Randomized trial of neoadjuvant vaccination with tumor-cell lysate induces T cell response in low-grade gliomas

BACKGROUND. Long-term prognosis of WHO grade II low-grade gliomas (LGGs) is poor, with a high risk of recurrence and malignant transformation into high-grade gliomas. Given the relatively intact immune system of patients with LGGs and the slow tumor growth rate, vaccines are an attractive treatment strategy. METHODS. We conducted a pilot study to evaluate the safety and immunological effects of vaccination with GBM6-AD, lysate of an allogeneic glioblastoma stem cell line, with poly-ICLC in patients with LGGs. Patients were randomized to receive the vaccines before surgery (arm 1) or not (arm 2) and all patients received adjuvant vaccines. Coprimary outcomes were to evaluate safety and immune response in the tumor. RESULTS. A total of 17 eligible patients were enrolled — 9 in arm 1 and 8 in arm 2. This regimen was well tolerated with no regimen-limiting toxicity. Neoadjuvant vaccination induced upregulation of type-1 cytokines and chemokines and increased activated CD8+ T cells in peripheral blood. Single-cell RNA/T cell receptor sequencing detected CD8+ T cell clones that expanded with effector phenotype and migrated into the tumor microenvironment (TME) in response to neoadjuvant vaccination. Mass cytometric analyses detected increased tissue resident–like CD8+ T cells with effector memory phenotype in the TME after the neoadjuvant vaccination. CONCLUSION. The regimen induced effector CD8+ T cell response in peripheral blood and enabled vaccine-reactive CD8+ T cells to migrate into the TME. Further refinements of the regimen may have to be integrated into future strategies

Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging

Defects in the DNA repair mechanism nucleotide excision repair (NER) may lead to tumors in xeroderma pigmentosum (XP) or to premature aging with loss of subcutaneous fat in Cockayne syndrome (CS). Mutations of mitochondrial (mt)DNA play a role in aging, but a link between the NER-associated CS proteins and base excision repair (BER)-associated proteins in mitochondrial aging remains enigmatic. We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress. MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csbm/m and Csa−/− mice. Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging

Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor

Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L⁻/⁻ and wild type cells are equally resistant to ionising radiation, whereas 53Bp1⁻/⁻/Dot1L⁻/⁻ cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1⁻/⁻ cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents

Complete Amino Acid Sequences of a Pair of Fish (Tilapia) Prolactins, tPRL\u3csub\u3e177\u3c/sub\u3e and tPRL\u3csub\u3e188\u3c/sub\u3e

The complete amino acid sequences of a pair of tilapia (Oreochromis mossambicus) prolactins (PRLs) were determined. The larger PRL of molecular mass 20,836 Da consists of 188 amino acid residues. The smaller PRL of molecular mass 19,584 Da is 11 residues shorter. On alignment of the two sequences, the 19.6-kDa PRL (tPRL177) has two conspicuous deletions on the NH2- terminal side of the disulfide bond which connects the first and second cysteine residues. The degree of similarity between the two PRL sequences is unexpectedly low (130 identical residues, 69%) compared with that between the variants of other teleostean PRLs. Circular dichroism spectra and hydropathy profiles suggest structural similarity of the two PRLs. The sequence of the 20.8-kDa PRL (tPRL188) has 69% identity with that of salmon PRL. The sequence of tPRL177 is 56% identical with that of salmon PRL. Each tilapia PRL is equally similar to mammalian PRLs (about 30% identical residues). Regions highly conserved among teleostean and mammalian PRLs were identified on the COOH-terminal side of the disulfide bond connecting the first and second cysteine residues

Aromatase is concentrated in the proximal pars distalis of tilapia pituitary

Aromatase has been identified in the teleostean, avian, and mammalian pituitaries, although its cellular location(s) is not yet certain. To address this question, experiments were performed in tilapia (Oreochromis mossambicus), a species which has been well characterized with respect to the intraglandular distribution of the different pituitary cell types. To estimate aromatase, glands were microdissected into rostral pars distalis (RPD), proximal pars distalis (PPD), and neurointermediate lobe (NIL) and organs were cultured in the presence of [3H]androstenedione for 16-24 hr. [3H]Estrogen products were isolated and quantified after ether extraction, hydrolysis with glucuronidase-sulfatase, thin-layer chromatography, and phenolic partition. Authentic estrone or estradiol-17β were produced by all pituitary regions and also by the urophyseal region of the spinal cord. Aromatase was two to five times higher in PPD than in RPD or NIL and similar to activity in adjacent hypothalamus-preoptic area (HPOA). Much lower estrogen yields were obtained in cultures of cerebellum, urophysis, and other cord regions. Since the PPD contains most of the somatotropes, these data are consistent with earlier studies implicating GH3/GH4 cell strains as an enriched enzyme source, although its presence in other cell types cannot be ruled out. The unusually high and localized aromatase in tilapia pituitary renders this species a useful model for studying the targets and functional importance of estrogen as a parahormone in the pituitary. © 1988

Amino acid sequence of growth hormone isolated from medium of incubated pituitary glands of tilapia (Oreochromis mossambicus)

The amino acid sequence of tilapia (Oreochromis mossambicus) growth hormone (GH) was determined directly by Edman degradation of peptide fragments generated by lysyl endopeptidase and trypsin digestion. The N-terminal residue was deduced to be pyroglutamic acid through the use of pyroglutamyl aminopeptidase; its removal allowed amino acid sequence determination of the remainder of the N-terminal trypsin peptide by Edman degradation. Tilapia GH is composed of 187 amino acid residues and shows high similarity to other perciform GHs. Sequence identities are: 89% with tuna GH, 83% with bonito GH, 85% with yellowtail GH, 89% with red sea bream GH, and 34% with bovine GH. The two asparagine residues (Asn-148 and Asn-184) were recovered by Edman degradation, suggesting the absence of N-glycosylation. © 1991