Author Correction: Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Parameter Optimization of RB-SiC Polishing by Femtosecond Laser.

Reaction-boned silicon carbide (RB-SiC) is considered a new material for large lightweight ground-based space telescopes due to its high specific stiffness, low thermal deformation, and excellent optical quality. The excellent mechanical properties of RB-SiC result in the low efficiency of traditional polishing and mechanical polishing. In this paper, a polishing method for RB-SiC based on a femtosecond laser is proposed to improve surface quality. A theoretical heat conduction model was established in the process of femtosecond laser irradiation of SiC. We analyzed the ablation type and calculated the single-pulse ablation threshold of SiC, which verified the feasibility of femtosecond laser polishing. Further, the effects of polishing parameters on the polished surface quality were analyzed by a series of experiments, and the optimal parameters were selected. It was observed to improve polishing efficiency and can replace the intermediate steps of traditional mechanical polishing.

DNAJA2 deficiency activates cGAS-STING pathway via the induction of aberrant mitosis and chromosome instability.

Molecular chaperone HSP70s are attractive targets for cancer therapy, but their substrate broadness and functional non-specificity have limited their role in therapeutical success. Functioning as HSP70's cochaperones, HSP40s determine the client specificity of HSP70s, and could be better targets for cancer therapy. Here we show that tumors defective in HSP40 member DNAJA2 are benefitted from immune-checkpoint blockade (ICB) therapy. Mechanistically, DNAJA2 maintains centrosome homeostasis by timely degrading key centriolar satellite proteins PCM1 and CEP290 via HSC70 chaperone-mediated autophagy (CMA). Tumor cells depleted of DNAJA2 or CMA factor LAMP2A exhibit elevated levels of centriolar satellite proteins, which causes aberrant mitosis characterized by abnormal spindles, chromosome missegregation and micronuclei formation. This activates the cGAS-STING pathway to enhance ICB therapy response in tumors derived from DNAJA2-deficient cells. Our study reveals a role for DNAJA2 to regulate mitotic division and chromosome stability and suggests DNAJA2 as a potential target to enhance cancer immunotherapy, thereby providing strategies to advance HSPs-based cancer therapy.

Concurrent delivery of immune checkpoint blockade modulates T cell dynamics to enhance neoantigen vaccine-generated antitumor immunity.

Neoantigen vaccines aiming to induce tumor-specific T cell responses have achieved promising antitumor effects in early clinical trials. However, the underlying mechanism regarding response or resistance to this treatment is unclear. Here we observe that neoantigen vaccine-generated T cells can synergize with the immune checkpoint blockade for effective tumor control. Specifically, we performed single-cell sequencing on over 100,000 T cells and uncovered that combined therapy induces an antigen-specific CD8 T cell population with active chemokine signaling (Cxcr3+/Ccl5+), lower co-inhibitory receptor expression (Lag3-/Havcr2-) and higher cytotoxicity (Fasl+/Gzma+). Furthermore, generation of neoantigen-specific T cells in the draining lymph node is required for combination treatment. Signature genes of this unique population are associated with T cell clonal frequency and better survival in humans. Our study profiles the dynamics of tumor-infiltrating T cells during neoantigen vaccine and immune checkpoint blockade treatments and high-dimensionally identifies neoantigen-reactive T cell signatures for future development of therapeutic strategies.

Dual targeting of CTLA-4 and CD47 on Treg cells promotes immunity against solid tumors.

Blockade of CD47, the "do not eat me" signal, has limited effects in solid tumors despite its potent antitumor effects in hematopoietic malignancies. Taking advantage of the high expression of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) on Treg cells and abundant Fc receptor-expressing active phagocytes inside the tumor microenvironment (TME), we designed and tested a heterodimer combining an anti-CTLA-4 antibody, which targets Treg cells, with the CD47 ligand, signal regulatory protein α (SIRPα), to selectively block CD47 on intratumoral Treg cells. We hypothesized that heterodimer treatment would increase antibody-dependent cellular phagocytosis of the targeted Treg cells. We found that anti-CTLA-4×SIRPα preferentially depleted ICOShigh immunosuppressive Treg cells in the TME and enhanced immunity against solid tumors, including MC38 and CT26 murine colon cancers. Mechanistically, we found that CD47 expression on Treg cells limited anti-CTLA-4-mediated depletion and Fc on the heterodimer-enhanced depletion. Furthermore, anti-human CTLA-4×SIRPα depleted tumor Treg cells and exhibits less toxicity than anti-human CTLA-4 in a humanized mouse model. Collectively, these results demonstrate that simultaneously modulating both "eat me" and do not eat me signals induces Treg cell depletion inside the TME and may be an effective strategy for treating solid tumors.

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway.

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

Hypofractionated EGFR tyrosine kinase inhibitor limits tumor relapse through triggering innate and adaptive immunity.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a first-line therapy for rapidly killing tumors such as those associated with non-small cell lung cancer by blocking oncogenic receptor signaling, but tumor relapse often occurs. Here, we have observed that hypofractionated EGFR TKI treatment (HypoTKI) is more potent than standard hyperfractionated EGFR TKI treatment (HyperTKI), and its antitumor effect associated with preventing tumor relapse depends on T cells. HypoTKI triggers greater innate sensing for type I IFN and CXCL10 production through the Myd88 signaling pathway to enhance tumor-specific T cell infiltration and reactivation. We also demonstrate that timely programmed cell death ligand-1 (PD-L1) blockade can synergize with HypoTKI to control advanced large tumors and effectively limit tumor relapse without severe side effects. Our study provides evidence for exploring the potential of a proper combination of EGFR TKIs and immunotherapy as a first-line treatment for treating EGFR-driven tumors.

Polymerase-mediated ultramutagenesis in mice produces diverse cancers with high mutational load.

Mutations underlie all cancers, and their identification and study are the foundation of cancer biology. We describe what we believe to be a novel approach to mutagenesis and cancer studies based on the DNA polymerase ε (POLE) ultramutator phenotype recently described in human cancers, in which a single amino acid substitution (most commonly P286R) in the proofreading domain results in error-prone DNA replication. We engineered a conditional PoleP286R allele in mice. PoleP286R/+ embryonic fibroblasts exhibited a striking mutator phenotype and immortalized more efficiently. PoleP286R/+ mice were born at Mendelian ratios but rapidly developed lethal cancers of diverse lineages, yielding the most cancer-prone monoallelic model described to date, to our knowledge. Comprehensive whole-genome sequencing analyses showed that the cancers were driven by high base substitution rates in the range of human cancers, overcoming a major limitation of previous murine cancer models. These data establish polymerase-mediated ultramutagenesis as an efficient in vivo approach for the generation of diverse animal cancer models that recapitulate the high mutational loads inherent to human cancers.

Increased differentiation capacity of bone marrow-derived mesenchymal stem cells in aquaporin-5 deficiency.

Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5(-/-) mice showed significantly lower plasma membrane water permeability than those from AQP5(+/+) mice. In characterizing the cultured BMMSCs from AQP5(-/-) and AQP5(+/+) mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5(-/-) than AQP5(+/+) BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5(-/-) -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5(-/-) BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5(-/-) mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5(-/-) than AQP5(+/+) BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5(+/+) than AQP5(-/-) BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration.