Mitochondrial ACSS1-K635 acetylation knock-in mice exhibit altered metabolism, cell senescence, and nonalcoholic fatty liver disease.

Acetyl-CoA synthetase short-chain family member 1 (ACSS1) uses acetate to generate mitochondrial acetyl-CoA and is regulated by deacetylation by sirtuin 3. We generated an ACSS1-acetylation (Ac) mimic mouse, where lysine-635 was mutated to glutamine (K635Q). Male Acss1K635Q/K635Q mice were smaller with higher metabolic rate and blood acetate and decreased liver/serum ATP and lactate levels. After a 48-hour fast, Acss1K635Q/K635Q mice presented hypothermia and liver aberrations, including enlargement, discoloration, lipid droplet accumulation, and microsteatosis, consistent with nonalcoholic fatty liver disease (NAFLD). RNA sequencing analysis suggested dysregulation of fatty acid metabolism, cellular senescence, and hepatic steatosis networks, consistent with NAFLD. Fasted Acss1K635Q/K635Q mouse livers showed increased fatty acid synthase (FASN) and stearoyl-CoA desaturase 1 (SCD1), both associated with NAFLD, and increased carbohydrate response element-binding protein binding to Fasn and Scd1 enhancer regions. Last, liver lipidomics showed elevated ceramide, lysophosphatidylethanolamine, and lysophosphatidylcholine, all associated with NAFLD. Thus, we propose that ACSS1-K635-Ac dysregulation leads to aberrant lipid metabolism, cellular senescence, and NAFLD.

Ketogenic diet induces p53-dependent cellular senescence in multiple organs.

A ketogenic diet (KD) is a high-fat, low-carbohydrate diet that leads to the generation of ketones. While KDs improve certain health conditions and are popular for weight loss, detrimental effects have also been reported. Here, we show mice on two different KDs and, at different ages, induce cellular senescence in multiple organs, including the heart and kidney. This effect is mediated through adenosine monophosphate-activated protein kinase (AMPK) and inactivation of mouse double minute 2 (MDM2) by caspase-2, leading to p53 accumulation and p21 induction. This was established using p53 and caspase-2 knockout mice and inhibitors to AMPK, p21, and caspase-2. In addition, senescence-associated secretory phenotype biomarkers were elevated in serum from mice on a KD and in plasma samples from patients on a KD clinical trial. Cellular senescence was eliminated by a senolytic and prevented by an intermittent KD. These results have important clinical implications, suggesting that the effects of a KD are contextual and likely require individual optimization.

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks.

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair.

Manganese Superoxide Dismutase Acetylation and Regulation of Protein Structure in Breast Cancer Biology and Therapy.

The loss and/or dysregulation of several cellular and mitochondrial antioxidants' expression or enzymatic activity, which leads to the aberrant physiological function of these proteins, has been shown to result in oxidative damage to cellular macromolecules. In this regard, it has been surmised that the disruption of mitochondrial networks responsible for maintaining normal metabolism is an established hallmark of cancer and a novel mechanism of therapy resistance. This altered metabolism leads to aberrant accumulation of reactive oxygen species (ROS), which, under specific physiological conditions, leads to a potential tumor-permissive cellular environment. In this regard, it is becoming increasingly clear that the loss or disruption of mitochondrial oxidant scavenging enzymes may be, in specific tumors, either an early event in transformation or exhibit tumor-promoting properties. One example of such an antioxidant enzyme is manganese superoxide dismutase (MnSOD, also referred to as SOD2), which detoxifies superoxide, a ROS that has been shown, when its normal physiological levels are disrupted, to lead to oncogenicity and therapy resistance. Here, we will also discuss how the acetylation of MnSOD leads to a change in detoxification function that leads to a cellular environment permissive for the development of lineage plasticity-like properties that may be one mechanism leading to tumorigenic and therapy-resistant phenotypes.

Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.

Lysine 68 acetylation directs MnSOD as a tetrameric detoxification complex versus a monomeric tumor promoter.

Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSODK68Q is accompanied with a change of MnSOD's stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSODK68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression.

Loss of Sirt2 increases and prolongs a caerulein-induced pancreatitis permissive phenotype and induces spontaneous oncogenic Kras mutations in mice.

