Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53.

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

hMOF, a KAT(8) with many lives.

Dynamic lysine acetylation regulates proteins involved in diverse cellular processes, with individual enzymes often acetylating multiple substrates. Here, Li et al. (2009) show that the substrate specificity of hMOF/MYST1/KAT8 is controlled by differential interaction with two mutually exclusive partners.

MYST protein acetyltransferase activity requires active site lysine autoacetylation.

The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

Biochemical pathways that regulate acetyltransferase and deacetylase activity in mammalian cells.

Protein phosphorylation is regulated dynamically in eukaryotic cells via modulation of the enzymatic activity of kinases and phosphatases. Like phosphorylation, acetylation has emerged as a critical regulatory protein modification that is altered dynamically in response to diverse cellular cues. Moreover, acetyltransferases and deacetylases are tightly linked to cellular signaling pathways. Recent studies provide clues about the mechanisms utilized to regulate acetyltransferases and deacetylases. The therapeutic value of deacetylase inhibitors suggests that understanding acetylation pathways will directly impact our ability to rationally target these enzymes in patients. Recently discovered mechanisms that directly regulate the catalytic activity of acetyltransferases and deacetylases provide exciting new insights about these enzymes.

Deacetylation of the DNA-binding domain regulates p53-mediated apoptosis.

In unstressed cells, the p53 tumor suppressor is highly unstable. DNA damage and other forms of cellular stress rapidly stabilize and activate p53. This process is regulated by a complex array of post-translational modifications that are dynamically deposited onto p53. Recent studies show that these modifications orchestrate p53-mediated processes such as cell cycle arrest and apoptosis. Cancer cells carry inherent genetic damage, but avoid arrest and apoptosis by inactivating p53. Defining the enzymatic machinery that regulates the stress-induced modification of p53 at single-residue resolution is critical to our understanding of the biochemical mechanisms that control this critical tumor suppressor. Specifically, acetylation of p53 at lysine 120, a DNA-binding domain residue mutated in human cancer, is essential for triggering apoptosis. Given the oncogenic properties of deacetylases and the success of deacetylase inhibitors as anticancer agents, we investigated the regulation of Lys(120) deacetylation using pharmacologic and genetic approaches. This analysis revealed that histone deacetylase 1 is predominantly responsible for the deacetylation of Lys(120). Furthermore, treatment with the clinical-grade histone deacetylase inhibitor entinostat enhances Lys(120) acetylation, an event that is mechanistically linked to its apoptotic effect. These data expand our understanding of the mechanisms controlling p53 function and suggest that regulation of p53 modification status at single-residue resolution by targeted therapeutics can selectively alter p53 pathway function. This knowledge may impact the rational application of deacetylase inhibitors in the treatment of human cancer.

Regulation of microRNA-145 by growth arrest and differentiation.

MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.

Analytic and Clinical Validation of a Pan-Cancer NGS Liquid Biopsy Test for the Detection of Copy Number Amplifications, Fusions and Exon Skipping Variants.

Liquid biopsies are an integral part of the diagnosis of cancer. Here, we have extended previous validation studies of a new targeted NGS panel to include the detection of copy number amplifications (CNAs), fusions, and exon skipping variants. Detection of these gene classes included specimens from clinical and healthy donors and cell lines (fusions: ROS1, EML4-ALK, NTRK1; exon skipping: MET exon 14; CNAs: HER2, CDK6, EGFR, MYC, and MET). The limit of detection (LOD) for fusion/skipping was 42 copies (QC threshold was three copies) and was verified using three additional fusion/skipping variants. LOD for CNAs was 1.40-fold-change (QC threshold = 1.15-fold change) and was verified with three additional CNAs. In repeatability and intermediate precision (within lab) studies, all fusion/skipping variants were detected in all runs and all days of testing (n = 18/18; 100%); average CV for repeatability was 20.5% (range 8.7-34.8%), and for intermediate precision it was 20.8% (range 15.7-30.5%). For CNAs, 28/29 (96.6%) copy gains were detected. For CNAs, the average CV was 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for intermediate precision. The test panel meets the criteria for being highly sensitive and specific and extends its utility for the serial detection of clinically relevant variants in cancer.

Targeted Next-Generation Sequencing of Liquid Biopsy Samples from Patients with NSCLC.

