Irreparable DNA Damage Research Articles

Electron-Beam-Killed Staphylococcus Vaccine Reduced Lameness in Broiler Chickens

Broiler chicken lameness caused by bacterial chondronecrosis with osteomyelitis (BCO) is presently amongst the most important economic and animal welfare issues faced by the poultry industry, and the estimated economic loss is around USD 150 million. BCO lameness is associated with multiple opportunistic bacterial pathogens inhabiting the respiratory and gastrointestinal tracts. In cases of immune deficiency resulting from stress, injury, or inflammation of the tissue, opportunistic pathogens, mainly Staphylococcus spp., can infiltrate the respiratory or gastrointestinal mucosa and migrate through the bloodstream to eventually colonize the growth plates of long bones, causing necrosis that leads to lameness. This is the first report of developing a Staphylococcus vaccine against BCO lameness disease in broiler chickens. Electron beam (eBeam) technology causes irreparable DNA damage, preventing bacterial multiplication, while keeping the epitopes of the cell membrane intact, helping the immune system generate a more effective response. Our results show a 50% reduction of lameness incidence in the eBeam-vaccinated chicken group compared to the control. Additionally, the eBeam-vaccinated chickens present higher titer of anti-Staphylococcus IgA, signifying the development of an efficient and more specific humoral immune response. Our data establish the eBeam-killed Staphylococcus vaccine as an effective approach to reducing the incidence of lameness in broiler chickens.

1G6-D7 Inhibits Homologous Recombination Repair by Targeting Extracellular HSP90α to Promote Apoptosis in Non-Small Cell Lung Cancer.

Despite recent advances in treatment, non-small cell lung cancer (NSCLC) continues to have a high mortality rate. Currently, NSCLC pathogenesis requires further investigation, and therapeutic drugs are still under development. Homologous recombination repair (HRR) repairs severe DNA double-strand breaks. Homologous recombination repair deficiency (HRD) occurs when HRR is impaired and causes irreparable double-strand DNA damage, leading to genomic instability and increasing the risk of cancer development. Poly(ADP-ribose) polymerase (PARP) inhibitors can effectively treat HRD-positive tumors. Extracellular heat shock protein 90α (eHSP90α) is highly expressed in hypoxic environments and inhibits apoptosis, thereby increasing cellular tolerance. Here, we investigated the relationship between eHSP90α and HRR in NSCLC. DNA damage models were established in NSCLC cell lines (A549 and H1299). The activation of DNA damage and HRR markers, apoptosis, proliferation, and migration were investigated. In vivo tumor models were established using BALB/c nude mice and A549 cells. We found that human recombinant HSP90α stimulation further activated HRR and reduced DNA damage extent; however, eHSP90α monoclonal antibody, 1G6-D7, effectively inhibited HRR. HRR inhibition and increased apoptosis were observed after LRP1 knockdown; this effect could not be reversed with hrHSP90α addition. The combined use of 1G6-D7 and olaparib caused significant apoptosis and HRR inhibition in vitro and demonstrated promising anti-tumor effects in vivo. Extracellular HSP90α may be involved in HRR in NSCLC through LRP1. The combined use of 1G6-D7 and PARP inhibitors may exert anti-tumor effects by inhibiting DNA repair and further inducing apoptosis of NSCLC cells.

DNA Damage and Senescence in the Aging and Alzheimer's Disease Cortex Are Not Uniformly Distributed.

Alzheimer's disease (AD) is a neurodegenerative illness with a typical age of onset exceeding 65 years of age. The age dependency of the condition led us to track the appearance of DNA damage in the frontal cortex of individuals who died with a diagnosis of AD. The focus on DNA damage was motivated by evidence that increasing levels of irreparable DNA damage are a major driver of the aging process. The connection between aging and the loss of genomic integrity is compelling because DNA damage has also been identified as a possible cause of cellular senescence. The number of senescent cells has been reported to increase with age, and their senescence-associated secreted products are likely contributing factors to age-related illnesses. We tracked DNA damage with 53BP1 and cellular senescence with p16 immunostaining of human post-mortem brain samples. We found that DNA damage was significantly increased in the BA9 region of the AD cortex compared with the same region in unaffected controls (UCs). In the AD but not UC cases, the density of cells with DNA damage increased with distance from the pia mater up to approximately layer V and then decreased in deeper areas. This pattern of DNA damage was overlaid with the pattern of cellular senescence, which also increased with cortical depth. On a cell-by-cell basis, we found that the intensities of the two markers were tightly linked in the AD but not the UC brain. To test whether DNA damage was a causal factor in the emergence of the senescence program, we used etoposide treatment to damage the DNA of cultured mouse primary neurons. While DNA damage increased after treatment, after 24 h, no change in the expression of senescence-associated markers was observed. Our work suggests that DNA damage and cellular senescence are both increased in the AD brain and increasingly coupled. We propose that in vivo, the relationship between the two age-related processes is more complex than previously thought.

Multiplex imaging reveals spatially resolved DNA-damage response neighborhoods in TP53-mutated myelodysplastic neoplasms.

While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.

Oxidative stress-induced DNA damage and DNA repair mechanisms in mangrove bacteria exposed to climatic and heavy metal stressors

Bacteria thriving in the mangrove ecosystem are major drivers of elemental cycles. Climate change and environmental stressors (heavy metals) influence the performance of these microorganisms, thereby affecting the biogeochemical cycle. The present study reports the genotoxic effect of climatic and heavy metal stressors on mangrove bacteria and their adaptation strategies. Comparative analysis between two bacterial strains, Bacillus stercoris GST-03 and Pseudomonas balearica DST-02 isolated from the Bhitarkanika mangrove ecosystem, Odisha, India, showed cellular injuries in response to various stressors as evident by declined growth, elevated levels of reactive oxygen species (ROS) and resulted DNA damage. B. stercoris GST-03 showed more tolerance towards acidic pH, whereas P. balearica DST-02 showed higher tolerance towards UV exposure and heavy metals (Lead and Cadmium). The adaptation strategies of the strains revealed a significant role of GST in ROS scavenging activity and the involvement of Nucleotide excision repair or SOS response pathways. However, irreparable DNA damage was observed at pH 9 and 200 ppm Cd in B. stercoris GST-03, and at pH 4, 1000 ppm of Pb and 200 ppm of Cd in P. balearica DST-02. The current findings provide a broad overview of bacterial response and adaptability concerning future climate and environmental changes.

IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

MYC up-regulation confers vulnerability to dual inhibition of CDK12 and CDK13 in high-risk Group 3 medulloblastoma

BackgroundMedulloblastoma (MB) is the most common cerebellar malignancy during childhood. Among MB, MYC-amplified Group 3 tumors display the worst prognosis. MYC is an oncogenic transcription factor currently thought to be undruggable. Nevertheless, targeting MYC-dependent processes (i.e. transcription and RNA processing regulation) represents a promising approach.MethodsWe have tested the sensitivity of MYC-driven Group 3 MB cells to a pool of transcription and splicing inhibitors that display a wide spectrum of targets. Among them, we focus on THZ531, an inhibitor of the transcriptional cyclin-dependent kinases (CDK) 12 and 13. High-throughput RNA-sequencing analyses followed by bioinformatics and functional analyses were carried out to elucidate the molecular mechanism(s) underlying the susceptibility of Group 3 MB to CDK12/13 chemical inhibition. Data from International Cancer Genome Consortium (ICGC) and other public databases were mined to evaluate the functional relevance of the cellular pathway/s affected by the treatment with THZ531 in Group 3 MB patients.ResultsWe found that pharmacological inhibition of CDK12/13 is highly selective for MYC-high Group 3 MB cells with respect to MYC-low MB cells. We identified a subset of genes enriched in functional terms related to the DNA damage response (DDR) that are up-regulated in Group 3 MB and repressed by CDK12/13 inhibition. Accordingly, MYC- and CDK12/13-dependent higher expression of DDR genes in Group 3 MB cells limits the toxic effects of endogenous DNA lesions in these cells. More importantly, chemical inhibition of CDK12/13 impaired the DDR and induced irreparable DNA damage exclusively in MYC-high Group 3 MB cells. The augmented sensitivity of MYC-high MB cells to CDK12/13 inhibition relies on the higher elongation rate of the RNA polymerase II in DDR genes. Lastly, combined treatments with THZ531 and DNA damage-inducing agents synergically suppressed viability of MYC-high Group 3 MB cells.ConclusionsOur study demonstrates that CDK12/13 activity represents an exploitable vulnerability in MYC-high Group 3 MB and may pave the ground for new therapeutic approaches for this high-risk brain tumor.

Current Understanding of the Role of Senescent Melanocytes in Skin Ageing.