Mice lacking Sirt2 spontaneously develop tumors in multiple organs, as well as when expressed in combination with oncogenic KrasG12D, leading to pancreatic tumors. Here, we report that after caerulein-induced pancreatitis, Sirt2-deficient mice exhibited an increased inflammatory phenotype and delayed pancreatic tissue recovery. Seven days post injury, the pancreas of Sirt2-/- mice display active inflammation, whereas wild-type mice had mostly recovered. In addition, the pancreas from the Sirt2-/- mice exhibited extensive tissue fibrosis, which was still present at six weeks after exposure. The mice lacking Sirt2 also demonstrated an enhanced whole body pro-inflammatory phenotype that was most obvious with increasing age. Importantly, an accumulation of a cell population with spontaneous cancerous KrasG12D mutations was observed in the Sirt2-/- mice that is enhanced in the recovering pancreas after exposure to caerulein. Finally, transcriptome analysis of the pancreas of the Sirt2-/- mice exhibited a pro-inflammatory genomic signature. These results suggest that loss of Sirt2, as well as increased age, enhanced the immune response to pancreatic injury and induced an inflammatory phenotype permissive for the accumulation of cells carrying oncogenic Kras mutations.

Emerging evidence for targeting mitochondrial metabolic dysfunction in cancer therapy.

Mammalian cells use a complex network of redox-dependent processes necessary to maintain cellular integrity during oxidative metabolism, as well as to protect against and/or adapt to stress. The disruption of these redox-dependent processes, including those in the mitochondria, creates a cellular environment permissive for progression to a malignant phenotype and the development of resistance to commonly used anticancer agents. An extension of this paradigm is that when these mitochondrial functions are altered by the events leading to transformation and ensuing downstream metabolic processes, they can be used as molecular biomarkers or targets in the development of new therapeutic interventions to selectively kill and/or sensitize cancer versus normal cells. In this Review we propose that mitochondrial oxidative metabolism is altered in tumor cells, and the central theme of this dysregulation is electron transport chain activity, folate metabolism, NADH/NADPH metabolism, thiol-mediated detoxification pathways, and redox-active metal ion metabolism. It is proposed that specific subgroups of human malignancies display distinct mitochondrial transformative and/or tumor signatures that may benefit from agents that target these pathways.

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination.

The nitric oxide (NO)-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR) to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR) repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1). Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18), which causes stem cell differentiation has no effect on double-strand break (DSB) repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.

A multifaceted role for MOF histone modifying factor in genome maintenance.

MOF (males absent on the first) was initially identified as a dosage compensation factor in Drosophila that acetylates lysine 16 of histone H4 (H4K16ac) and increased gene transcription from the single copy male X-chromosome. In humans, however, the ortholog of Drosophila MOF has been shown to interact with a range of proteins that extend its potential significance well beyond transcription. For example, recent results indicate MOF is an upstream regulator of the ATM (ataxia-telangiectasia mutated) protein, the loss of which is responsible for ataxia telangiectasia (AT). ATM is a key regulatory kinase that interacts with and phosphorylates multiple substrates that influence critical, cell-cycle control and DNA damage repair pathways in addition to other pathways. Thus, directly or indirectly, MOF may be involved in a wide range of cellular functions. This review will focus on the contribution of MOF to cellular DNA repair and new results that are beginning to examine the in vivo physiological role of MOF.

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes.

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

ß2-spectrin depletion impairs DNA damage repair.

ß2-Spectrin (ß2SP/SPTBN1, gene SPTBN1) is a key TGF-ß/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-ß signaling and can contribute to liver cancer development. Here we report that cells deficient in ß2-Spectrin (ß2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), ß2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, ß2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in ß2SP deficient cells. We propose that ß2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination.

Torin2 Suppresses Ionizing Radiation-Induced DNA Damage Repair.

Several classes of inhibitors of the mammalian target of rapamycin (mTOR) have been developed based on its central role in sensing growth factor and nutrient levels to regulate cellular metabolism. However, its ATP-binding site closely resembles other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, resulting in reactivity with these targets that may also be therapeutically useful. The ATP-competitive mTOR inhibitor, Torin2, shows biochemical activity against the DNA repair-associated proteins ATM, ATR and DNA-PK, which raises the possibility that Torin2 and related compounds might radiosensitize cancerous tumors. In this study Torin2 was also found to enhance ionizing radiation-induced cell killing in conditions where ATM was dispensable, confirming the requirement for multiple PIKK targets. Moreover, Torin2 did not influence the initial appearance of γ-H2AX foci after irradiation but significantly delayed the disappearance of radiation-induced γ-H2AX foci, indicating a DNA repair defect. Torin2 increased the number of radiation-induced S-phase specific chromosome aberrations and reduced the frequency of radiation-induced CtIP and Rad51 foci formation, suggesting that Torin2 works by blocking homologous recombination (HR)-mediated DNA repair resulting in an S-phase specific DNA repair defect. Accordingly, Torin2 reduced HR-mediated repair of I-Sce1-induced DNA damage and contributed to replication fork stalling. We conclude that radiosensitization of tumor cells by Torin2 is associated with disrupting ATR- and ATM-dependent DNA damage responses. Our findings support the concept of developing combination cancer therapies that incorporate ionizing radiation therapy and Torin2 or compounds with similar properties.