Liquid biopsy tests have become an integral part of the molecular diagnosis of patients with non-small cell lung cancer (NSCLC). We describe a new test panel that uses very low input (20 ng) of cell-free nucleic acids extracted from human plasma, which is designed to yield results in less than 72 h. In this study, we performed novel amplicon-based targeted next-generation sequencing with a semiconductor-based system, the Ion GeneStudio S5 Prime. The analytic performance of the assay was evaluated using contrived and retrospectively collected clinical specimens. The cumulative percent coefficient of variation for the new test process was very precise at 8.4% for inter-day, 4.0% for inter-operator and 3.4% for inter-instrument. We also observed significant agreement (95.7-100%) with an orthogonal, high-sensitivity droplet digital™ Polymerase Chain Reaction (ddPCR) test. This method offers a valuable supplement to assessing targeted mutations from blood while conserving specimens and maintaining sensitivity, with rapid turn-around times to actionable results.

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks.

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.

Design and Characterization of a Novel Blood Collection and Transportation Device for Proteomic Applications.

A major hurdle for blood-based proteomic diagnostics is efficient transport of specimens from the collection site to the testing laboratory. Dried blood spots have shown utility for diagnostic applications, specifically those where red blood cell hemolysis and contamination of specimens with hemoglobin is not confounding. Conversely, applications that are sensitive to the presence of the hemoglobin subunits require blood separation, which relies on centrifugation to collect plasma/serum, and then cold-chain custody during shipping. All these factors introduce complexities and potentially increased costs. Here we report on a novel whole blood-collection device (BCD) that efficiently separates the liquid from cellular components, minimizes hemolysis in the plasma fraction, and maintains protein integrity during ambient transport. The simplicity of the design makes the device ideal for field use. Whole blood is acquired through venipuncture and applied to the device with an exact volume pipette. The BCD design was based on lateral-flow principles in which whole blood was applied to a defined area, allowing two minutes for blood absorption into the separation membrane, then closed for shipment. The diagnostic utility of the device was further demonstrated with shipments from multiple sites (n = 33) across the U.S. sent to two different centralized laboratories for analyses using liquid chromatography/mass spectrometry (LC/MS/MS) and matrix assisted laser desorption/ionization-time of flight (MALDI-ToF) commercial assays. Specimens showed high levels of result label concordance for the LC/MS/MS assay (Negative Predictive Value = 98%) and MALDI-ToF assay (100% result concordance). The overall goal of the device is to simplify specimen transport to the laboratory and produce clinical test results equivalent to established collection methods.

Development of a TCR beta repertoire assay for profiling liquid biopsies from NSCLC donors.

Aim: The aim of this study was to demonstrate the utility of T-Cell receptor beta (TCRß) sequencing as a robust method for assessing T-cell repertoire changes in donors with non-small cell lung cancer (NSCLC). We further demonstrated the use of the assay by monitoring repertoire modulation in a defined model antigen system, cytomegalovirus (CMV). Methods: Peripheral blood mononuclear cells from four healthy donors were challenged with a 1-week exposure to whole-cell lysate from CMV-infected cells or CMVpp65495-503 peptide (NLVPMVATV). T-cell repertoire perturbations were assessed using the Oncomine TCR Beta-SR Assay and Ion GeneStudio S5 Plus Sequencer. A pp65 tetramer flow cytometry assay was used as an orthogonal method to assess clonal expansion of a subset of CMV-specific T-cells. For evaluation of the assay in peripheral blood lymphocytes from NSCLC donors, five whole blood specimens were evaluated using the same sequencing workflow. Results: The TCR beta assay identified 6,683-61,936 unique clones from 1-2 million reads per sample, and an average of 80% of the total reads were usable for TCR profiling. In the NSCLC donors, TCR convergence and clonality values were consistent with published results and ranged 0.016-0.033 for convergence and 0.09-0.48 for clonality. In the CMV study, TCR sequencing detected the expansion of a common family of clones in all 4 samples in response to antigen stimulation. This expansion corresponded to an increase in pp65 tetramer staining by flow cytometry. Baseline TCR convergence scores ranged 0.009-0.041 and increased 5-fold in one sample as a result of pp65 antigen stimulation. Conclusion: The results of this study demonstrated the utility of profiling of the TCRß repertoire in a model system and in donors with NSCLC. Additionally, we demonstrated the correlation between RNA-seq methods and protein-tetramer analysis using flow cytometry. These techniques represent an emerging solution that could complement other liquid and tissue diagnostic assays in the clinic and will be of value in predicting host response/resistance and adverse events to immunotherapies. Prospective clinical studies are on-going in which the developed TCR beta assay will undergo further validation.

