Published papers

Ionizing radiation activates cytoprotective pathways in cancer cells. Fibroblast growth factor receptor (FGFR) is a key player in these pathways. Thus, FGFR signaling is a potential target to induce radiosensitization. LY2874455 is an orally administrable selective pan-FGFR inhibitor. However, the radiosensitizing effects of LY2874455 remain unclear. In this study, we addressed this issue by using radioresistant human cancer cell lines H1703 (FGFR1 mutant), A549 (FGFR1-4 wild-type), and H1299 (FGFR1-4 wild-type). At an X-ray dose corresponding to 50%-clonogenic survival as the endpoint, 100 nM LY2874455 increased the sensitivity of H1703, A549, and H1299 cells by 31%, 62%, and 53%, respectively. The combination of X-rays and LY2874455 led to a marked induction of mitotic catastrophe, a hallmark of radiation-induced cell death. Furthermore, combination treatment suppressed the growth of A549 xenografts to a significantly greater extent than either X-rays or the drug alone without noticeable toxicity. This is the first report to show the radiosensitizing effect of a selective pan-FGFR inhibitor. These data suggest the potential efficacy of LY2874455 as a radiosensitizer, warranting clinical validation.

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

Purpose: Understanding the immune response during radiation therapy (RT) in a clinical setting is imperative for maximizing the efficacy of combined RT and immunotherapy. Calreticulin, a major damage-associated molecular pattern that is exposed on the cell surface after RT, is presumed to be associated with the tumor-specific immune response. Here, we examined changes in calreticulin expression in clinical specimens obtained before and during RT and analyzed its relationship with the density of CD8+ T cells in the same patient set. Methods and Materials: This retrospective analysis evaluated 67 patients with cervical squamous cell carcinoma who were treated with definitive RT. Tumor biopsy specimens were collected before RT and after 10 Gy irradiation. Calreticulin expression in tumor cells was evaluated via immunohistochemical staining. Subsequently, the patients were divided into 2 groups according to the level of calreticulin expression, and the clinical outcomes were compared. Finally, the correlation between calreticulin levels and density of stromal CD8+ T cells was evaluated. Results: The calreticulin expression significantly increased after 10 Gy (82% of patients showed an increase; P <.01). Patients with increased calreticulin levels tended to show better progression-free survival, but this was not statistically significant (P =.09). In patients with high expression of calreticulin, a positive trend was observed between calreticulin and CD8+ T cell density, but the association was not statistically significant (P =.06). Conclusions: Calreticulin expression increased after 10 Gy irradiation in tissue biopsies of patients with cervical cancer. Higher calreticulin expression levels are potentially associated with better progression-free survival and greater T cell positivity, but there was no statistically significant relationship between calreticulin upregulation and clinical outcomes or CD8+ T cell density. Further analysis will be required to clarify mechanisms underlying the immune response to RT and to optimize the RT and immunotherapy combination approach.

Abstract The strong cell killing effect of high linear energy transfer (LET) carbon ions is dependent on lethal DNA damage. Our recent studies suggest that induction of clusters of double-strand breaks (DSBs) in close proximity is one of the potential mechanisms. However, the relationship between LET, the degree of DSB clustering and the cell killing effect of carbon ions remains unclear. Here, we used high-resolution imaging technology to analyze the volume of γH2AX foci induced by monoenergetic carbon ions with a clinically-relevant range of LET (13–100 keV/μm). We obtained data from 3317 γH2AX foci and used a gaussian function to approximate the probability (p) that 1 Gy-carbon ions induce γH2AX foci of a given volume (vth) or greater per nucleus. Cell killing effects were assessed in clonogenic assays. The cell killing effect showed high concordance with p at vth = 0.7 μm3 across various LET values; the difference between the two was 4.7% ± 2.2%. This relationship was also true for clinical carbon ion beams harboring a mixed LET profile throughout a spread-out Bragg peak width (30–120 mm), with the difference at vth = 0.7 μm3 being 1.6% ± 1.2% when a Monte Carlo simulation-derived dose-averaged LET was used to calculate p. These data indicate that the cell killing effect of carbon ions is predictable by the ability of carbon ions to induce γH2AX foci containing clustered DSBs, which is linked to LET, providing the biological basis for LET modulation in the planning of carbon ion radiotherapy.