Androgen drives melanoma invasiveness and metastatic spread by inducing tumorigenic fucosylation.

Melanoma incidence and mortality rates are historically higher for men than women. Although emerging studies have highlighted tumorigenic roles for the male sex hormone androgen and its receptor (AR) in melanoma, cellular and molecular mechanisms underlying these sex-associated discrepancies are poorly defined. Here, we delineate a previously undisclosed mechanism by which androgen-activated AR transcriptionally upregulates fucosyltransferase 4 (FUT4) expression, which drives melanoma invasiveness by interfering with adherens junctions (AJs). Global phosphoproteomic and fucoproteomic profiling, coupled with in vitro and in vivo functional validation, further reveal that AR-induced FUT4 fucosylates L1 cell adhesion molecule (L1CAM), which is required for FUT4-increased metastatic capacity. Tumor microarray and gene expression analyses demonstrate that AR-FUT4-L1CAM-AJs signaling correlates with pathological staging in melanoma patients. By delineating key androgen-triggered signaling that enhances metastatic aggressiveness, our findings help explain sex-associated clinical outcome disparities and highlight AR/FUT4 and its effectors as potential prognostic biomarkers and therapeutic targets in melanoma.

Differential network analysis of ROS1 inhibitors reveals lorlatinib polypharmacology through co-targeting PYK2.

Multiple tyrosine kinase inhibitors (TKIs) are often developed for the same indication. However, their relative overall efficacy is frequently incompletely understood and they may harbor unrecognized targets that cooperate with the intended target. We compared several ROS1 TKIs for inhibition of ROS1-fusion-positive lung cancer cell viability, ROS1 autophosphorylation and kinase activity, which indicated disproportionately higher cellular potency of one TKI, lorlatinib. Quantitative chemical and phosphoproteomics across four ROS1 TKIs and differential network analysis revealed that lorlatinib uniquely impacted focal adhesion signaling. Functional validation using pharmacological probes, RNA interference, and CRISPR-Cas9 knockout uncovered a polypharmacology mechanism of lorlatinib by dual targeting ROS1 and PYK2, which form a multiprotein complex with SRC. Rational multi-targeting of this complex by combining lorlatinib with SRC inhibitors exhibited pronounced synergy. Taken together, we show that systems pharmacology-based differential network analysis can dissect mixed canonical/non-canonical polypharmacology mechanisms across multiple TKIs enabling the design of rational drug combinations.

Brain metastasis-associated fibroblasts secrete fucosylated PVR/CD155 that induces breast cancer invasion.

Brain metastasis cancer-associated fibroblasts (bmCAFs) are emerging as crucial players in the development of breast cancer brain metastasis (BCBM), but our understanding of the underlying molecular mechanisms is limited. In this study, we aim to elucidate the pathological contributions of fucosylation (the post-translational modification of proteins by the dietary sugar L-fucose) to tumor-stromal interactions that drive the development of BCBM. Here, we report that patient-derived bmCAFs secrete high levels of polio virus receptor (PVR), which enhance the invasive capacity of BC cells. Mechanistically, we find that HIF1α transcriptionally upregulates fucosyltransferase 11, which fucosylates PVR, triggering its secretion from bmCAFs. Global phosphoproteomic analysis of BC cells followed by functional verification identifies cell-cell junction and actin cytoskeletal signaling as modulated by bmCAF-secreted, -fucosylated PVR. Our findings delineate a hypoxia- and fucosylation-regulated mechanism by which bmCAFs contribute to the invasiveness of BCBM in the brain.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease.

Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer-Associated Stellate Cell Small Extracellular Vesicles.

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography-tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1-like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.

Plasma cell dependence on histone/protein deacetylase 11 reveals a therapeutic target in multiple myeloma.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

Cell Type-specific Adaptive Signaling Responses to KRASG12C Inhibition.

PURPOSE: Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. EXPERIMENTAL DESIGN: We performed mass spectrometry-based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. RESULTS: Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. CONCLUSIONS: Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma.

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. In vivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

Managing a Large-Scale Multiomics Project: A Team Science Case Study in Proteogenomics.

