BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation

Summary: BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR. : Following induction of DNA double-strand break, a pro-end-joining environment is created in G2 by transient 53BP1 phosphorylation and RIF1 recruitment. Here, Isono et al. show that, if timely repair does not ensue, BRCA1 promotes 53BP1 dephosphorylation and RIF1 release, favoring repair by homologous recombination. Keywords: ATM, DNA-end resection, BRCA1, 53BP1, RIF1, PP4C, NHEJ, H

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

Background: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. Methods: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. Results: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. Conclusions: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression

A circulating subset of iNKT cells mediates antitumor and antiviral immunity

新規の循環型iNKT細胞を発見 --抗腫瘍・抗ウイルス感染効果の高い免疫細胞療法の開発への貢献に期待--. 京都大学プレスリリース. 2022-10-24.Invariant natural killer T (iNKT) cells are a group of innate-like T lymphocytes that recognize lipid antigens. They are supposed to be tissue resident and important for systemic and local immune regulation. To investigate the heterogeneity of iNKT cells, we recharacterized iNKT cells in the thymus and peripheral tissues. iNKT cells in the thymus were divided into three subpopulations by the expression of the natural killer cell receptor CD244 and the chemokine receptor CXCR6 and designated as C0 (CD244⁻CXCR6⁻), C1 (CD244⁻CXCR6⁺), or C2 (CD244⁺CXCR6⁺) iNKT cells. The development and maturation of C2 iNKT cells from C0 iNKT cells strictly depended on IL-15 produced by thymic epithelial cells. C2 iNKT cells expressed high levels of IFN-γ and granzymes and exhibited more NK cell–like features, whereas C1 iNKT cells showed more T cell–like characteristics. C2 iNKT cells were influenced by the microbiome and aging and suppressed the expression of the autoimmune regulator AIRE in the thymus. In peripheral tissues, C2 iNKT cells were circulating that were distinct from conventional tissue-resident C1 iNKT cells. Functionally, C2 iNKT cells protected mice from the tumor metastasis of melanoma cells by enhancing antitumor immunity and promoted antiviral immune responses against influenza virus infection. Furthermore, we identified human CD244⁺CXCR6⁺ iNKT cells with high cytotoxic properties as a counterpart of mouse C2 iNKT cells. Thus, this study reveals a circulating subset of iNKT cells with NK cell–like properties distinct from conventional tissue-resident iNKT cells

Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

Background and Purpose: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies.Copyright:Materials and Methods: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/ + and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA doublestrand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3.Results: The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring longretained DSBs at 24 h post-irradiation.Conclusions: Efficient induction of mitotic catastrophe in apoptosis-resistant p53- deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment

The Non-Cancer Specific Elevation of the Serum Squamous Cell Carcinoma Antigen during the Post-Radiotherapy Follow-Up of Cervical Cancer Patients

The elevation of the serum squamous cell carcinoma (SCC) antigen unrelated to disease progression occurs during the follow-up of patients with cervical cancer treated with radiotherapy. Although known empirically, the incidence and characteristics of this non-cancer specific elevation in SCC remain unclear. Here, we examined the post-treatment kinetics of SCC in 143 consecutive patients with squamous cell carcinoma of the cervix treated with definitive radiotherapy; in all patients, progression-free disease status was confirmed by periodic monitoring for at least 36 months (median, 61 months). We found that the 5-year cumulative incidence of post-treatment SCC elevation was unexpectedly high at 37.3% (59/143 patients), and that 59.3% (35/59) of event-positive patients experienced multiple events. The median peak SCC level for a given event was 2.0 ng/mL (interquartile range, 1.7–2.9 ng/mL). The multivariate analysis showed that renal dysfunction was associated significantly with a greater incidence of SCC elevation (p = 0.046). In addition, the 5-year cumulative incidence of SCC elevation was significantly greater in patients with renal dysfunction than in those without (54.8% vs. 32.9%, respectively; hazard ratio, 2.1 [95% confidence interval, 1.1–4.2]; p = 0.028). These data will be useful for monitoring cervical cancer patients treated with radiotherapy

Association between Tumor Mutational Burden, Stromal CD8⁺ Tumor-Infiltrating Lymphocytes, and Clinical Factors in Cervical Cancers Treated with Radiotherapy

