Blockade of novel immune checkpoints and new therapeutic combinations to boost antitumor immunity

Immunotherapy has emerged as a promising strategy for boosting antitumoral immunity. Blockade of immune checkpoints (ICs), which regulate the activity of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells has proven clinical benefits. Antibodies targeting CTLA-4, PD-1, and PD-L1 are IC-blockade drugs approved for the treatment of various solid and hematological malignancies. However, a large subset of patients does not respond to current anti-IC immunotherapy. An integrative understanding of tumor-immune infiltrate, and IC expression and function in immune cell populations is fundamental to the design of effective therapies. The simultaneous blockade of newly identified ICs, as well as of previously described ICs, could improve antitumor response. We review the potential for novel combinatory blockade strategies as antitumoral therapy, and their effects on immune cells expressing the targeted ICs. Preclinical evidence and clinical trials involving the blockade of the various ICs are reported. We finally discuss the rationale of IC co-blockade strategy with respect to its downstream signaling in order to improve effective antitumoral immunity and prevent an increased risk of immune-related adverse events (irAEs)

p27Kip1 regulates alpha-synuclein expression

Alpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies

The transcription factor C/EBPd represses a-synuclein transcription: potential pathogenic effects of C/EBPd deficiency in Parkinson's disease

Trabajo presentado en el XI Simposi de Neurobiologia: Future technical advances, organizado por la Socitat Catalana de Biologia, en Barcelona, los días 12 y 13 de noviembre de 2018α-Synuclein, one of the most abundant proteins in neuronal cytosol, plays an ill-defined role in neurotransmitter release and synaptic vesicle trafficking and is the main component of Lewy bodies, the intracellular protein aggregates that are considered the histological hallmark of Parkinson’s disease. High α-synuclein levels are associated with increased risk for Parkinson's disease. Surprisingly little is known about the regulation of transcription of the human αsynuclein (SNCA) gene. CCAAT/enhancer binding protein δ (C/EBPδ) is a b-zip transcription factor expressed in the CNS that plays distinct roles in neurons and glial cells. C/EBPδ binding boxes are present in the SNCA genomic region, suggesting that this transcription factor could regulate SNCA transcription. The aim of this study was to determine if C/EBPδ regulates the expression of SNCA. We first observed that α-synuclein expression was markedly increased in C/EBPδ-deficient mice in several brain regions, both at mRNA and protein level. α-synuclein levels were also increased in C/EBPδ-deficient primary neuronal, but not glial, cultures. In accordance, C/EBPδ overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced α-synuclein expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans. Finally, decreased C/EBPδ expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between α-synuclein and C/EBPδ levels. This study demonstrates for the first time that C/EBPδ is a potent repressor of SNCA transcription. These findings suggest that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson’s disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target to treat these neurological disordersSupported by: PI14/302 from the Instituto de Salud Carlos III, Spain, cofinanced with FEDER funds.Peer reviewe

Paper de la proteïna p27 en la regulació de l'expressió de l'α-sinucleïna: Implicacions en la malaltia de Parkinson

[cat] El nostre grup ha identificat prèviament diferents gens i programes transcripcionals regulats per la proteïna p27. En aquest treball ens centrem en el paper de la proteïna p27 en la regulació a nivell transcripcional de l’expressió del gen que codifica per l’α-sinucleïna (gen SNCA), una proteïna molt important en el desenvolupament de la malaltia de Parkinson. Específicament, l’α-sinucleïna forma agregats en regions concretes del cervell. En aquest treball hem comprovat que les cèl·lules deficients de p27 presenten nivells més elevats d’α-sinucleïna. Per altra banda, en cervells de ratolins KO de p27 també observem un augment de l’expressió d’α-sinucleïna. Addicionalment, mitjançant la tècnica de silenciament gènic CRISPR/Cas9 vam obtenir línies cèl·lules KO de p27 i E2F4 i en aquestes cèl·lules també s’observa un augment de l’α-sinucleïna. Posteriorment, hem descrit, mitjançant experiments de ChIP i de luciferases, la regulació a nivell transcripcional de p27 del gen SNCA en associació a diferents factors de transcripció, C/EBPδ i E2F4/p130. En ambdós casos l’expressió de p27 reprimeix l’expressió del gen SNCA. A més a més, mitjançant shRNAs i la tècnica de Proximity Ligation Assay (PLA) hem observat com la disminució dels nivells de p27 i p130 també augmenten l’agregació de l’α-sinucleïna. També hem identificat CDK5 com una possible quinasa encarregada de fosforil·lar a p27 i exportar-ho al citoplasma on no pot exercir la seva funció com a regulador transcripcional. Finalment, mitjançant shRNAs vam disminuir els nivells de CDK5, en aquestes cèl·lules i amb la tècnica de PLA hem aconseguit veure com la supressió de CDK5 provoca un augment en l’agregació de l’α-sinucleïna. Com està descrit que CDK5 és capaç d’interaccionar amb els complexes E2F1/pRB, hem caracteritzat la unió de p27 a E2F1 mitjançant columnes cromatogràfiques i hem establert que E2F1 s’uneix a al extrem final de p27, entre els aminoàcids 160-198, de manera similar a la que s’uneix amb E2F4.[eng] Our group has identified different transcriptional programs regulated by the protein p27. In this work we focus on the role of p27 in the transcriptional regulation of the gene that encodes for α-synuclein (SNCA gene), a very important protein involved in Parkinson’s disease. Specifically, α-synuclein forms aggregates in specific regions of the brain. In this thesis, we found that p27-deficient cells have higher levels of α-synuclein. On the other hand, in brains of p27 KO mice we also observed an increase in α-synuclein levels in some brain regions. Additionally, using the CRISPR/Cas9 gene silencing technique we obtained KO cell lines of p27 and E2F4 and in these cell lines we also observe an increase of α-synuclein. Subsequently, through ChIP and luciferase assays, we described the transcriptional regulation of p27 of the SNCA gene in association with two different transcription factors, C/EBPδ and E2F4/p130. In both cases, p27 expression supresses SNCA expression. Furthermore, using shRNAs and the Proximity Ligation Assay (PLA) technique, we observed how the decrease of p27 and p130 protein levels also increased α-synuclein aggregation levels. We have also identified CDK5 as a kinase that phosphorylates p27 and cause it exportation to cytoplasm where it cannot function as a transcriptional regulator. Finally, using shRNAs we decreased the levels of CDK5 in these cells and with PLA we were able to see how the decrease of CDK5 levels causes an increase in α-synuclein aggregation levels. As it is described that CDK5 can interact with E2F1/pRb complexes, we have characterized the binding of p27 to E2F1 by affinity chromatography assays and we established that, as occurs with E2F4, E2F1 binds to the carboxyl moiety of p27, between aminoacids 160-170

p27^Kip1 regulates alpha-synuclein expression

International audienceAlpha-synuclein (α-SYN) is the main component of anomalous protein aggregates (Lewy bodies) that play a crucial role in several neurodegenerative diseases (synucleinopathies) like Parkinson's disease and multiple system atrophy. However, the mechanisms involved in its transcriptional regulation are poorly understood. We investigated here the role of the cyclin-dependent kinase (Cdk) inhibitor and transcriptional regulator p27Kip1 (p27) in the regulation of α-SYN expression. We observed that selective deletion of p27 by CRISPR/Cas9 technology in neural cells resulted in increased levels of α-SYN. Knock-down of the member of the same family p21Cip1 (p21) also led to increased α-SYN levels, indicating that p27 and p21 collaborate in the repression of α-SYN transcription. We demonstrated that this repression is mediated by the transcription factor E2F4 and the member of the retinoblastoma protein family p130 and that it is dependent of Cdk activity. Chromatin immunoprecipitation analysis revealed specific binding sites for p27, p21 and E2F4 in the proximal α-SYN gene promoter. Finally, luciferase assays revealed a direct action of p27, p21 and E2F4 in α-SYN gene expression. Our findings reveal for the first time a negative regulatory mechanism of α-SYN expression, suggesting a putative role for cell cycle regulators in the etiology of synucleinopathies

CCAAT/enhancer binding protein δ is a transcriptional repressor of α-synuclein

α-Synuclein is the main component of Lewy bodies, the intracellular protein aggregates representing the histological hallmark of Parkinson’s disease. Elevated α-synuclein levels and mutations in SNCA gene are associated with increased risk for Parkinson’s disease. Despite this, little is known about the molecular mechanisms regulating SNCA transcription. CCAAT/enhancer binding protein (C/EBP) β and δ are b-zip transcription factors that play distinct roles in neurons and glial cells. C/EBPβ overexpression increases SNCA expression in neuroblastoma cells and putative C/EBPβ and δ binding sites are present in the SNCA genomic region suggesting that these proteins could regulate SNCA transcription. Based on these premises, the goal of this study was to determine if C/EBPβ and δ regulate the expression of SNCA. We first observed that α-synuclein CNS expression was not affected by C/EBPβ deficiency but it was markedly increased in C/EBPδ-deficient mice. This prompted us to characterize further the role of C/EBPδ in SNCA transcription. C/EBPδ absence led to the in vivo increase of α-synuclein in all brain regions analyzed, both at mRNA and protein level, and in primary neuronal cultures. In agreement with this, CEBPD overexpression in neuroblastoma cells and in primary neuronal cultures markedly reduced SNCA expression. ChIP experiments demonstrated C/EBPδ binding to the SNCA genomic region of mice and humans and luciferase experiments showed decreased expression of a reporter gene attributable to C/EBPδ binding to the SNCA promoter. Finally, decreased CEBPD expression was observed in the substantia nigra and in iPSC-derived dopaminergic neurons from Parkinson patients resulting in a significant negative correlation between SNCA and CEBPD levels. This study points to C/EBPδ as an important repressor of SNCA transcription and suggests that reduced C/EBPδ neuronal levels could be a pathogenic factor in Parkinson’s disease and other synucleinopathies and C/EBPδ activity a potential pharmacological target for these neurological disorders.This study was supported by grants PI10/378, PI12/709, and PI14/302 from the Instituto de Salud Carlos III, Spain, cofinanced with FEDER funds