Improving CAR T-Cell Persistence

Over the last decade remarkable progress has been made in enhancing the efficacy of CAR T therapies. However, the clinical benefits are still limited, especially in solid tumors. Even in hematological settings, patients that respond to CAR T therapies remain at risk of relapsing due to several factors including poor T-cell expansion and lack of long-term persistence after adoptive transfer. This issue is even more evident in solid tumors, as the tumor microenvironment negatively influences the survival, infiltration, and activity of T-cells. Limited persistence remains a significant hindrance to the development of effective CAR T therapies due to several determinants, which are encountered from the cell manufacturing step and onwards. CAR design and ex vivo manipulation, including culture conditions, may play a pivotal role. Moreover, previous chemotherapy and lymphodepleting treatments may play a relevant role. In this review, the main causes for decreased persistence of CAR T-cells in patients will be discussed, focusing on the molecular mechanisms underlying T-cell exhaustion. The approaches taken so far to overcome these limitations and to create exhaustion-resistant T-cells will be described. We will also examine the knowledge gained from several key clinical trials and highlight the molecular mechanisms determining T-cell stemness, as promoting stemness may represent an attractive approach to improve T-cell therapies

Nouveau mécanisme de régulation de la recombinaison homologue par le complexe d'assemblage des nucléosomes caf-1

The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.La réplication des chromosomes est altérée par les facteurs endogènes et/ou exogènes qui perturbent la progression des fourches de réplication. Les cellules doivent donc coordonner la synthèse d’ADN avec des mécanismes assurant la stabilité et le rétablissement des fourches bloquées. La recombinaison homologue (RH) est un mécanisme universel qui permet la réparation de l’ADN et participe au maintien de la réplication des chromosomes. Néanmoins, les mécanismes qui régulent la RH, notamment la RH ectopique versus la RH allélique, restent mal compris. Un autre mécanisme essentiel assurant la stabilité des génomes est l’assemblage de l’ADN néo-synthétisé autour de nucléosomes, conduisant à la constitution de fibres chromatiniennes nécessaires à l’organisation structurale du matériel génétique. Chez Saccharomyces cerevisiae, des défauts d’assemblage de la chromatine conduisent à une instabilité des fourches de réplication et augmentent le taux de RH. Sachant que les chaperonnes d’histones jouent un rôle crucial durant l’assemblage de la chromatine, j'ai décidé de me concentrer sur le rôle de la chaperonne d’histones H3-H4 appelé Chromatin Assembly Factor 1 (CAF-1) dans les mécanismes de RH, chez Schizosaccharomyces pombe. En effet, la RH est associée à une étape de synthèse de l’ADN, et peu de choses sont connues sur l’assemblage de la chromatine au cours de cette synthèse. Mes résultats ont exclu un rôle de CAF-1 dans la recombinaison allelique et le maintien de la stabilité des fourches de réplication. Par contre, CAF-1 joue un rôle important dans les mécanismes de recombinaisons ectopique et dans la formation de réarrangements chromosomiques induits par des blocages de fourches. Mes données suggèrent un modèle selon lequel CAF-1 permet la stabilisation d’intermédiaires de recombinaison précoces (D-loop), via le dépôt de nucleosomes au cours de l’extension par polymérisation de ces intermédiaires. Ainsi CAF-1 neutralise la dissociation des intermédiaires de recombinaison précoces par l’ADN helicase Rqh1. CAF-1 ferait partie d'un équilibre qui règle la stabilité/dissociation des intermédiaires de recombinaison précoces. J'ai montré que le rôle de CAF-1 dans cet équilibre a une importance toute particulière pendant la recombinaison non-allelique, révélant ainsi un nouveau niveau de régulation des mécanismes de RH par l'assemblage de la chromatine

Espressione eterologa, purificazione e saggio di attività della Fe-idrogenasi di Enterobacter cloacae

Lavoro svolto durante il tirocinio che è consistito nell’espressione della ferro idrogenasi di Enterobacter cloacae in Escherichia coli e nella sua purificazione

Nouveau mécanisme de régulation de la recombinaison homologue par le complexe d'assemblage des nucléosomes caf-1

