Selective Inhibition of ATM-dependent Double-strand Break Repair and Checkpoint Control Synergistically Enhances the Efficacy of ATR Inhibitors.

Ataxia telangiectasia and Rad3-related protein (ATR) kinase regulate a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double-strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell-cycle checkpoints controlled by ataxia telangiectasia-mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1-phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell-cycle checkpoints, and DSB repair lowered the p53 protective barrier and extended the life of ATR inhibitor-induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Furthermore, a combination study in 26 patient-derived xenograft models of triple-negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared with single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.

Selective ATM inhibition augments radiation-induced inflammatory signaling and cancer cell death.

Over half of all cancer patients undergo radiation therapy but there is an unmet need for more efficacious combination strategies with molecular targeted drugs. DNA damage response has emerged as an important intervention point for improving anti-tumor effects of radiation and several inhibitors are currently in development. Ataxia telangiectasia mutated (ATM) kinase is a key regulator of cellular response to DNA double strand breaks and a potential target for radiosensitization. We recently reported two new potent and selective ATM inhibitors, M3541 and M4076, that effectively sensitize cancer cells to radiation and regress human xenografts in clinically relevant animal models. Here, we dive deeper into the cellular events in irradiated cancer cells exposed to ATM inhibitors. Suppression of ATM activity inhibited radiation-induced ATM signaling and abrogated G1 checkpoint activation resulting in enhanced cell death. Our data indicated that entry into mitosis with gross structural abnormalities in multiple chromosomes is the main mechanism behind the increased cell killing. Misalignment and mis-segregation led to formation of multiple micronuclei and robust activation of the interferon response and inflammatory signaling via the cGAS/STING/TBK1 pathway. Cancer cells exposed to radiation in the presence of M3541 were more susceptible to killing in co-culture with NK cells from healthy donors. In addition, strong upregulation of PD-L1 expression was observed in the surviving irradiated cancer cells exposed to M3541. Simultaneous activation of the STING pathway and PD-L1 suggested that combination of radiation, ATM inhibitors and PD-L1 targeted therapy may offer a novel approach to radio-immunotherapy of locally advanced tumors.

Colocalized targeting of TGF-ß and PD-L1 by bintrafusp alfa elicits distinct antitumor responses.

BACKGROUND: Bintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-ß (TGF-ß) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-ß trap (TGF-ßRII) to the TME, enabling it to sequester TGF-ß in the tumor more effectively than systemic TGF-ß blockade, thereby enhancing antitumor activity. METHODS: Multiple technologies were used to characterize the TGF-ß trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-ß trap or the pan-TGF-ß antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-ß and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-ß were investigated in vitro. RESULTS: BA and fresolimumab had comparable intrinsic binding to TGF-ß1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-ß-dependent and PD-L1-expressing cells more potently than TGF-ß trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-ß trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-ß-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-ß1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-ß1 co-expression was also seen in human tumors. Finally, BA induced TGF-ß1 internalization and degradation in the lysosomes. CONCLUSION: BA more effectively blocks TGF-ß by targeting TGF-ß trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.

A New Class of Selective ATM Inhibitors as Combination Partners of DNA Double-Strand Break Inducing Cancer Therapies.

Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer therapeutics today. Double-strand breaks (DSB) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia-mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell-cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth, and potentiated antitumor activity of ionizing radiation in cancer cell lines. Oral administration of M3541 and M4076 to immunodeficient mice bearing human tumor xenografts with a clinically relevant radiotherapy regimen strongly enhanced the antitumor activity, leading to complete tumor regressions. The efficacy correlated with the inhibition of ATM activity and modulation of its downstream targets in the xenograft tissues. In vitro and in vivo experiments demonstrated strong combination potential with PARP and topoisomerase I inhibitors. M4076 is currently under clinical investigation.

DNA-PK Inhibitor Peposertib Amplifies Radiation-Induced Inflammatory Micronucleation and Enhances TGFß/PD-L1 Targeted Cancer Immunotherapy.

Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFß "trap"/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFß and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. IMPLICATIONS: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFß/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.

DNA-PK Inhibitor, M3814, as a New Combination Partner of Mylotarg in the Treatment of Acute Myeloid Leukemia.

