded caching. The considered problem includes K users and a content server with access to N files. Only r users, known as requesters, request a file each at the beginning of the delivery phase. The objective is to minimize the average and worst-case delivery rate, i.e., the average and worst-case number of broadcast bits from all users among all possible demands. For this novel D2D coded caching problem, we propose a scheme based on uncoded cache placement and exploiting common demands and one-shot delivery. We also propose information-theoretic converse results under the assumption of uncoded cache placement. Furthermore, we adapt the scheme proposed by Yapar et al. for uncoded cache placement and one-shot delivery to the request-robust D2D coded caching problem and prove that the performance of the adapted scheme is order optimal within a factor of two under uncoded cache placement and within a factor of four in general. Finally, through numerical evaluations, we show that the proposed scheme outperforms known D2D coded caching schemes applied to the request-robust scenario for most cache size ranges.

Targeting m6A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer.

OBJECTIVE: The role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME. DESIGN: Clinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. YTHDF1 function was determined in syngeneic tumours, intestine-specific Ythdf1 knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated YTHDF1-siRNA was used for YTHDF1 silencing in vivo. RESULTS: YTHDF1 expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8+ T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of Ythdf1 augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while Ythdf1 knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or ApcMin/+ models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in Ythdf1 knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8+ T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-siYTHDF1 boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC. CONCLUSION: YTHDF1 impairs antitumour immunity via an m6A-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.

Clonal Dissemination of Antifungal-Resistant Candida haemulonii, China.

Candida haemulonii, a relative of C. auris, frequently shows antifungal resistance and is transmissible. However, molecular tools for genotyping and investigating outbreaks are not yet established. We performed genome-based population analysis on 94 C. haemulonii strains, including 58 isolates from China and 36 other published strains. Phylogenetic analysis revealed that C. haemulonii can be divided into 4 clades. Clade 1 comprised strains from China and other global strains; clades 2-4 contained only isolates from China, were more recently evolved, and showed higher antifungal resistance. Four regional epidemic clusters (A, B, C, and D) were identified in China, each comprising ≥5 cases (largest intracluster pairwise single-nucleotide polymorphism differences <50 bp). Cluster A was identified in 2 hospitals located in the same city, suggesting potential intracity transmissions. Cluster D was resistant to 3 classes of antifungals. The emergence of more resistant phylogenetic clades and regional dissemination of antifungal-resistant C. haemulonii warrants further monitoring.

Out of the cycle: Impact of cell cycle aberrations on cancer metabolism and metastasis.

The use of cell cycle inhibitors has necessitated a better understanding of the cell cycle in tumor biology to optimize the therapeutic approach. Cell cycle aberrations are common in cancers, and it is increasingly acknowledged that these aberrations exert oncogenic effects beyond the cell cycle. Multiple facets such as cancer metabolism, immunity and metastasis are also affected, all of which are beyond the effect of cell proliferation alone. This review comprehensively summarized the important recent findings and advances in these interrelated processes. In cancer metabolism, cell cycle regulators can modulate various pathways in aerobic glycolysis, glucose uptake and gluconeogenesis, mainly through transcriptional regulation and kinase activities. Amino acid metabolism is also regulated through cell cycle progression. On cancer metastasis, metabolic plasticity, immune evasion, tumor microenvironment adaptation and metastatic site colonization are intricately related to the cell cycle, with distinct regulatory mechanisms at each step of invasion and dissemination. Throughout the synthesis of current understanding, knowledge gaps and limitations in the literature are also highlighted, as are new therapeutic approaches such as combinational therapy and challenges in tackling emerging targeted therapy resistance. A greater understanding of how the cell cycle modulates diverse aspects of cancer biology can hopefully shed light on identifying new molecular targets by harnessing the vast potential of the cell cycle.

Targeting YAP1/TAZ in nonsmall-cell lung carcinoma: From molecular mechanisms to precision medicine.

Accumulating evidence has underscored the importance of the Hippo-YAP1 signaling in lung tissue homeostasis, whereas its deregulation induces tumorigenesis. YAP1 and its paralog TAZ are the key downstream effectors tightly controlled by the Hippo pathway. YAP1/TAZ exerts oncogenic activities by transcriptional regulation via physical interaction with TEAD transcription factors. In solid tumors, Hippo-YAP1 crosstalks with other signaling pathways such as Wnt/ß-catenin, receptor tyrosine kinase cascade, Notch and TGF-ß to synergistically drive tumorigenesis. As YAP1/TAZ expression is significantly correlated with unfavorable outcomes for the patients, small molecules have been developed for targeting YAP1/TAZ to get a therapeutic effect. In this review, we summarize the recent findings on the deregulation of Hippo-YAP1 pathway in nonsmall cell lung carcinoma, discuss the molecular mechanisms of its dysregulation in leading to tumorigenesis, explore the therapeutic strategies for targeting YAP1/TAZ, and provide the research directions for deep investigation. We believe that detailed delineation of Hippo-YAP1 regulation in tumorigenesis provides novel insight for accurate therapeutic intervention.

CD47-targeted immunotherapy unleashes antitumour immunity in Epstein-Barr virus-associated gastric cancer.

The aims of this study were to enhance the antitumour immunity in Epstein-Barr virus-associated gastric cancer (EBVaGC). We performed RNA-seq analysis to compare the differential expression genes between EBVaGC and EBV-negative gastric cancer (EBVnGC) patients. The expression levels of CD68, CD163 and CD47 were analyzed by immunohistochemistry. Different subsets of macrophages were investigated by a coincubation model. The effects of CD47 blockade were also detected. The expression levels of CD68, CD163 and CD47 were significantly higher in EBVaGC, and were associated with poor prognoses. Macrophages coincubated with EBV+ AGS cells tended to be immunosuppressed, which could be reversed by CD47 deficiency or blocking CD47. EBV resulted in cGAS-STING pathway activation, which stimulated CD47 expression and inhibited macrophage phagocytosis. Anti-CD47 therapy activated cGAS-STING signaling, which was responsible for production of IFN-ß, resulting in activation of antitumour immunity. Our results provide a promising new strategy for CD47-targeted immunotherapy in EBVaGC.

METTL3 Inhibits Antitumor Immunity by Targeting m6A-BHLHE41-CXCL1/CXCR2 Axis to Promote Colorectal Cancer.

BACKGROUND & AIMS: N6-Methyladenosine (m6A) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic m6A modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC. METHODS: Mettl3 knockout mice, CD34+ humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. M6A sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration. RESULTS: We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4+ and CD8+ T cells, and eventually suppressed CRC in ApcMin/+Mettl3+/- mice, CD34+ humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the m6A-BHLHE41-CXCL1 axis by analysis of m6A sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an m6A-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment. CONCLUSIONS: Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the m6A-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer.

BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.