La réplication des chromosomes est altérée par les facteurs endogènes et/ou exogènes qui perturbent la progression des fourches de réplication. Les cellules doivent donc coordonner la synthèse d ADN avec des mécanismes assurant la stabilité et le rétablissement des fourches bloquées. La recombinaison homologue (RH) est un mécanisme universel qui permet la réparation de l ADN et participe au maintien de la réplication des chromosomes. Néanmoins, les mécanismes qui régulent la RH, notamment la RH ectopique versus la RH allélique, restent mal compris. Un autre mécanisme essentiel assurant la stabilité des génomes est l assemblage de l ADN néo-synthétisé autour de nucléosomes, conduisant à la constitution de fibres chromatiniennes nécessaires à l organisation structurale du matériel génétique. Chez Saccharomyces cerevisiae, des défauts d assemblage de la chromatine conduisent à une instabilité des fourches de réplication et augmentent le taux de RH. Sachant que les chaperonnes d histones jouent un rôle crucial durant l assemblage de la chromatine, j'ai décidé de me concentrer sur le rôle de la chaperonne d histones H3-H4 appelé Chromatin Assembly Factor 1 (CAF-1) dans les mécanismes de RH, chez Schizosaccharomyces pombe. En effet, la RH est associée à une étape de synthèse de l ADN, et peu de choses sont connues sur l assemblage de la chromatine au cours de cette synthèse. Mes résultats ont exclu un rôle de CAF-1 dans la recombinaison allelique et le maintien de la stabilité des fourches de réplication. Par contre, CAF-1 joue un rôle important dans les mécanismes de recombinaisons ectopique et dans la formation de réarrangements chromosomiques induits par des blocages de fourches. Mes données suggèrent un modèle selon lequel CAF-1 permet la stabilisation d intermédiaires de recombinaison précoces (D-loop), via le dépôt de nucleosomes au cours de l extension par polymérisation de ces intermédiaires. Ainsi CAF-1 neutralise la dissociation des intermédiaires de recombinaison précoces par l ADN helicase Rqh1. CAF-1 ferait partie d'un équilibre qui règle la stabilité/dissociation des intermédiaires de recombinaison précoces. J'ai montré que le rôle de CAF-1 dans cet équilibre a une importance toute particulière pendant la recombinaison non-allelique, révélant ainsi un nouveau niveau de régulation des mécanismes de RH par l'assemblage de la chromatine.The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Multifaceted Interplay between Hormones, Growth Factors and Hypoxia in the Tumor Microenvironment

Hormones and growth factors (GFs) are signaling molecules implicated in the regulation of a variety of cellular processes. They play important roles in both healthy and tumor cells, where they function by binding to specific receptors on target cells and activating downstream signaling cascades. The stages of tumor progression are influenced by hormones and GF signaling. Hypoxia, a hallmark of cancer progression, contributes to tumor plasticity and heterogeneity. Most solid tumors contain a hypoxic core due to rapid cellular proliferation that outgrows the blood supply. In these circumstances, hypoxia-inducible factors (HIFs) play a central role in the adaptation of tumor cells to their new environment, dramatically reshaping their transcriptional profile. HIF signaling is modulated by a variety of factors including hormones and GFs, which activate signaling pathways that enhance tumor growth and metastatic potential and impair responses to therapy. In this review, we summarize the role of hormones and GFs during cancer onset and progression with a particular focus on hypoxia and the interplay with HIF proteins. We also discuss how hypoxia influences the efficacy of cancer immunotherapy, considering that a hypoxic environment may act as a determinant of the immune-excluded phenotype and a major hindrance to the success of adoptive cell therapies

Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

<div><p>Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.</p> </div

Preventing D-loop disassembly by CAF-1 requires its ability to interact with PCNA.

