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Coal and gas out-bursts are a major risk in deep gassy coal mines. In order to minimize the out-burst potential during the mining process, we propose a novel blast-injection integrated (BII) technology. In this technology, a group of de-stress blasts are first used to de-stress the area near the active mining face and push the stress peak further away from the mining face. The cracks generated by the blasting improve the connectivity between the pre-existing fractures and promote gradual release of gas at the mining face. This is followed by water injection directly into the coal seam through the blast holes. The injection of water can greatly improve coal plasticity and reduce the initial speed of gas diffusion, ultimately reducing the potential for violent out-bursts. To implement this technology, a set of devices were developed that include hole packers and transport and safety accessories. The BII technology was subsequently tested at an active mine. It was found that the stress relief zone in front of the coal face increased longitudinally by ∼5 m, and the rate of gas emission at the mining face doubled within 30 min after the de-stress blasting. The injection water volume after the de-stress blasting also increased by 25 times compared to the un-blasted condition, and the monthly gas concentration in the return airway decreased from 0.58% to 0.36%. Additionally, the concentration of dust in the return airway was found to decrease. Based on the field test results, it was concluded that BII technology is an effective measure to limit coal and gas out-burst potential.

Cancer cells express high levels of programmed death ligand 1 (PD-L1), a ligand of the programmed cell death protein 1 (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 a first-line therapy for patients with lung cancer. 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, allowing bypass of inhibitory upstream open reading frames in the PD-L1 5′ untranslated region, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrate that integrated stress-response-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. O’Donnell and colleagues report that activation of the integrated stress response in non-small cell lung cancer cells by impairing heme production leads to enhanced PD-L1 translation in an eIF5B-dependent manner.

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.

BackgroundAlthough approved by FDA, anti-CTLA-4 treatment has severe side effect that limits its clinical usage. Blockade of CD47, the “don't eat me” signal, has limited effects in solid tumors despite its potent anti-tumor effects in hematopoietic malignancies. Targeted delivery of immune blockers into tumor tissues are desireble.MethodsTaking advantage of the high expression of CTLA-4 on Treg cells and abundant Fc receptor+ active phagocytes inside the tumor microenvironment (TME), we design and test an anti-CTLA-4×SIRPα (CD47 ligand)-Fc heterodimer that selectively blocks CD47 on intratumoral Treg cells and increases antibody-dependent cellular phagocytosis (the “eat me” signal).ResultsAnti-CTLA-4×SIRPα preferentially depletes ICOShigh immunosuppressive Treg cells in the TME (figure 1–3) and enhances immunity against solid tumors. Mechanistically, we discovered that CD47 expression on Treg cells limits anti-CTLA-4 mediated depletion while Fc on the heterodimer enhances the depletion. Furthermore, anti-human CTLA-4×SIRPα depletes tumor Treg cells (figure 4–6) and exhibits less toxicity than anti-human CTLA-4 in a humanized mouse model.ConclusionsCollectively, these results highlight coordinatively modulating “eat me” and “don't eat me” signals for depleting Treg cells inside the TME as a unique strategy for solid tumor treatment.Abstract 800 Figure 1Anti-CTLA-4×SIRPα selectively targets intratumor Treg. (A) Expression of CTLA-4 transcripts in colorectal cancer patient tissues based on single cell sequencing online database (29). (B and C) CTLA-4 expression on Treg cells from PBMC, spleen and tumor cells in MC38 tumor-bearing mice on day 14. (D) Schematic diagram of anti-CTLA-4×SIRPα heterodimer. (E) Kinetic association (ka), dissociation (kd), and calculated affinity (KD) for binding of the indicated analyte to mouse CTLA-4 or CD47 antigen were measured by surface plasmon resonance using OpenSPR instrument. (F) Binding of SIRPα, CTLA-4 and anti-CTLA-4×SIRPα to RBC from C57BL/6 mice, n=4. (G) Anti-CTLA-4×SIRPα binding on Treg of PBMC and tumor cells from MC38 tumor-bearing mice. (H) C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells. After 13 days, 20 µg anti-CTLA-4×SIRPα was injected intraperitoneally. Five days later, mice were perfused with PBS, and tumor and other tissues were harvested and homogenized. Anti-CTLA-4×SIRPα protein abundance was determined with ELISA, n=4.Abstract 800 Figure 2Anti-CTLA-4×SIRPα preferentially depletes ICOShi Treg. (A–C) C57BL/6 mice (n=4) were inoculated with 5 × 105 MC38 tumor cells and treated with Combo or anti-CTLA-4×SIRPα on day 13. Five days later, Treg cells from tumor, spleen and draining lymph node (dLN) were analyzed. The representative flow cytometric gating was shown in (A). Treg cells frequency from different groups was shown in (B). The ratio of CD8+ T cells to Treg was shown in (C). (D–F) Foxp3-EGFP reporter mice (n=3) were inoculated with 5 × 105 MC38 tumor cells and treated with Combo or anti-CTLA-4×SIRPα on day 13. Five days later, Foxp3-EGFP+ cells from tumor, spleen and dLN were analyzed. The representative flow cytometric gating was shown in (D). Foxp3-EGFP+ cell frequency from different groups was shown in (E). The ratio of CD8+ T cells to Foxp3-EGFP+ cells was shown in (F). (G–I) Same experiment scheme as (A–F). Representative ICOS expression on Treg cells was shown in (G). (H) ICOS++ frequency among Treg cells (H) and ICOS++ frequency among YFP+ cells (I) were shown.Abstract 800 Figure 3Anti-CTLA-4×SIRPα reduces tumor in T cell dependent way. (A) C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with equal moles of anti-CTLA-4 plus SIRPα (Combo, 12 µg + 8 µg) or anti-CTLA-4×SIRPα (20 µg) on day 14. Tumor growth was measured every 3 days, n=5. (B) BALB/C mice were inoculated with 5 × 105 CT26 tumor cells and treated with Combo (30 µg+ 20 µg) or anti-CTLA-4×SIRPα (50 µg) on day 6. Tumor growth was measured every 3 days, n=5. (C) Rag1-/- mice were inoculated with 1 × 105 MC38 tumor cells and treated with Combo (12 µg+ 8 µg) or anti-CTLA-4×SIRPα (20 µg) on day 13. Tumor growth was measured every 3 days, n=5. (D) MuMT mice were inoculated with 1.5 × 106 MC38 tumor cells and treated with anti-CTLA-4×SIRPα (20 µg) on day 14. Tumor growth was measured every 3 days, n=4. (E and F) C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 13. 