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The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5’ HIV LTR. Consequently, “block and lock” or “shock and kill” strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the T ATA box and a djacent s equences of H IV e ssential for T at trans -activation (TASHET) of the core promoter by host cell p re- i nitiation c omplexes of H IV (PICH) has been shown to be necessary for Tat trans -activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo . Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.

BackgroundImmunocytokines can strengthen anti-PD-(L)1 therapy by promoting T-cell survival, but their shortened half-life and systemic toxicity limit their clinical development. We developed a bifunctional anti-PD-1/IL7v (BICKI®IL7v) to selectively Cis-deliver IL-7 to PD-1+tumor-specific T-cells. RNAseq and TILs scRNAseq analyses demonstrate that IL-7R and IL-7R pathway gene expression prior ICI treatment is significantly correlated with better OS and/or PFS across several cancers. IL-7R expression is correlated with higher stemness and lower apoptosis markers, providing a strong rationale of co-targeting IL-7 & PD-1 to sustain durable tumor-specific-T-cell response. The Anti-PD-1/IL-7v aims to cis-deliver IL-7 and sensitizes PD-1+tumor-specific T-cells to antagonizes PD-1 inhibitory signal and provide long-term survival and proliferative signals.MethodsEfficacy of anti-PD1/IL7v was evaluated in Ectopic tumor (MC38) or orthotopic HCC (Hepa1.6) in hPD1KI mice. Anti-PD1/IL7v effect was evaluated on chronically-stimulated human T-cells by scRNAseq and FACs.ResultsAnti-PD-1/IL7v was designed using a high-affinity antagonist anti-PD-1, fused to a single point IL-7 mutein (IL7v) having lower affinity to IL-7R complex, to allow an optimal cis-potentiation of PD-1+T-cells and synergistic activation. Using an in vitro chronic stimulation model of human T cells, we demonstrated that anti-PD-1/IL7v promotes long-term reinvigoration proliferation/survival of stem-like-memory-TCF1+CD8+T-cells (>5 weeks), whereas IL-2/IL-15 promote short-term T-cell survival and differentiation into exhausted phenotype.Anti-PD1/IL-7v showed significant anti-tumor efficacy in-vivo in responsive and refractory mouse models in monotherapy or combination (8 different orthotopic or ectopic models). In the orthotopic HCC model, anti-PD-1/IL7v induced >60% complete response while anti-PD-1 or IL-7 alone has no effect. Using FTY720 agent blocking ingress of new T-cells within TME, we demonstrated that anti-PD1/IL7v anti-tumor efficacy is mediated by amplification of TILs but also involves migration of fresh T-cells into TME. Further analyses demonstrated that anti-PD1/IL7v enhances quality and biodistribution of T-cells by promoting intratumoral TCF1+stem-like-CD8+T-cell proliferation and T-cell migration into the tumor nest whereas anti-PD-1 induced mostly T-cell exclusion. These data correlate with capacity of anti-PD1/IL7v to induce integrins and adhesion molecules surface expression. Finally, high synergistic efficacy in combination therapy with Sorafenib in HCC model (p=0.023), Oxaliplatin in MC38 model(85%CR) has been observed illustrating the potential of combining chemotherapy with ICI and anti-apoptotic cytokine therapy.ConclusionsOur data validate the rational of selective delivery of IL-7 to PD-1+tumor-specific-T-cells to limit risk of I-O/I-O immunotoxicity and sustain long-lasting proliferation and survival of stem-like CD8+T-cells to strengthen PD-(L)1 therapy. Synergistic anti-tumor efficacy with tyrosine-kinase-inhibitor or chemotherapeutics agents was demonstrated highlighting the potential clinical benefit of combination therapy with anti-PD-1/IL7v.

BackgroundThe c-type lectin receptor CLEC-1 is a pattern recognition receptor1 expressed by endothelial and myeloid cells in mice, non-human primates, and humans. While genetic deletion of CLEC-1 in mice does not lead to any developmental defect, CLEC-1 deletion or CLEC-1 targeting using monoclonal antibodies increases damaged or necrotic cell antigen cross-presentation by cDC1 dendritic cells, as well as subsequent T-cell activation and anti-tumor response.2 However, the identification of CLEC-1 endogenous ligands and their relative involvement in the immune checkpoint activity of CLEC-1 remained to be fully investigated.MethodsEndogenous CLEC-1 ligand candidates were identified by affinity capture assays followed by LC/MS analysis. Ligand candidates were validated through protein-protein binding assays and Biacore affinity measurements. Through an immunization campaign, a library of anti-CLEC-1 monoclonal antibodies was generated and screened for CLEC-1 protein binding. Monoclonal antibodies were also assessed for their abilities to inhibit the binding of CLEC-1 to its newly identified endogenous ligands. Different classes of antagonist anti-CLEC-1 antibodies were thereby identified and subsequently evaluated for their anti-tumor efficacies in hepatocellular carcinoma (Hepa1.6) and colorectal cancer (MC38) preclinical models, using human CLEC-1 knock-in mice.ResultsWhile we confirm CLEC-1 specific binding to the E3 ubiquitin ligase TRIM212 and to the secreted histidine rich glycoprotein (HRG),3 we also identify several novel intra-cellular and cell surface CLEC-1 ligands. We show that the binding of CLEC-1 to these newly identified ligands is protein-specific, as deglycosylation does not impair CLEC-1 binding to its ligands. Finally, we document the antitumoral activities of several classes of antagonist anti-CLEC-1 monoclonal antibodies: while blocking of CLEC-1 binding to its secreted ligand HRG moderately increases anti-tumor responses, inhibition of CLEC-1 binding to its cytoplasmic membrane ligands significantly impairs MC38 tumor growth (n=12, p=0.04) and increases the overall survival of Hepa1.6-bearing mice (n=12, p=0.002), as compared to corresponding isotype control treatment.ConclusionsAltogether, by shedding new light on the role of CLEC-1/CLEC-1 ligand interactions, our results further dissect the mechanism of action of the myeloid checkpoint CLEC-1 in its ability to impair anti-tumor immunity and support its use as a novel and highly promising target for cancer immunotherapy.ReferencesDrouin M, Saenz J, Chiffoleau E. C-Type Lectin-Like Receptors: Head or Tail in Cell Death Immunity. Front Immunol. 2020;11:251.Drouin M, Saenz J, Gauttier V, et al. CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy. Sci Adv. 2022;8(46):eabo7621.Gao S, Wake H, Sakaguchi M, et al. Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A. iScience. 2020;23(6):101180.