Highly collaborative scientists are often called on to extend their expertise to different types of projects and to expand the scope and scale of projects well beyond their previous experience. For a large-scale project involving "big data" to be successful, several different aspects of the research plan need to be developed and tested, which include but are not limited to the experimental design, sample collection, sample preparation, metadata recording, technical capability, data acquisition, approaches for data analysis, methods for integration of different data types, recruitment of additional expertise as needed to guide the project, and strategies for clear communication throughout the project. To capture this process, we describe an example project in proteogenomics that built on our collective expertise and experience. Key steps included definition of hypotheses, identification of an appropriate clinical cohort, pilot projects to assess feasibility, refinement of experimental designs, and extensive discussions involving the research team throughout the process. The goal of this chapter is to provide the reader with a set of guidelines to support development of other large-scale multiomics projects.

Lowering Sample Requirements to Study Tyrosine Kinase Signaling Using Phosphoproteomics with the TMT Calibrator Approach.

Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile tyrosine kinase substrates. A typical protocol requests 10 mg of total protein from ≈108 cells or 50-100 mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemical labeling reduces the protein amount required for each sample, and newer approaches use a material-rich reference channel as a calibrator to trigger detection and quantification for smaller samples. Here, it is demonstrated that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling tenfold (to ≈1 mg total protein per sample from 107 cells grown in one plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. Data are available through PRIDE (PXD019764 for label-free and PXD018952 for TMT). This strategy opens more opportunities for pY profiling of large sample cohorts and samples with limited protein quantity such as immune cells, xenograft models, and human tumors.

Network of Interactions between ZIKA Virus Non-Structural Proteins and Human Host Proteins.

The Zika virus (ZIKV) is a mosquito-borne Flavivirus and can be transmitted through an infected mosquito bite or through human-to-human interaction by sexual activity, blood transfusion, breastfeeding, or perinatal exposure. After the 2015-2016 outbreak in Brazil, a strong link between ZIKV infection and microcephaly emerged. ZIKV specifically targets human neural progenitor cells, suggesting that proteins encoded by ZIKV bind and inactivate host cell proteins, leading to microcephaly. Here, we present a systematic annotation of interactions between human proteins and the seven non-structural ZIKV proteins corresponding to a Brazilian isolate. The interaction network was generated by combining tandem-affinity purification followed by mass spectrometry with yeast two-hybrid screens. We identified 150 human proteins, involved in distinct biological processes, as interactors to ZIKV non-structural proteins. Our interacting network is composed of proteins that have been previously associated with microcephaly in human genetic disorders and/or animal models. Further, we show that the protein inhibitor of activated STAT1 (PIAS1) interacts with NS5 and modulates its stability. This study builds on previously published interacting networks of ZIKV and genes related to autosomal recessive primary microcephaly to generate a catalog of human cellular targets of ZIKV proteins implicated in processes related to microcephaly in humans. Collectively, these data can be used as a resource for future characterization of ZIKV infection biology and help create a basis for the discovery of drugs that may disrupt the interaction and reduce the health damage to the fetus.

Fascin Controls Metastatic Colonization and Mitochondrial Oxidative Phosphorylation by Remodeling Mitochondrial Actin Filaments.

The deregulation of the actin cytoskeleton has been extensively studied in metastatic dissemination. However, the post-dissemination role of the actin cytoskeleton dysregulation is poorly understood. Here, we report that fascin, an actin-bundling protein, promotes lung cancer metastatic colonization by augmenting metabolic stress resistance and mitochondrial oxidative phosphorylation (OXPHOS). Fascin is directly recruited to mitochondria under metabolic stress to stabilize mitochondrial actin filaments (mtF-actin). Using unbiased metabolomics and proteomics approaches, we discovered that fascin-mediated mtF-actin remodeling promotes mitochondrial OXPHOS by increasing the biogenesis of respiratory Complex I. Mechanistically, fascin and mtF-actin control the homeostasis of mtDNA to promote mitochondrial OXPHOS. The disruption of mtF-actin abrogates fascin-mediated lung cancer metastasis. Conversely, restoration of mitochondrial respiration by using yeast NDI1 in fascin-depleted cancer cells is able to rescue lung metastasis. Our findings indicate that the dysregulated actin cytoskeleton in metastatic lung cancer could be targeted to rewire mitochondrial metabolism and to prevent metastatic recurrence.

Proteogenomic landscape of squamous cell lung cancer.