Role of the Exocyst Complex Component Sec6/8 in Genomic Stability.

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.

Targeted inhibition of histone deacetylases and hedgehog signaling suppress tumor growth and homologous recombination in aerodigestive cancers.

Standard combined modality therapies for aerodigestive tract malignancies have suboptimal outcomes, and targeting cancer-specific molecular pathways in combination with radiation could improve the therapeutic ratio. Dysregulation of epigenetic modulators such as histone deacetylases (HDACs), and developmental morphogens such as the hedgehog (HH) pathway have been implicated in aerodigestive tumor progression and metastasis. We hypothesized that simultaneous targeting of HDACs and the HH-pathway mediator Smoothened (Smo) represents an opportunity to overcome therapeutic resistance in these cancers. We evaluated the effects of the HDAC inhibitor SAHA and Smo inhibitor GDC-0449 with radiation in multiple aerodigestive cancer cell lines. Isobologram analyses showed that SAHA and GDC-0449 synergistically suppressed cancer cell proliferation in vitro. SAHA and GDC-0449 cooperatively enhanced G0/G1 cell cycle arrest which was associated with up-regulation of p21(waf). GDC-0449 prevented SAHA-induced up-regulation of Gli-1 and Gli-2. Both Smo and Ptc-1 expression was cooperatively suppressed by SAHA and GDC-0449. The combination of SAHA and GDC-0449 induced radiation sensitization with 2 Gy as determined by colony formation assays and cytogenetic analyses, which correlated with higher residual γ-H2AX and 53BP1 foci. In mouse tumor xenografts of the SqCC/Y1 cell line, SAHA and GDC-0449 delayed tumor growth longer and prolonged survival more than either agent alone. In summary, we have identified synergistic effect of HDAC and HH signaling for radiosensitization to improve therapeutic outcomes for aerodigestive malignancies.

Detecting ATM-dependent chromatin modification in DNA damage response.

Loss of function or mutation of the ataxia-telangiectasia mutated gene product (ATM) results in inherited genetic disorders characterized by neurodegeneration, immunodeficiency, and cancer. Ataxia-telangiectasia mutated (ATM) gene product belongs to the PI3K-like protein kinase (PIKKs) family and is functionally implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control, and telomere maintenance. The ATM protein kinase is primarily activated in response to DNA double strand breaks (DSBs), the most deleterious form of DNA damage produced by ionizing radiation (IR) or radiomimetic drugs. It is detected at DNA damage sites, where ATM autophosphorylation causes dissociation of the inactive homodimeric form to the activated monomeric form. Interestingly, heat shock can activate ATM independent of the presence of DNA strand breaks. ATM is an integral part of the sensory machinery that detects DSBs during meiosis, mitosis, or DNA breaks mediated by free radicals. These DNA lesions can trigger higher order chromatin reorganization fuelled by posttranslational modifications of histones and histone binding proteins. Our group, and others, have shown that ATM activation is tightly regulated by chromatin modifications. This review summarizes the multiple approaches used to discern the role of ATM and other associated proteins in chromatin modification in response to DNA damage.

Identification and characterization of a cell division-regulating kinase AKB1 (associated kinase of Trypanosoma brucei 14-3-3) through proteomics study of the Tb14-3-3 binding proteins.

We used a proteomics approach to identify the binding partners of Trypanosoma brucei 14-3-3 (Tb14-3-3) which led to the identification of a novel kinase, AKB1. The binding between these two proteins was mediated by an amphipathic groove structure in Tb14-3-3 and 1-438 amino acid sequence of AKB1. Recombinant AKB1 but not its ATP-binding-deficient mutant (DFG to NFG) possessed an auto-phosphorylation activity as well as a kinase activity towards a peptide substrate in vitro. However, the autophosphorylation was not required for the binding of AKB1 to Tb14-3-3. Interestingly, the kinase activity of AKB1 was inhibited by calcium, and the kinase was found to utilize GTP, and dATP in addition to ATP as phospho-donors. AKB1 formed homodimers through a leucine-zipper structure. Either knockdown of AKB1 or overexpression of AKB1, but not kinase-dead AKB1 mutant, deregulated cytokinesis and cell division, suggesting that kinase activity of AKB1 is crucial for its function. Furthermore, we showed that AKB1 exists in a detergent insoluble fraction. Laser confocal microscopy revealed that the majority of AKB1 is co-localized with α-tubulin. Taken together, these findings suggest that AKB1 might regulate cytokinesis and cell division by phosphorylating cytoskeleton-associated proteins.

Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR.

MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.