Longitudinal monitoring for the emergence of epidermal growth factor C797S resistance mutations in non-small cell lung cancer using blood-based droplet digital PCR.

Validation of assays for the C797S mutation as a biomarker for osimertinib resistance is promising in guiding treatment decision-making for multidrug resistant non-small cell lung cancer. A newly developed droplet digital PCR (ddPCR) assay was used to retrospectively evaluate the emergence of the C797S mutation in six remnant plasma samples in this case report. It was found that the detected emergence of C797S clearly correlated with clinical signs of treatment resistance. Had these data been available to aid treatment selection in real time, there would have been hope for recaptured disease response and control instead of treatment cessation. The results of this study show that highly sensitive ddPCR methods can be used for the monitoring of emergent epidermal growth factor somatic variant mutations in circulation.

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction.

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non-Small Cell Lung Carcinoma.

Nearly 80% of cancer patients do not have genetic mutation results available at initial oncology consultation; up to 25% of patients begin treatment before receiving their results. These factors hinder the ability to pursue optimal treatment strategies. This study validates a blood-based genome-testing service that provides accurate results within 72 hours. We focused on targetable variants in advanced non-small cell lung carcinoma-epidermal growth factor receptor gene (EGFR) variant L858R, exon 19 deletion (ΔE746-A750), and T790M; GTPase Kirsten ras gene (KRAS) variants G12C/D/V; and echinoderm microtubule associated protein like and 4 anaplastic lymphoma receptor tyrosine kinase fusion (EML4-ALK) transcripts 1/2/3. Test development included method and clinical validation using samples from donors with (n = 219) or without (n = 30) cancer. Clinical sensitivity and specificity for each variant ranged from 78.6% to 100% and 94.2% to 100%, respectively. We also report on 1643 non-small cell lung carcinoma samples processed in our CLIA-certified laboratory. Mutation results were available within 72 hours for 94% of the tests evaluated. We detected 10.5% mutations for EGFR sensitizing (n = 2801 samples tested), 13.8% mutations for EGFR resistance (n = 1055), 13.2% mutations in KRAS (n = 3477), and 2% mutations for EML4-ALK fusion (n = 304). This rapid, highly sensitive, and actionable blood-based assay service expands testing options and supports faster treatment decisions.

A rare DNA contact mutation in cancer confers p53 gain-of-function and tumor cell survival via TNFAIP8 induction.

The p53 tumor suppressor gene encodes a sequence-specific transcription factor. Mutations in the coding sequence of p53 occur frequently in human cancer and often result in single amino acid substitutions (missense mutations) in the DNA binding domain (DBD), blocking normal tumor suppressive functions. In addition to the loss of canonical functions, some missense mutations in p53 confer gain-of-function (GOF) activities to tumor cells. While many missense mutations in p53 cluster at six "hotspot" amino acids, the majority of mutations in human cancer occur elsewhere in the DBD and at a much lower frequency. We report here that mutations at K120, a non-hotspot DNA contact residue, confer p53 with the previously unrecognized ability to bind and activate the transcription of the pro-survival TNFAIP8 gene. Mutant K120 p53 binds the TNFAIP8 locus at a cryptic p53 response element that is not occupied by wild-type p53. Furthermore, induction of TNFAIP8 is critical for the evasion of apoptosis by tumor cells expressing the K120R variant of p53. These findings identify induction of pro-survival targets as a mechanism of gain-of-function activity for mutant p53 and will likely broaden our understanding of this phenomenon beyond the limited number of GOF activities currently reported for hotspot mutants.

Multi-focal control of mitochondrial gene expression by oncogenic MYC provides potential therapeutic targets in cancer.

Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt's Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms.

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001.

The retinoblastoma tumor suppressor modulates DNA repair and radioresponsiveness.

PURPOSE: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance. EXPERIMENTAL DESIGN: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts. RESULTS: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05). CONCLUSIONS: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy.

Tumor suppression by p53: is apoptosis important or not?

The mechanisms by which p53 suppresses tumor growth remain ill defined. In this issue of Cell Reports, Timofeev et al. (2013) and Valente et al. (2013) reveal context-dependent contributions of p53-dependent apoptosis to its tumor-suppressive function.

Inhibition of the single downstream target BAG1 activates the latent apoptotic potential of MYC.

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.