200 μg of anti-CD4 (E) or anti-CD8 (F) was administrated one day before treatment initiation and then twice a week for 2 weeks. Tumor growth was measured every 3 days, n=5. (G–I) C57BL/6 mice (n=4) were inoculated with 5 × 105 MC38 tumor cells and treated with Combo or anti-CTLA-4×SIRPα on day 14. Five days later, tumor-infiltrating lymphocytes (TILs) were analyzed for total CD45 in tumor (E), Ki67 expression on CD4 and CD8 T cells from different groups (F and G) after treatment.Abstract 800 Figure 4Anti-CTLA-4×SIR enhances tumor-specific T cell IFN-gamma. (A) C57BL/6 mice (n=4) were inoculated with 5 × 105 MC38 tumor cells and treated with Combo or anti-CTLA-4×SIRPα on day 13. Five days later, TILs were analyzed for tumor specific T cells. (B and C) C57BL/6 mice (n=4) were inoculated with 5 × 105 MC38 tumor cells and treated with Combo or anti-CTLA-4×SIRPα on day 13. Five days later, TILs were purified with CD45+ positive selection magnetic beads. TILs (A) and dLN (B) were re-stimulated with irradiated MC38 tumor cells or irrelevant control Lewis lung cancer (LLC) cells for 48 h. IFN-γ producing cells were determined by ELISPOT assay. (D) WT or Ifng-/- C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 13. Tumor growth was measured every 3 days, n=4. (E) C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 13. 150 μg of anti-IFN-γ was administrated one day before treatment initiation and then twice a week for 2 weeks. Tumor growth was measured every 3 days, n=5. (F and G) C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 14. Six weeks later, anti-CTLA-4×SIRPα cured mice and control naïve mice were re-challenged with 5 × 106 MC38 tumor cells on the left flank (opposite to the original injection flank) (F), and 5 × 105 LLC tumor cells were injected on the right flank (G). Tumor growth was measured every 3 days, n=5.Abstract 800 Figure 5CD47 expression on Treg cells is essential. (A) Cd47-/- tumor bearing C57BL/6 mice were treated with Combo or anti-CTLA-4×SIRPα on day 13. Tumor growth was measured every 3 days, n=5. (B) WT or Cd47-/- C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 14. Tumor growth was measured every 3 days, n=5. (C) WT or Cd47-/- T cells were purified by negative selection magnetic beads and intravenously transferred to Rag1-/- mice. 2 × 105 MC38 tumor cells were inoculated into the recipient mice the next day. Mice were treated with 40 µg anti-CTLA-4×SIRPα on day 7 and day 12. Experiment scheme was shown in upper panel and tumor growth was shown in lower panel, n=5. (D) C57BL/6 mice were inoculated with 5 × 105 MC38-cEGFR tumor cells and treated with 30 µg anti-EGFR×SIRPα or anti-CTLA-4×SIRPα on day 14. Tumor growth was measured every 3 days, n=3–4. (E) WT or Fcer1g-/- C57BL/6 mice were inoculated with 1 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα on day 13. Tumor growth was measured every 3 days, n=5. (F) WT C57BL/6 mice were inoculated with 5 × 105 MC38 tumor cells and treated with anti-CTLA-4×SIRPα or anti-CTLA-4×SIRPα with mutant Fc (Anti-CTLA-4×SIRPα-InFc) on day 14. Tumor growth was measured every 3 days, n=5. (G) Bone marrow derived macrophages from WT or Fcer1g-/-mice were mixed with CFSE labelled in vitro differentiated Treg cells from WT or Cd47-/- mice for 3 h. Phagocytosis was determined with flow cytometry. Phagocytosis index was defined as the percentage of CFSE+ macrophages among total macrophages.Abstract 800 Figure 6A human version anti-CTLA-4×SIRPα heterodimer depletes Treg. (A and B) CD47 and CTLA-4 expression level on Treg cells of PBMC and tumor from NSCLC patients. The representative flow cytometric gating was shown in (A), pooled data from different patients was show in (B). (C) Anti-hCTLA-4, SIRPα and anti-hCTLA-4×SIRPα binding on Jurkat and Jurkat-hCTLA-4 expressing cells. (D) Comparison of anti-hCTLA-4×SIRPα binding on Jurkat and Jurkat-hCTLA-4 cells based on (C). (E and F) PBMC-humanized NSG mice were inoculated with 2.5 × 106 A549 tumor cells and treated with anti-hCTLA-4 plus SIRPα (h-Combo, 18 µg +12 µg) or anti-hCTLA-4×SIRPα (30 µg) on day 12. Two days later, the frequency of tumor-infiltrating Treg cells (G) and ICOS expression level on Treg cells (F) were analyzed, n=4. (G) PBMC-humanized NSG mice were treated with 200 μg anti-hCTLA-4 or anti-hCTLA-4×SIRPα twice a week for 4 times. Mouse body weight was monitored every 3 days, n=5. Data are shown as mean ± SEM from two independent experiments. P value was determined by paired t test (B), unpaired t test (E and F) and two-way ANOVA with Geisser-Greenhouse's correction (G). The normality of data was confirmed by Shapiro-Wilk test.

