Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines.
Human PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of IFNB1 and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation.
We demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons.
Our findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.
Copyright © 2024 Kerschbaum-Gruber, Kleinwächter, Popova, Kneringer, Appel, Stasny, Röhrer, Dias, Benedum, Walch, Postl, Barna, Kratzer, Pickl, Akalin, Horvat, Franke, Widder, Georg and Slade.

Paroxysmal nocturnal haemoglobinuria (PNH) is a disorder resulting from erythrocyte membrane deficiencies caused by PIG-A gene mutations. While current treatments alleviate symptoms, they fail to address the underlying cause of the disease-the pathogenic PNH clones. In this study, we found that the expression of carbamoyl phosphate synthetase 1 (CPS1) was downregulated in PNH clones, and the level of CPS1 was negatively correlated with the proportion of PNH clones. Using PIG-A knockout K562 (K562 KO) cells, we demonstrated that CPS1 knockdown increased cell proliferation and altered cell metabolism, suggesting that CPS1 participates in PNH clonal proliferation through metabolic reprogramming. Furthermore, we observed an increase in the expression levels of the histone demethylase JMJD1C in PNH clones, and JMJD1C expression was negatively correlated with CPS1 expression. Knocking down JMJD1C in K562 KO cells upregulated CPS1 and H3K36me3 expression, decreased cell proliferation and increased cell apoptosis. Chromatin immunoprecipitation analysis further demonstrated that H3K36me3 regulated CPS1 expression. Finally, we demonstrated that histone demethylase inhibitor JIB-04 can suppressed K562 KO cell proliferation and reduced the proportion of PNH clones in PNH mice. In conclusion, aberrant regulation of the JMJD1C-H3K36me3-CPS1 axis contributes to PNH clonal proliferation. Targeting JMJD1C with a specific inhibitor unveils a potential strategy for treating PNH patients.
© 2024 British Society for Haematology and John Wiley & Sons Ltd.

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease involving hematopoietic stem cell membrane defects caused by acquired phosphatidylinositol glycan anchor biosynthesis class A (PIGA) mutations. In this study, 97 target genes were selected as a target gene panel and screened in 23 PNH patients via the sequencing of specific DNA target regions. Through functional analysis, we identified that suppressor-of-Zeste 12 (SUZ12) may be involved in the proliferation of PNH clones. mRNA and protein expression levels of SUZ12 and the trimethylation level of histone H3 at lysine 27 (H3K27) in CD59- peripheral blood leukocytes from PNH patients were higher than those in CD59+ cells from PNH patients and peripheral blood leukocytes from healthy controls. In addition, the relative expression of SUZ12 in PNH patients was positively correlated with Ret% and the proportion of PNH clones. When we knocked down SUZ12 expression in a PIGA knockdown THP-1 cell line (THP-1 KD cells), the trimethylation of histone H3K27(H3K27me3) and cell proliferation decreased, apoptosis increased, and cell cycle arrest occurred in G0/G1 phase. In conclusion, SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels. Our results may provide new therapeutic targets and possibilities for PNH patients.
©2022 Society for Leukocyte Biology.

Drug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin's lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.
Immunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.
We demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.
Our findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.
Copyright © 2021 Ge, Du, Chen, Zhu, Yao, Zhang, Wang, Sun, Gao, Hu and Hou.

The PIG-A gene mutation assay is a valuable tool for measuring in vivo gene mutations in blood cells. The human PIG-A assay, used as a potential genotoxicity biomarker, is minimally invasive, sensitive, and cost-efficient; however, the relationship between carcinogen exposure and PIG-A mutations is not well understood.
We investigated the genotoxic effect of red blood cells using PIG-A assay and lymphocyte cytokinesis-block micronucleus test in barbecue restaurant workers (N = 70) exposed to polycyclic aromatic hydrocarbons (PAHs) and self-identified healthy control subjects (N = 56). Urinary PAH metabolites were measured to evaluate internal exposure levels.
Multivariate Poisson regression showed that the PAH-exposed workers exhibited significantly higher PIG-A mutant frequency (MF) (8.04 ± 6.81 × 10- 6) than did the controls (5.56 ± 5.26 × 10- 6) (RR = 0.707, 95% CI: 0.615-0.812, P < 0.001). These results indicate that PAH exposure is a risk factor for elevated PIG-A MF. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in the PAH-exposed workers (MN: 3.06 ± 2.07 ‰, NBUD: 1.38 ± 1.02 ‰) were also significantly higher than in the controls (MN: 1.46 ± 0.64 ‰, P < 0.001; NBUD: 0.70 ± 0.60 ‰, P < 0.001). Additionally, PIG-A MFs showed better associations with several urinary hydroxylated PAH metabolites (P2-OH-Flu = 0.032, r2-OH-Flu = 0. 268; P2-OH-Phe = 0.022, r2-OH-Phe = 0.286; P3-OH-Phe = 0.0312, r3-OH-Phe = 0.270; P4-OH-Phe = 0.018, r4-OH-Phe = 0.296), while the increase in MN, NPB, and NBUD frequencies was not associated with any OH-PAH metabolites; and high-PAH-exposed workers showed the highest PIG-A MFs. Furthermore, there was a significant association between PIG-A MF and PAH exposure levels (Chi-square test for trend, P = 0.006).
Our results indicate that an increase in PIG-A MF in barbecue workers could reflect the response to PAH exposure, providing evidence of its potential as a genotoxicity biomarker in human risk assessment.
© 2021. The Author(s).