How genomic and transcriptomic alterations affect the functional proteome in lung cancer is not fully understood. Here, we integrate DNA copy number, somatic mutations, RNA-sequencing, and expression proteomics in a cohort of 108 squamous cell lung cancer (SCC) patients. We identify three proteomic subtypes, two of which (Inflamed, Redox) comprise 87% of tumors. The Inflamed subtype is enriched with neutrophils, B-cells, and monocytes and expresses more PD-1. Redox tumours are enriched for oxidation-reduction and glutathione pathways and harbor more NFE2L2/KEAP1 alterations and copy gain in the 3q2 locus. Proteomic subtypes are not associated with patient survival. However, B-cell-rich tertiary lymph node structures, more common in Inflamed, are associated with better survival. We identify metabolic vulnerabilities (TP63, PSAT1, and TFRC) in Redox. Our work provides a powerful resource for lung SCC biology and suggests therapeutic opportunities based on redox metabolism and immune cell infiltrates.

K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling.

BRAF plays an indispensable role in activating the MEK/ERK pathway to drive tumorigenesis. Receptor tyrosine kinase and RAS-mediated BRAF activation have been extensively characterized, however, it remains undefined how BRAF function is fine-tuned by stimuli other than growth factors. Here, we report that in response to proinflammatory cytokines, BRAF is subjected to lysine 27-linked poly-ubiquitination in melanoma cells by the ITCH ubiquitin E3 ligase. Lysine 27-linked ubiquitination of BRAF recruits PP2A to antagonize the S365 phosphorylation and disrupts the inhibitory interaction with 14-3-3, leading to sustained BRAF activation and subsequent elevation of the MEK/ERK signaling. Physiologically, proinflammatory cytokines activate ITCH to maintain BRAF activity and to promote proliferation and invasion of melanoma cells, whereas the ubiquitination-deficient BRAF mutant displays compromised kinase activity and reduced tumorigenicity. Collectively, our study reveals a pivotal role for ITCH-mediated BRAF ubiquitination in coordinating the signals between cytokines and the MAPK pathway activation in melanoma cells.

Hyper-phosphorylation of Rb S249 together with CDK5R2/p39 overexpression are associated with impaired cell adhesion and epithelial-to-mesenchymal transition: Implications as a potential lung cancer grading and staging biomarker.

Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.

Functional Proteomics and Deep Network Interrogation Reveal a Complex Mechanism of Action of Midostaurin in Lung Cancer Cells.

Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation.

MPP8 and SIRT1 crosstalk in E-cadherin gene silencing and epithelial-mesenchymal transition.

As a critical developmental process, epithelial-mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E-cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Consequently, disabling either MPP8 methyl-H3K9 binding or SIRT1 interaction de-represses E-cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.

Ubiquitinated sirtuin 1 (SIRT1) function is modulated during DNA damage-induced cell death and survival.

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR.

hMOF acetylation of DBC1/CCAR2 prevents binding and inhibition of SirT1.

The NAD(+)-dependent deacetylase SirT1 regulates gene silencing and genomic stability in response to nutrient deprivation and DNA damage. An important regulator of SirT1 in mammalian cells is DBC1 (deleted in breast cancer 1; KIAA1967 or CCAR2), which binds to SirT1 and inhibits the deacetylation of substrates. Recent studies have revealed that ATM/ATR-mediated phosphorylation of DBC1 promotes binding to SirT1. Here we show that DBC1 is modified by acetylation on two N-terminal lysine residues (K112 and K215). The MYST family histone acetyltransferase hMOF (human MOF) is responsible for DBC1 acetylation. Acetylation of K112 and K215 inhibits DBC1-SirT1 binding and increases SirT1 deacetylase activity. SirT1 also promotes DBC1 deacetylation, suggesting the presence of a negative-feedback mechanism that stabilizes the SirT1-DBC1 complex and limits SirT1 activity. hMOF binding and acetylation of DBC1 are inhibited after DNA damage in an ATM-dependent fashion, contributing to increased SirT1-DBC1 binding after DNA damage. Furthermore, a DBC1 mutant that mimics the acetylated state fails to promote apoptosis after DNA damage. These results suggest that acetylation of DBC1 inhibits binding to SirT1 and serves as a mechanism that connects DNA damage signaling to SirT1 and cell fate determination.