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 T(reg) 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 T(reg) cells, with the CD47 ligand, signal regulatory protein α (SIRPα), to selectively block CD47 on intratumoral T(reg) cells. We hypothesized that heterodimer treatment would increase antibody-dependent cellular phagocytosis of the targeted T(reg) cells. We found that anti–CTLA-4×SIRPα preferentially depleted ICOS(high) immunosuppressive T(reg) cells in the TME and enhanced immunity against solid tumors, including MC38 and CT26 murine colon cancers. Mechanistically, we found that CD47 expression on T(reg) cells limited anti–CTLA-4–mediated depletion and Fc on the heterodimer-enhanced depletion. Furthermore, anti-human CTLA-4×SIRPα depleted tumor T(reg) 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 T(reg) cell depletion inside the TME and may be an effective strategy for treating solid tumors.

Gas outburst is a serious disaster in deep mining. In this paper, we first study the effect of water content on gas outburst, and found that increasing the water content of coal can significantly reduce the elastic modulus and uniaxial compressive strength, which is beneficial in reducing the peak stress concentration in the roadway and pushing the peak stress deeper. We also found that by increasing the water content of the coal seam, the firmness coefficient increases, while the initial speed of gas diffusion decreases simultaneously. Increasing the water content of the coal seam can also change the Langmuir adsorption constant of coal, reduce the a value of the limit adsorption capacity, increase the b value of the adsorption capacity, and reduce the amount of desorption gas in the coal. These studies indicate that increasing the water content of coal can significantly reduce the risk of gas outbursts. To increase the water injection volume, the technology of promoting coal seam infusion by blasting has been proposed. Blasting promotes equilibrium distribution in the stress field and generate new fractures, which promotes the water injection. In the 8th coal mine in the Pingdingshan coal field, the test results indicate that after loosening blasting, the water injection of a single borehole increased by an average of 54.6 times under the injection pressure of 3–7 MPa. The water content of coal is improved significantly, and the prediction index value of the gas outburst is reduced significantly. This technology is important for gas control in similar coal mines.

Coal and gas outburst is greatly affected by the stress distribution in front of the heading face when tunnelling in high gassy coal seam. In this paper, the FLAC3D software and Mohr-Coulomb failure criterion are used to study the stress differences in front of the heading face. A total of 31 homogeneous models are built to study the stress differences affected by the coal seam mechanical properties. And another 8 kinds of heterogeneous models are built to study the stress differences affected by the abruptly changes of coal seam mechanical properties. When tunnelling in homogeneous coal seam, the large elastic modulus of coal seam will increase the stress peak value in front of the heading face, but do not change the stress peak position. The small internal friction angle will push the stress peak far away from the heading face. Therefore, the range of stress-relief zone in front of the heading face could significantly increase. The small cohesion of the coal seam will also push the stress peak far away from the heading face, but will result in an increase in the stress peak value. The Poisson ratio and tensile strength of the coal seam have little influence on the value and position of stress peak. As the elastic modulus decreases, cohesion and internal friction angle can greatly increase the displacement of the coal in front of the heading face. Taking into account both the mechanical properties of coal seam and the corresponding stress peaks, gas outburst is not easy to occur in the homogeneous coal seam. When tunnelling in heterogeneous coal seam, the abrupt change of coal seam elastic modulus greatly affects the stress concentration peak. If the cohesion of the coal seams also changes greatly with the elastic modulus, the stress peak will be enhanced. The abrupt change of coal seam cohesion and Poisson ratio have a slight influence on the stress concentration, while the abrupt change of internal friction angle of coal seam have little effect on the stress concentration. An outburst accident is most likely triggered when the cohesion and elastic modulus of the coal seams change greatly at the same time. Outcomes of this study is very beneficial for the prediction and prevention of coal and gas outburst. Meanwhile, this study provides a reference for the setting of mechanical properties of the coal seam and rock layers in the numerical simulation of mining.

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

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-Pole(P286R) 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.

Bispecific T-cell engagers (BiTEs) that preferentially target tumor-associated antigens (TAA) to reengage CD3 signaling have been approved to treat acute B-cell lymphoblastic leukemia. However, their applications in solid tumors have been hampered due to short half-life, weak anti-tumor activity, and severe toxicity at therapeutic doses. To explore new targets, we designed a bispecific antibody (BsAb) which simultaneously targets CD3 and immune checkpoint PD-L1. Compared with conventional TAA based targeting, PDL1xCD3 generates far superior anti-tumor immune responses in vivo. Mechanistically, blockade of PD-L1 on dendritic cells instead of tumor cells can potently rejuvenate preexisting tumor reactive CD8 T cells in a B7-1/2 dependent manner for a durable anti-tumor responses. This study argues that targeting DC-T cell instead of current tumor-T cell can achieve much better T cell rejuvenation in BsAb therapy.

Summary 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-β 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.

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.

Hydraulic fracturing can increase coal seam fractures and reduce gas outburst risks, but the main challenge of its implementation in coal mines is to achieve uniform fracturing and gas reduction. In this paper, we studied the mechanism of water-driven gas release through two aspects: the H2O molecule is more easily adsorbed by coal than CH4, and water can decrease the gas adsorption capacity of coal. Comparative experiments of conventional hydraulic fracturing and blasting-fracturing technologies were carried out in #8 coal mine in Pingdingshan, China. We found that fractures and water contents around the blasting-based fracturing hole were more uniform than the conventional fracturing hole. After hydraulic fracturing, low water content coal usually has high gas content, which confirmed that gas in wet areas was displaced by water and driven to dry areas. To improve the uniformity of hydraulic fracturing, we proposed the uniform fracturing technology by combining blasting-fracturing holes and control holes. Blasting before hydraulic fracturing will increase the uniformity of fracturing and water distribution, and the control holes are used to monitor and release gas driven by water injection. The effective wetting range of conventional hydraulic fracturing was from −6 to 6 m along the strike direction and from −3 to 9 m along the dip direction with 94.71 m3 water injected. While the effective wetting range using uniform fracturing was increased to from −21 to 18 m along the strike direction and from −12 to 18 m along the dip direction with78.13 m3 water injected. Furthermore, number of boreholes to be drilled was largely reduced from 1300 to 132 when using the uniform fracturing technology to replace the dense hole technology in a 300 m long roadway. This research has suggested that uniform fracturing can effectively reduce gas outburst risk in gassy mines in China.

Cancer is characterized by increased mutation rate, and recent work has led to a deeper understanding of mutator phenotypes and underlying molecular mechanisms. Differences in the mutational landscape of individual cancers underlie key aspects of clinical behavior. For example, overall base substitution rate is the best predictor of immune therapy response. Most genetically engineered mouse models of cancer (GEMMs) reiterate a few driver events but fail to recapitulate the high mutational loads that are the ultimate cause of most human cancer, making GEMMs inadequate for many aspects of tumor behavior, such as immune responses. The highest base substitution rates in cancers (≥100/Mb) result from specific heterozygous single amino acid substitutions (usually P286R) in the proofreading domain of DNA polymerase epsilon (POLE), rendering POLE highly error prone. POLE mutations are most common in endometrial cancer but occur with lower incidence in a wide range of cancers. We hypothesized that GEMMs could be generated by recapitulating PoleP286R via conditional knockin. Previously, we showed that constitutive expression of PoleP286R throughout the body triggered malignancies of diverse lineages. Mice harboring LSL-PoleP286R and the endometrial BAC-Sprr2f-Cre developed endometrial cancers starting at 1 year. Endometrial cancers exhibited histologic features previously reported in human POLE tumors, and metastasized in all mice (p This abstract is also being presented as Poster A34. Citation Format: Hao-Dong Li, Changzheng Lu, He Zhang, Ileana C. Cuevas, Subhransu S. Sahoo, Yang-Xin Fu, Diego H. Castrillon. Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR13.

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.

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 e (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.

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.