Despite significant advances in the treatment of acute myeloid leukemia (AML) the long-term prognosis remains relatively poor and there is an urgent need for improved therapies with increased potency and tumor selectivity. Mylotarg is the first AML-targeting drug from a new generation of antibody drug conjugate (ADC) therapies aiming at the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic payload, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSB by the non-homologous end joining pathway driven by DNA-dependent protein kinase (DNA-PK) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg and a potential new combination treatment option for AML patients.

Bacteria-to-Human Protein Networks Reveal Origins of Endogenous DNA Damage.

DNA damage provokes mutations and cancer and results from external carcinogens or endogenous cellular processes. However, the intrinsic instigators of endogenous DNA damage are poorly understood. Here, we identify proteins that promote endogenous DNA damage when overproduced: the DNA "damage-up" proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six function clusters, demonstrating DDP mechanisms in three: reactive oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis. Half of the tested human homologs promote DNA damage and mutation when overproduced in human cells, with DNA damage-elevating mechanisms like those in E. coli. Our work identifies networks of DDPs that provoke endogenous DNA damage and may reveal DNA damage-associated functions of many human known and newly implicated cancer-promoting proteins.

Bromodomain proteins: repairing DNA damage within chromatin.

Genome surveillance and repair, termed the DNA damage response (DDR), functions within chromatin. Chromatin-based DDR mechanisms sustain genome and epigenome integrity, defects that can disrupt cellular homeostasis and contribute to human diseases. An important chromatin DDR pathway is acetylation signalling which is controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes, which regulate acetylated lysines within proteins. Acetylated proteins, including histones, can modulate chromatin structure and provide molecular signals that are bound by acetyl-lysine binders, including bromodomain (BRD) proteins. Acetylation signalling regulates several DDR pathways, as exemplified by the preponderance of HATs, HDACs and BRD proteins that localize at DNA breaks to modify chromatin for lesion repair. Here, we explore the involvement of acetylation signalling in the DDR, focusing on the involvement of BRD proteins in promoting chromatin remodelling to repair DNA double-strand breaks. BRD proteins have widespread DDR functions including chromatin remodelling, chromatin modification and transcriptional regulation. We discuss mechanistically how BRD proteins read acetylation signals within chromatin to trigger DDR and chromatin activities to facilitate genome-epigenome maintenance. Thus, DDR pathways involving BRD proteins represent key participants in pathways that preserve genome-epigenome integrity to safeguard normal genome and cellular functions.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.

Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination.

How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary "readers" of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.

Identification of differentially expressed microRNAs in human hepatocellular adenoma associated with type I glycogen storage disease: a potential utility as biomarkers.

BACKGROUND: It is known that malignant transformation to hepatocellular carcinoma (HCC) occurs at a higher frequency in hepatocellular adenoma (HCA) from type I glycogen storage disease (GSD I) compared to HCA from other etiologies. In this study, we aimed to identify differentially expressed miRNAs in GSD Ia HCA as candidates that could serve as putative biomarkers for detection of GSD Ia HCA and/or risk assessment of malignant transformation. METHODS: Utilizing massively parallel sequencing, the miRNA profiling was performed for paired adenomas and normal liver tissues from seven GSD Ia patients. Differentially expressed miRNAs were validated in liver tumor tissues, HCC cell lines and serum using quantitative RT-PCR. RESULTS: miR-34a, miR-34a, miR-224, miR-224, miR-424, miR-452 and miR-455-5p were found to be commonly deregulated in GSD Ia HCA, general population HCA, and HCC cell lines at compatible levels. In comparison with GSD Ia HCA, the upregulation of miR-130b and downregulation of miR-199a-5p, miR-199b-5p, and miR-214 were more significant in HCC cell lines. Furthermore, serum level of miR-130b in GSD Ia patients with HCA was moderately higher than that in either GSD Ia patients without HCA or healthy individuals. CONCLUSION: We make the first observation of distinct miRNA deregulation in HCA associated with GSD Ia. We also provide evidence that miR-130b could serve as a circulating biomarker for detection of GSD Ia HCA. This work provides prominent candidate miRNAs worth evaluating as biomarkers for monitoring the development and progress of liver tumors in GSD Ia patients in the future.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.