Background: Tumor mutational burden (TMB) and stromal CD8-positive tumor-infiltrating lymphocytes (CD8+TILs) serve important roles in antitumor immune responses to radiotherapy. This study aimed to elucidate the association between TMB, CD8+TILs, and clinical factors in patients with cervical cancer treated with radiotherapy. Methods: Patients with squamous cell carcinoma of the uterine cervix treated with definitive radiotherapy, and with available somatic mutation data and immunohistochemical staining data from identical tumor tissues, were enrolled retrospectively. The association between TMB and/or CD8+TIL density and patient characteristics, mutation profiles, and treatment outcome was analyzed. Results: The study analyzed 44 patients (median follow-up period, 61 months). There was no significant correlation between TMB and CD8+TIL density, or between TMB or CD8+TIL density and patient characteristics. TMB-high or CD8+TIL density-low status was associated with worse overall survival and distant metastasis-free survival; the predictive value of these factors became greater when used in combination. TMB-high or CD8+TIL density-high status was associated with ARID1A mutations. Conclusions: These data indicate independence of TMB and CD8+TIL density and the involvement of ARID1A alterations in antitumor immune responses in patients with cervical cancers treated with radiotherapy, warranting further mechanistic research and prospective validation

Mutation Analysis of Radioresistant Early-Stage Cervical Cancer

Radiotherapy is a definitive treatment for early-stage cervical cancer; however, a subset of this disease recurs locally, necessitating establishment of predictive biomarkers and treatment strategies. To address this issue, we performed gene panel-based sequencing of 18 stage IB cervical cancers treated with definitive radiotherapy, including two cases of local recurrence, followed by in vitro and in silico analyses. Simultaneous mutations in KRAS and SMAD4 (KRASmt/SMAD4mt) were detected only in a local recurrence case, indicating potential association of this mutation signature with radioresistance. In isogenic cell-based experiments, a combination of activating KRAS mutation and SMAD4 deficiency led to X-ray resistance, whereas either of these factors alone did not. Analysis of genomic data from 55,308 cancers showed a significant trend toward co-occurrence of mutations in KRAS and SMAD4. Gene Set Enrichment Analysis of the Cancer Cell Line Encyclopedia dataset suggested upregulation of the pathways involved in epithelial mesenchymal transition and inflammatory responses in KRASmt/SMAD4mt cancer cells. Notably, irradiation with therapeutic carbon ions led to robust killing of X-ray-resistant KRASmt/SMAD4mt cancer cells. These data indicate that the KRASmt/SMAD4mt signature is a potential predictor of radioresistance, and that carbon ion radiotherapy is a potential option to treat early-stage cervical cancers with the KRASmt/SMAD4mt signature

Use of a Si/CdTe Compton Camera for In vivo Real-Time Monitoring of Annihilation Gamma Rays Generated by Carbon Ion Beam Irradiation

Real-time monitoring of C-ion-induced annihilation gamma-rays was implemented using a Compton camera in a mouse model. An adult C57BL/6 mouse was anesthetized, and C-ion beams were directed into the abdomen at 1 × 109 particles/s for 20 s. The 511 keV annihilation gamma-rays, generated by the interaction between the irradiated C-ion beam and the target mouse, were detected using a silicon/cadmium telluride (Si/CdTe) Compton camera for 20 min immediately after irradiation. The irradiated site and the peak intensity position of 511 keV gamma emissions due to C-ion beam irradiation on a mouse were observed at the abdomen of the mouse by developing Compton images. Moreover, the positron emitter transport was observed by evaluating the range of gamma-ray emission after the C-ion beam irradiation on the mouse. Our data suggest that by confirming the peak intensity and beam range of C-ion RT with Si/CdTe-based Compton camera, it would be possible to reduce the intra-fractional and inter-fractional dose distribution degradation. Therefore, the results of this study would contribute to the future development of adaptive therapy with C-ion RT for humans

Characteristics of PSA Bounce after Radiotherapy for Prostate Cancer: A Meta-Analysis

The rate and characteristics of prostate-specific antigen (PSA) bounce post-radiotherapy remain unclear. To address this issue, we performed a meta-analysis. Reports of PSA bounce post-radiotherapy with a cutoff of 0.2 ng/mL were searched by using Medline and Web of Science. The primary endpoint was the occurrence rate, and the secondary endpoints were bounce characteristics such as amplitude, time to occurrence, nadir value, and time to nadir. Radiotherapy modality, age, risk classification, androgen deprivation therapy, and the follow-up period were extracted as clinical variables. Meta-analysis and univariate meta-regression were performed with random-effect modeling. Among 290 search-positive studies, 50 reports including 26,258 patients were identified. The rate of bounce was 31%; amplitude was 1.3 ng/mL; time to occurrence was 18 months; nadir value was 0.5 ng/mL; time to nadir was 33 months. Univariate meta-regression analysis showed that radiotherapy modality (29.7%), age (20.2%), and risk classification (12.2%) were the major causes of heterogeneity in the rate of bounce. This is the first meta-analysis of PSA bounce post-radiotherapy. The results are useful for post-radiotherapy surveillance of prostate cancer patients