Melanocytes reside within the basal epidermis of human skin, and function to protect the skin from ultraviolet light through the production of melanin. Prolonged exposure of the skin to UV light can induce irreparable DNA damage and drive cells into senescence, a sustained cell cycle arrest that prevents the propagation of this damage. Senescent cells can also be detrimental and contribute to skin ageing phenotypes through their senescence-associated secretory phenotype. Senescent cells can act in both an autocrine and paracrine manner to produce widespread tissue inflammation and skin ageing. Recently, melanocytes have been identified as the main senescent cell population within the epidermis and have been linked to a variety of skin ageing phenotypes, such as epidermal thinning and the presence of wrinkles. However, the literature surrounding melanocyte senescence is limited and tends to focus on the role of senescence in the prevention of melanoma. Therefore, this review aims to explore the current understanding of the contribution of senescent melanocytes to human skin ageing.

Sirtuin5 protects colorectal cancer from DNA damage by keeping nucleotide availability

In our previous study, we reported that sirtuin5 (SIRT5), a member of the NAD+-dependent class III histone deacetylase family, is highly expressed in colorectal cancer (CRC). Herein we show that SIRT5 knockdown impairs the production of ribose-5-phosphate, which is essential for nucleotide synthesis, resulting in continuous and irreparable DNA damage and consequently leading to cell cycle arrest and enhanced apoptosis in CRC cells. These SIRT5 silencing-induced effects can be reversed by nucleoside supplementation. Mechanistically, SIRT5 activates transketolase (TKT), a key enzyme in the non-oxidative pentose phosphate pathway, in a demalonylation-dependent manner. Furthermore, TKT is essential for SIRT5-induced malignant phenotypes of CRC both in vivo and in vitro. Altogether, SIRT5 silencing induces DNA damage in CRC via post-translational modifications and inhibits tumor growth, suggesting that SIRT5 can serve as a promising target for CRC treatment.

Analysis of Skin Cancer and Patient Healthcare Using Data Mining Techniques.

Skin cancer is the uncontrolled growth of irregular cancer cells in the human-skin's outer layer. Skin cells commonly grow in an uneven pattern on exposed skin surfaces. The majority of melanomas, aside from this variety, form in areas that are rarely exposed to sunlight. Harmful sunlight, which results in a mutation in the DNA and irreparable DNA damage, is the primary cause of skin cancer. This demonstrates a close connection between skin cancer and molecular biology and genetics. Males and females both experience the same incidence rate. Avoiding revelation to ultraviolet (UV) emissions can lower the risk rate. This needed to be known about in order to be prevented from happening. To identify skin cancer, an improved image analysis technique was put forth in this work. The skin alterations are routinely monitored by this proposed skin cancer categorization approach. Therefore, early detection of suspicious skin changes can aid in the early discovery of skin cancer, increasing the likelihood of a favourable outcome. Due to the blessing of diagnostic technology and recent advancements in cancer treatment, the survival rate of patients with skin cancer has grown. The strategy for detecting skin cancer using image processing technologies is presented in this paper. The system receives the image of the skin lesion as an input and analyses it using cutting-edge image processing methods to determine whether skin cancer is present. The Lesion Image Analysis Tools use texture, size, and shape assessment for image segmentation and feature phases to check for various cancer criteria including asymmetries, borders, pigment, and diameter. The image is classified as Normal skin and a lesion caused by skin cancer using the derived feature parameters.

R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline.

Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.

Simultaneous Imaging and Flow-cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-induced Senescent Cancer Cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restrainingpropagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated β-galactosidase (SA-β-gal), the DNA synthesis marker 5-ethynyl-2'-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Abstract 5432: Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL

Abstract Introduction: CD37 has shown promise as a new therapeutic target for B-cell malignancies. CD37 is highly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Alpha-emitting radionuclides have demonstrated potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm) causing localized and irreparable DNA damage while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-generating radioisotope 212Pb for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Approximately 90,000 cases of CLL and NHL are expected annually in the US. Standard of care includes chemotherapy combined with anti-CD20 antibodies (obinutuzumab and rituximab) and Bruton’s tyrosine kinase inhibitor ibrutinib. While these therapies are initially effective, most patients inevitably relapse. Materials and methods: The efficacy and tolerability of a single dose 212Pb-NNV003 treatment were evaluated in disseminated models using CD37-expressing human CLL (MEC-2) and Burkitt’s lymphoma (Daudi) cells. Comparison to therapeutic doses of ibrutinib was investigated in the MEC-2 model. R2G2 or CB17-SCID mice were injected intravenously with 2.5×106 MEC-2 or 10×106 Daudi cells on day 0, followed by intravenous injection of 212Pb-NNV003 (2.5, 5 or 7.5 µCi in Daudi model and 5, 10, 15 or 20 µCi in MEC-2 model) or daily oral administration of ibrutinib (12.5 or 25 mg/kg) starting on day 2 (n=10-12 per group). 212Pb-cetuximab (unspecific isotype control), unlabelled NNV003 and NaCl were used as controls. Results: In vitro testing of the antibody showed that MEC-2 cells were 5 times more resistant to ibrutinib than Daudi cells. Treatment of mice injected intravenously with Daudi cells with 2.5 μCi 212Pb-NNV003 resulted in survival of 100% of the mice 100 days after start of treatment, while 100 days after start of treatment with 100 μg obinutuzumab only 40% of the mice were still alive. Control mice had a median survival of 7-8 weeks. Treatment of mice injected intravenously with MEC-2 cells with 15 μCi 212Pb-NNV003 resulted in survival of 90-100% for more than 25 weeks in two independent experiments. Ibrutinib treated mice with intravenous MEC-2 cells presented a median survival of only 4.3-4.4 weeks, comparable to the unlabelled NNV003 and saline groups (4.1 and 4.4 weeks). Mice receiving non-specific 212Pb-cetuximab presented a median survival of 7-9 weeks. Conclusion: The study shows that a single injection 212Pb-NNV003 is safe and effective for the treatment of CD37 positive CLL and NHL in preclinical models, with promising efficacy in both ibrutinib-resistant and sensitive CLL models. Treatment with 212Pb-NNV003 was superior to both ibrutinib and obinutuzumab. Further clinical testing is warranted. Citation Format: Jostein Dahle, Amal Saidi, Tania Stallons, Helen Heyerdahl, Ada Repetto-Llamazares, Julien Torgue. Targeted alpha therapy with 212Pb-NNV003 in treatment of NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5432.

Mathematical modeling of radiotherapy and its impact on tumor interactions with the immune system

Radiotherapy is a primary therapeutic modality widely utilized with curative intent. Traditionally tumor response was hypothesized to be due to high levels of cell death induced by irreparable DNA damage. However, the immunomodulatory aspect of radiation is now widely accepted. As such, interest into the combination of radiotherapy and immunotherapy is increasing, the synergy of which has the potential to improve tumor regression beyond that observed after either treatment alone. However, questions regarding the timing (sequential vs concurrent) and dose fractionation (hyper-, standard-, or hypo-fractionation) that result in improved anti-tumor immune responses, and thus potentially enhanced tumor inhibition, remain. Here we discuss the biological response to radiotherapy and its immunomodulatory properties before giving an overview of pre-clinical data and clinical trials concerned with answering these questions. Finally, we review published mathematical models of the impact of radiotherapy on tumor-immune interactions. Ranging from considering the impact of properties of the tumor microenvironment on the induction of anti-tumor responses, to the impact of choice of radiation site in the setting of metastatic disease, these models all have an underlying feature in common: the push towards personalized therapy.

DNA damage, cell cycle perturbation and cell death by naphthalene diimide derivative in gastric cancer cells

Stomach cancer causes the third-highest cancer-related deaths worldwide. Limited availability of anticancer measures with higher efficiency and low unwanted toxicities necessitates the development of better cancer chemotherapeutics. Naphthalene diimide (NDI) derivatives have gained significant attention owing to their excellent anticancer potential. We evaluated the anticancer properties of NDI derivatives, 1a and 2a in cancer cell lines and found that 1a showed higher efficacy as compared to 2a exhibiting a remarkable difference in activity upon single atom substitution of C with N. Particularly, NDI 1a showed potent inhibitory activity against gastric cancer cell line AGS with IC50 of 2.0 μM. NDI 1a induced remarkable morphological changes and reduced clonogenicity as well as the migratory ability of AGS cells. The reduction in AGS cell migration was mediated through inhibition of Tyr397 p-FAK dephosphorylation at focal adhesion points leading to enhanced attachment of cells at contact points. NDI 1a caused extensive DNA double-strand-breaks (DSBs) leading to activation of p53 and its transcriptional target p21. Reduced nuclear BRCA1 but enhanced nuclear p53BP1 foci formation upon 1a treatment suggests that DNA DSB repair is mediated through error-prone NHEJ which led to the accumulation of extensive DNA damage. Combinatorial effects mediated by interactions of 1a with double-stranded DNA through minor groove binding as well as induction of intracellular ROS exacerbated the loss of genomic integrity induced by 1a. NDI 1a mediated DNA damage-induced S phase arrest; however, cells experiencing extensive and irreparable DNA damage underwent mitochondrial apoptosis through downregulation of anti-apoptotic protein p21. Furthermore, proliferation inhibitory activity of 1a is also attributed to inhibition of β−catenin/c-Myc axis in AGS cells with constitutively active β−catenin pathway. In vivo toxicity analysis of 1a revealed minimal systemic toxicity suggesting that compound 1a is a safe and potential candidate for the development of gastric cancer chemotherapeutics.

Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy.

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Cell cycle control in cancer.

Cancer is a group of diseases in which cells divide continuously and excessively. Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms, to ensure the production of two genetically identical cells. Cell cycle checkpoints operate as DNA surveillance mechanisms that prevent the accumulation and propagation of genetic errors during cell division. Checkpoints can delay cell cycle progression or, in response to irreparable DNA damage, induce cell cycle exit or cell death. Cancer-associated mutations that perturb cell cycle control allow continuous cell division chiefly by compromising the ability of cells to exit the cell cycle. Continuous rounds of division, however, create increased reliance on other cell cycle control mechanisms to prevent catastrophic levels of damage and maintain cell viability. New detailed insights into cell cycle control mechanisms and their role in cancer reveal how these dependencies can be best exploited in cancer treatment.

Synergy of HSP90 Inhibition and Radiation Therapy for cGAS/STING Pathway Activation

Even though immunotherapy is a common part in cancer treatment, only a small subset of patients profits long term. Since radiation therapy (RT) has been shown to improve tumor immunogenicity, treatment strategies combining RT and immunotherapy are an emerging field in cancer research. The inhibition of DNA damage repair (DDR) mechanisms is a potential target, because its impairment enhances, like RT alone, type-I interferon induction by activating the cGAS/STING pathway. Type-I interferons mediate the immunomodulatory effects following RT. Therefore, combination of RT and DDR inhibition appears to be a promising approach to enhance tumor immunogenicity. To induce broad DDR impairment, we here employ TAS-116 (Taiho Pharmaceutical Co., Ltd.), an inhibitor of Heat Shock Protein 90 (HSP90). First, we determined the tumor type-dependent toxicity by titering TAS-116 in six murine tumor cell lines and measured cell death via flow cytometry after 24h. Treatment with subtoxic concentrations of TAS-116 one hour prior to RT (up to 8Gy) resulted in increased γH2AX foci, indicating DNA damage, both in RP157.8 SCLC and B16F10 melanoma cells. Declining γH2AX foci were seen upon 12Gy in vitro. Our data suggest that irreparable DNA damage may occur which leads to cell death. The induction of DNA damage following TAS-116 treatment in combination with 8Gy RT was confirmed in vivo using a transplantable mouse tumor model. To confirm our hypothesis that the observed DNA damage mediates STING pathway activation in vitro, we performed Western blot analysis for pSTING at timepoints up to 72h after RT. While TAS-116 alone led to an increase up to 72h, highest pSTING levels were observed after 3h in combination with 8Gy RT, demonstrating synergistic effects for STING activation by accelerating and enhancing STING phosphorylation. While our results display an enhanced specific CD8+ T-cell-mediated killing in vitro using the transfected lymphoma cell line E.G7 after either treatment alone, combination of TAS-116 and RT up to 8Gy led to a significant reduction of this effect. We hypothesize that an observed upregulation of PD-L1 on the tumor cell surface, a known adverse effect of STING activation and suppressor of CD8+ T-cell-mediated killing, causes this phenomenon. Here, we demonstrate a synergy of HSP90 inhibition and RT to activate the immunogenic cGAS/STING pathway. However, to harness this effect therapeutically, anti-PD-1 therapy seems to be required.

Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer

ObjectivePoly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. MethodsWe assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. ResultsEntinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. ConclusionEntinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

Phycocyanin-functionalized black phosphorus quantum dots enhance PDT/PTT therapy by inducing ROS and irreparable DNA damage.

To achieve synergistic photodynamic-photothermic therapy, we fabricate the novel phycocyanin (PC)-functionalized black phosphorus quantum dots (BPQDs) referred as PC@BPQDs through a one-step stirring method. PC@BPQDs are characterized by the feature of possessing both near-infrared (NIR) induced photothermal and photodynamic activity. The PC layer not only effectively alleviates plasma protein adsorption onto BPQDs, but also functionally boosts the photothermal therapy efficiency by enhanced ROS release, resulting in increased apoptosis in vitro. Moreover, PC@BPQDs eradicate tumors with high efficacy and low toxicity in vivo. Thus, PC@BPQDs have a promising potential in future therapeutic implications.