Targeting DNA Repair to Drive Immune Responses: It's Time to Reconsider the Strategy for Clinical Translation.

PARP inhibition induces robust local and systemic antitumor immune responses and curative responses when combined with immune checkpoint blockade in many preclinical studies. However, the combination has not markedly improved antitumor effect compared with individual agents in clinical trials to date. We propose that the data from these trials indicate a lack of synergistic interaction of PARP inhibition and immune checkpoint blockade, with implications for reexamining our current strategies for clinical translation. As current mouse models do not recapitulate the genomic heterogeneity or tumor microenvironment of human cancers, better models are urgently needed. Tumor-extrinsic factors modulate immune checkpoint blockade response and they may be better assessed in early-phase clinical trials with frequent tissue and blood sampling. Further work is also needed to uncover the dose and schedule dependency of DNA repair modulation on the immune system. In homologous recombination repair-deficient tumors, randomized trials should be prioritized to address whether the benefit is superior to that of PARP inhibitor monotherapy. In tumors that are not homologous recombination repair deficient, research biopsies should be integrated to early-phase clinical trials to discover biomarkers that can predict clinical benefit. These considerations are relevant to the variety of adjunctive therapeutics being combined with immune checkpoint blockade to improve probability, duration, and potency of antitumor activity.

The right angle on IL-2 therapy.

Engineered cytokines are able to improve immunotherapy in mouse tumor models.

Recent advances in the use of therapeutic cancer vaccines in genitourinary malignancies.

INTRODUCTION: Despite a recent increase in US FDA-approved treatments, genitourinary malignancies remain a source of significant morbidity and mortality. One focus of research is the use of therapeutic cancer vaccines in these diseases, and a significant body of clinical trial experience now exists for refining vaccine strategies to enhance antitumor efficacy and develop immune-based combination regimens. AREAS COVERED: In recent years, clinical data from multiple trials in genitourinary malignancies have enhanced our understanding of the potential for immunotherapy in these cancers. There are also emerging clinical strategies that combine cancer vaccines with chemotherapy, radiation, androgen-deprivation therapy and immune checkpoint inhibitors. This review is based on a search of relevant literature for data presented over the past 5 years from clinical trials of cancer vaccines in prostate, bladder and renal carcinomas. EXPERT OPINION: In the coming years, clinical trials informed by decades of preclinical data and emerging clinical data will help to define the role of immunotherapy in genitourinary malignancies. Combination strategies that capitalize on the immune properties of standard treatments will bring greater clinical benefits, and immune-based combinations will likely be moved to the neoadjuvant setting, where they may have optimal clinical impact.

Functionally defective germline variants of sialic acid acetylesterase in autoimmunity.

Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signalling and is required for the maintenance of immunological tolerance in mice. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 subjects of European origin with relatively common autoimmune disorders and in 2/648 controls of European origin. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in eight autoimmune subjects but in no control subjects. The odds ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

Concurrence of Danish dementia and cataract: insights from the interactions of dementia associated peptides with eye lens alpha-crystallin.

Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens alpha-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease.

Form and dimensions of aggregates dictate cytotoxicities of Danish dementia peptides.

Familial Danish dementia (FDD) is a neurodegenerative disease which results due to alterations in the BRI2 gene. The pathological symptoms of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. The ADan peptide is a 34 amino acid long peptide which is thought to be the major cause of amyloid deposition in brains of patients suffering from FDD. Due to the presence of two cysteine residues viz. cys5 and cys23, this peptide exists in two forms: a cyclic oxidized form where the two cysteines form a disulfide bridge and a linear reduced form where the sulphydryl groups of cysteine are free. The relationship between toxicity and structure of the reduced and oxidized forms of ADan peptides has been elucidated by a combination of biophysical and cellular toxicity assays. It is observed that the reduced peptide has a stronger lethal effect on neuronal cell lines compared to its oxidized counterparts at all stages of aggregation. Further, it is observed that the fresh reduced peptide induced greater cell death as compared to its aged counterpart.

Fibrillogenesis in ADan peptides is inhibited by biphenyl ethers.

In this study, biphenyl ethers of diverse functionality were used to assess their effect on fibrillogenesis of both the oxidized and reduced ADan peptides, in vitro. It was noted that these compounds not only stalled fibrillogenesis but were also able to disrupt pre-formed fibers. The EC(50) values for the inhibition of this process lie in the nanomolar range for 50 microM of peptide concentration, indicating the high potency of these compounds as inhibitors. It was found that these compounds impart to the peptides, an alpha-helical conformation which does not allow them to aggregate and form fibrils. These studies also point out that the transition of peptides through alpha-helical conformation may be a prelude to the onset of fibrillogenesis for oxADan peptides.

A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate.

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1alpha subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1alpha protein expression were determined 10 days post-transduction. Both vectors increased E1alpha expression 40-60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1alpha. Exposure for 24h to 5mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1alpha protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.

Hierarchy and the mechanism of fibril formation in ADan peptides.

Familial Danish dementia is a neurodegenerative disease which is a consequence of alterations in the BRI gene. The pathological signatures of the disease are cerebral amyloidolysis, parenchymal protein deposits and neuronal degeneration. Synthetic Danish dementia (ADan) peptides are capable of forming fibrillar assemblies in vitro at pH 4.8. However, the morphology of the aggregates formed depends greatly on the form of the peptides (oxidized or reduced). In addition to long slender assemblies (2-5 nm in diameter and several micrometers in length) we report ring-like or annular masses (8-9 nm in diameter and 1-2 mm in perimeter) in the case of the oxidized form of the peptides. The reduced forms mainly aggregate to produce granular heaps. The biophysical and kinetic characterization of the process of aggregation was carried out using different spectroscopic and imaging techniques. Neurotoxicity assays performed on both the forms reveal that the toxicity bears proportionality with the aggregate size.

Assembly of alpha-hemolysin on A431 cells leads to clustering of Caveolin-1.

Assembly and penetration of 14-strand beta-barrel of staphylococcal alpha-hemolysin (alpha-HL) is an intriguing phenomenon due to its water soluble property. alpha-HL interacts with the Caveolin-1 of A431 cells for its rapid assembly. A nine amino acid, non-hydrophobic peptide derived from alpha-HL has been shown to block the interaction of alpha-HL with the scaffolding domain of Caveolin-1. alpha-HL's presence was also detected in the Caveolin-1 enriched membrane fractions isolated by ultracentrifugation. Moreover, alpha-HL co-precipitates with Caveolin-1 specifically. In a time-dependent process, alpha-HL associates with the Caveolin-1 and co-localizes with Caveolin-1 that results in an extensive clustering of Caveolin-1 at cell-cell contacts. Mutants of alpha-HL devoid of Caveolin-1 binding motif failed to assemble into heptameric oligomers on the surface of A431 cells. Our data suggest that the conformational changes required to form the heptameric assembly might be triggered at the Caveolin-1 binding motif of alpha-HL.

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.

Mechanism of membrane binding by the bovine seminal plasma protein, PDC-109: a surface plasmon resonance study.

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.

Suppression of DTT-induced aggregation of abrin by alphaA- and alphaB-crystallins: a model aggregation assay for alpha-crystallin chaperone activity in vitro.

The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.