Interferon signaling is enhanced by ATR inhibition in glioblastoma cells irradiated with X-rays, protons or carbon ions.

Background And Purpose: Interferon (IFN) signaling plays an important role in antitumor immune responses. Inhibitors of the DNA damage response, such as ATR inhibitors, can increase IFN signaling upon conventional radiotherapy with X-rays. However, it is not known whether such inhibitors also enhance IFN signaling after irradiation with high linear energy transfer (LET) particles.

Potential Benefits of Combining Proton or Carbon Ion Therapy with DNA Damage Repair Inhibitors.

The use of charged particle radiotherapy is currently increasing, but combination therapy with DNA repair inhibitors remains to be exploited in the clinic. The high-linear energy transfer (LET) radiation delivered by charged particles causes clustered DNA damage, which is particularly effective in destroying cancer cells. Whether the DNA damage response to this type of damage is different from that elicited in response to low-LET radiation, and if and how it can be targeted to increase treatment efficacy, is not fully understood.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Introduction: Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis.

Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry.

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells.

Caspase activation counteracts interferon signaling after G2 checkpoint abrogation by ATR inhibition in irradiated human cancer cells.

Recent studies suggest that inhibition of the ATR kinase can potentiate radiation-induced antitumor immune responses, but the extent and mechanisms of such responses in human cancers remain scarcely understood. We aimed to assess whether the ATR inhibitors VE822 and AZD6738, by abrogating the G2 checkpoint, increase cGAS-mediated type I IFN response after irradiation in human lung cancer and osteosarcoma cell lines. Supporting that the checkpoint may prevent IFN induction, radiation-induced IFN signaling declined when the G2 checkpoint arrest was prolonged at high radiation doses.

A novel, rapid and sensitive flow cytometry method reveals degradation of promoter proximal paused RNAPII in the presence and absence of UV.

RNA polymerase II (RNAPII) is emerging as an important factor in DNA damage responses, but how it responds to genotoxic stress is not fully understood. We have developed a rapid and sensitive flow cytometry method to study chromatin binding of RNAPII in individual human cells through the cell cycle. Indicating enhanced transcription initiation at early timepoints, levels of RNAPII were increased at 15-30min after UV-induced DNA damage.

Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.

Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor.

New link between the RNA polymerase II-CTD and replication stress.

Conflicts between transcription and replication are a major source of replication stress. Our recent findings show that proper dephosphorylation of Serine 5 in the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1 is needed to prevent such conflicts in human cells.

Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.

Purpose: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin.

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress.

The Dual Cell Cycle Kinase Inhibitor JNJ-7706621 Reverses Resistance to CD37-Targeted Radioimmunotherapy in Activated B Cell Like Diffuse Large B Cell Lymphoma Cell Lines.

The CD37 targeting radioimmunoconjugate Lu-lilotomab satetraxetan (Betalutin) is currently being evaluated in a clinical phase 2b trial for patients with follicular lymphoma (FL) and in a phase 1 trial for patients with diffuse large B-cell lymphoma (DLBCL). Herein we have investigated the effect of Lu-lilotomab satetraxetan in seven activated B-cell like (ABC) DLBCL cell lines. Although the radioimmunoconjugate showed anti-tumor activity, primary resistance was observed in a subset of cell lines.

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775.

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21, RPE p21, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells.

Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown.

A novel role for ATR/Rad3 in G1 phase.

Checkpoint kinases are important in cellular surveillance pathways that help cells to cope with DNA damage and protect their genomes. In cycling cells, DNA replication is one of the most sensitive processes and therefore all organisms carefully regulate replication initiation and progression. The checkpoint kinase ATR plays important roles both in response to DNA damage and replication stress, and ATR inhibitors are currently in clinical trials for cancer treatment.

TLR9 stimulation of B-cells induces transcription of p53 and prevents spontaneous and irradiation-induced cell death independent of DNA damage responses. Implications for Common variable immunodeficiency.

In the present study, we address the important issue of whether B-cells protected from irradiation-induced cell death, may survive with elevated levels of DNA damage. If so, such cells would be at higher risk of gaining mutations and undergoing malignant transformation. We show that stimulation of B-cells with the TLR9 ligands CpG-oligodeoxynucleotides (CpG-ODN) prevents spontaneous and irradiation-induced death of normal peripheral blood B-cells, and of B-cells from patients diagnosed with Common variable immunodeficiency (CVID).

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features.

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process.

Hypoxia-induced alterations of G2 checkpoint regulators.

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints.

Simultaneous measurement of passage through the restriction point and MCM loading in single cells.

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear.

Targeting lung cancer through inhibition of checkpoint kinases.

Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed.

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy.

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10-24 Gray (Gy) (n = 19).

PLK1-inhibition can cause radiosensitization or radioresistance dependent on the treatment schedule.

Background And Purpose: PLK1-inhibitors are emerging as new potential anticancer agents. It is therefore important to explore the combined effects of PLK1-inhibitors with conventional therapies. Based on the functional roles of PLK1 in both mitosis and the G2 checkpoint, we hypothesized that the treatment schedule might influence the combined effects of PLK1-inhibiton and radiation.

The efficacy of CHK1 inhibitors is not altered by hypoxia, but is enhanced after reoxygenation.

Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia).