An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.

A chemical genetics analysis of the roles of bypass polymerase DinB and DNA repair protein AlkB in processing N2-alkylguanine lesions in vivo.

DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N2-alkylguanine adducts in vitro, including N2-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N2-alkyguanines, including N2-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N2-alkylguanine lesions: N2-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N2-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N2-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N2-alkylguanine DNA adducts was DinB-dependent lesion bypass.

Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics.

A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

Efficient extension of slipped DNA intermediates by DinB is required to escape primer template realignment by DnaQ.

We show that Escherichia coli DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the -4 position. A DinB(Y79L) variant, which extends these substrates less efficiently in vitro, allows the proofreading function of polymerase III to reverse their formation in vivo.

UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs.

Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.

The SOS Regulatory Network.

All organisms possess a diverse set of genetic programs that are used to alter cellular physiology in response to environmental cues. The gram-negative bacterium, Escherichia coli, mounts what is known as the "SOS response" following DNA damage, replication fork arrest, and a myriad of other environmental stresses. For over 50 years, E. coli has served as the paradigm for our understanding of the transcriptional, and physiological changes that occur following DNA damage (400). In this chapter, we summarize the current view of the SOS response and discuss how this genetic circuit is regulated. In addition to examining the E. coli SOS response, we also include a discussion of the SOS regulatory networks in other bacteria to provide a broader perspective on how prokaryotes respond to DNA damage.

The SOS Regulatory Network.

All organisms possess a diverse set of genetic programs that are used to alter cellular physiology in response to environmental cues. The gram-negative bacterium Escherichia coli induces a gene regulatory network known as the "SOS response" following exposure to DNA damage, replication fork arrest, and a myriad of other environmental stresses. For over 50 years, E. coli has served as the paradigm for our understanding of the transcriptional and physiological changes that occur after DNA damage. In this chapter, we summarize the current view of the SOS response and discuss how this genetic circuit is regulated. In addition to examining the E. coli SOS response, we include a discussion of the SOS regulatory networks found in other bacteria to provide a broad perspective on the mechanism and diverse physiological responses that ensueto protect cells and maintain genome integrity.

Chromosome segregation control by Escherichia coli ObgE GTPase.

Escherichia coli cells depleted of the conserved GTPase, ObgE, show early chromosome-partitioning defects and accumulate replicated chromosomes in which the terminus regions are colocalized. Cells lacking ObgE continue to initiate replication, with a normal ratio of the origin to terminus. Localization of the SeqA DNA binding protein, normally seen as punctate foci, however, was disturbed. Depletion of ObgE also results in cell filamentation, with polyploid DNA content. Depletion of ObgE did not cause lethality, and cells recovered fully after expression of ObgE was restored. We propose a model in which ObgE is required to license chromosome segregation and subsequent cell cycle events.

A bacterial G protein-mediated response to replication arrest.

To define factors in E. coli promoting survival to replication fork stress, we isolated insertion mutants sensitive to replication inhibitors. One insertion caused partial loss of the universally conserved GTPase, obgE/yhbZ gene. Although obgE is essential for growth, our insertion allele supported viability until challenged with various replication inhibitors. A mutation designed to negate the GTPase activity of the protein produced similar phenotypes, but was genetically dominant. Synergistic genetic interactions with recA and recB suggested that chromosome breaks and regressed forks accumulate in obgE mutants. Mutants in obgE also exhibited asynchronous overreplication during normal growth, as revealed by flow cytometry. ObgE overexpression caused SeqA foci, normally localized to replication forks, to spread extensively within the cell. We propose that ObgE defines a pathway analogous to the replication checkpoint response of eukaryotes and acts in a complementary way to the RecA-dependent SOS response to promote bacterial cell survival to replication fork arrest.