The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ.

BackgroundMyeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering ‘don’t eat me’ and ‘don’t find me’ signals. Recent literature demonstrates that C-type lectin receptors (CLRs) normally constrain immune cell–mediated tissue damage by suppressing myeloid cell activation and then promote tumor immune evasion. We previously identified the orphan (CLRs) CLEC-1 as over-expressed in situation of established immune tolerance and reported that CLEC-1 expression by dendritic cells (DCs) and macrophages is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we reported that CLEC-1 is highly expressed by myeloid cells purified from human tumor micro-environment significantly more expressed by suppressive macrophages.MethodsAs DCs and macrophages are professional phagocytes of dying/dead cell, we evaluated whether CLEC-1 could be a receptor of damaged cells in the phagocytosis.ResultsWe found that CLEC-1 fusion protein, binds specifically to late apoptotic and secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray) or culture stress conditions. Importantly, we observed in vivo that CLEC-1 deficient mice, but not wild-type, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy (eg. Cyclophosphamide. We then generated, screened and identified different anti-human Clec-1 antagonist monoclonal antibodies (mAbs) with the capacity to block the CLEC-1/CLEC-1L interaction. We discovered that various antagonist CLEC-1 mAbs, but not non-antagonist CLEC-1 control mAbs, increase the phagocytosis of CLEC-1L-positive human tumor cells by human CLEC-1 expressing TGFβ-polarized DCs or macrophages. Indeed, TGFβ-polarized DCs phagocytosed more efficiently Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) as well as bare NSCLC cells (A549) when CLEC-1 is antagonized by antibodies. Furthermore, macrophages more productively engulfed Rituximab-opsonized Raji cells as well in the context of CLEC-1 blockade (2–3 fold increase). Moreover, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) and Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+) were likewise more phagocytosed by CLEC-1 blocked macrophages.ConclusionsAltogether, these data indicate illustrate that CLEC-1 broadly inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors.

Background HIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans -activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown. Results Using electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans -activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans -activation in living cells and correct PICH formation in vitro . The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat. Conclusion Our data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans -activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

AbstractImpaired inhibitory control contributes to the development, maintenance, and relapse of alcohol-dependence, but the neural correlates of this deficit are still unclear. Because inhibitory control has been labeled as an executive function, most studies have focused on prefrontal areas, overlooking the contribution of more “primary” structures, such as the motor system. Yet, appropriate neural inhibition of the motor output pathway has emerged as a central aspect of healthy behavior. Here, we tested the hypothesis that this motor inhibition is altered in alcohol-dependence. Neural inhibitory measures of motor activity were obtained in 20 detoxified alcohol-dependent (AD) patients and 20 matched healthy subjects, using a standard transcranial magnetic stimulation procedure whereby motor-evoked potentials (MEPs) are elicited in a choice reaction time task. Moreover, behavioral inhibition and trait impulsivity were evaluated in all participants. Finally, the relapse status of patients was assessed 1 year after the experiment. As expected, AD patients displayed poorer behavioral inhibition and higher trait impulsivity than controls. More importantly, the MEP data revealed a considerable shortage of neural motor inhibition in AD patients. Interestingly, this neural defect was strongest in the patients who ended up relapsing during the year following the experiment. Our data suggest a strong motor component in the neural correlates of altered inhibitory control in AD patients. They also highlight an intriguing relationship with relapse and the perspective of a new biomarker to follow strategies aiming at reducing relapse in AD patients.

Life and death decisions of metazoan cells hinge on the balance between the expression of pro- versus anti-apoptotic gene products. The general RNA polymerase II transcription factor, TFIID, plays a central role in the regulation of gene expression through its core promoter recognition and co-activator functions. The core TFIID subunit TAF6 acts in vitro as an essential co-activator of transcription for the p53 tumor suppressor protein. We previously identified a splice variant of TAF6, termed TAF6delta that can be induced during apoptosis. To elucidate the impact of TAF6delta on cell death and gene expression, we have employed modified antisense oligonucleotides to enforce expression of endogenous TAF6delta. The induction of endogenous TAF6delta triggered apoptosis in tumor cell lines, including cells devoid of p53. Microarray experiments revealed that TAF6delta activates gene expression independently of cellular p53 status. Our data define TAF6delta as a pivotal node in a signaling pathway that controls gene expression programs and apoptosis in the absence of p53.