<p>(A, Top panel) Alignment of the human (p150), <i>S. cerevisiae</i> (Cac1), and <i>S. pombe</i> (Pcf1) large subunit of CAF-1. Boxes indicate the PEST, KER, and E/D domains. The dashed line indicates the acidic region involved in histone binding. PIP1 and 2 indicate the PCNA interacting peptide. (Bottom panel) PIP1 sequence in Cac1 and Pcf1. Numbers refer to amino acids. Underlined amino acids indicate mutation introduced in the Pcf1-PIP^mut protein. (B) Analysis of RIs by 2DGE in indicated strains and conditions; ON and OFF refers to the <i>RTS1</i>-RFB being active or not, respectively. Top panels are diagrams of RIs within the A<i>se</i>1 restriction fragment analyzed by 2DGE in indicated conditions. Stars indicate additional signals resulting from partial digestion due to cross-linked DNA. (C) Chromosomes from indicated strains and conditions were separated by PFGE and analyzed by Southern blotting using <i>rng3</i> probe, located <i>tel</i> proximal from <i>ura4</i>. Cells were grown with (RFB OFF) or without thiamine (RFB ON) for 48 h. (D) Quantification of the amounts of acentric chromosomes seen in panel C. Values correspond to the mean of at least three independent experiments ±SEM. Refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001968#pbio.1001968.s011" target="_blank">Data S1</a>, sheet 7. (E) Representative PCR amplification from 5-FOA^R colonies from the indicated strain and condition. PCR products and their sizes are indicated on the figure. (F) Rate of genomic deletion and translocation for the strains indicated; ON and OFF refers to the <i>RTS1</i>-RFB being active or not, respectively. The percentage of deletion and translocation events, as determined by the PCR assay, was used to balance the rate of <i>ura4</i> loss. The values reported are means of at least three independent median rates ±SD. Statistically significant fold differences in the rates of deletion or translocation events from the <i>wt</i> strain are indicated with an asterisk (<i>p</i><0.01). Statistical significance was calculated using the nonparametric Mann–Whitney U test. Refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001968#pbio.1001968.s011" target="_blank">Data S1</a>, sheet 8. (G) Model of D-loop stabilization by CAF-1 during template switch. CAF-1 might prevent the disassembly of the D-loop by promoting histone deposition coupled to DNA synthesis. Nascent chromatin assembled on the D-loop then counteracts Rqh1 activity (black line). Alternatively, CAF-1 is targeting on the D-loop via its interaction with PCNA and counteracts the activity of Rqh1 directly or indirectly (dashed green line).</p

The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1

<div><p>At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.</p></div

CAF-1 promotes faulty template switch at blocked replication forks.

<p>(A) Diagram of the <i>t> ura4 locus, in which <i>t</i> refers to the telomere (gray lines), <i>ura4</i> refers to the <i>wt</i> gene (red lines),> <i>and</i> RTS1</i>-RFB (blue bars, the darkest blue one corresponds to the most efficient RFB), and <i>ori</i> refers to the replication origin (opened black circle) on the centromere-proximal side. A <i>RTS1</i>-RFB is naturally located on chromosome II. The green and black circles indicate the centromere of chromosomes II and III, respectively. Fork arrest at the <i>RTS1</i>-RFB on chromosome III leads to ectopic recombination with the <i>RTS1</i> sequence located on chromosome II, resulting in <i>ura4</i> loss and genomic deletion associated or not with a translocation between chromosomes II and III. The loss of <i>ura4</i> is genetically selected using the 5-FOA drug that allows the selection of cells exhibiting a loss of <i>ura4⁺</i> function (deletion or mutation). Primers used for amplifying the 1 Kb <i>ura4</i> fragment or the 650 bp <i>rng3</i> fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). (B) Representative PCR-amplification from 5-FOA–resistant colonies from indicated strains and conditions. PCR products and their sizes are indicated on the figure. (C) Survival of indicated strains upon fork arrest at the <i>t> ura4 locus. Serial 10-fold dilution from indicated strains spotted onto media containing thiamine (RFB OFF) or not (RFB ON). (D) Rate of genomic deletion and translocation for the strains indicated; ON and OFF refers to the <i>RTS1</i>-RFB being active or not, respectively. The percentage of deletion and translocation events, as determined by the PCR assay, was used to balance the rate of <i>ura4</i> loss. The values reported are means of at least three independent median rates ± standard deviation (SD). Statistically significant fold differences in the rates of deletion or translocation events from the <i>wt</i> strain are indicated with a asterisk (<i>p</i><0.01). Statistical significance was calculated using the nonparametric Mann–Whitney U test. Refer to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001968#pbio.1001968.s011" target="_blank">Data S1</a>, sheet 1.</i></p

Collapsed forks, but not stalled forks, induce replication slippage.

<p>A. Left panel: the frequency of Ura⁺ revertants as a function of time-contact with indicated drugs for the indicated <i>ura4</i> alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura⁺ revertants in response to UV-C irradiation as a function of dose for the indicated <i>ura4</i> alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura⁺ revertants between the treated and untreated control samples. For <i>ura4</i> alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura⁺ revertants is indicated on the figure. B. Serial tenfold-dilutions from <i>ura4-dup20</i> strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura⁺ revertants after the indicated treatments in the <i>ura4-dup20</i> strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM.</p